Arts and Sciences, Irving K. Barber School of (Okanagan)
Chemistry, Department of (Okanagan)
DSpace
UBCO
Alsaggaf, Wejdan Taha
Doctor of Philosophy - PhD
University of British Columbia
A survey of literature indicates an increased interest in developing direct analysis of samples with minimum preparation. Paper spray ionization mass spectrometry (PSI/MS) was developed for this purpose over the last four years. More than 50 manuscripts have been published describing this technique but several aspects remain to be characterized and optimized. The overall objectives of this work are as follows: (i) to construct a lab-made PSI/MS set up and optimize parameters for the orientation of the paper substrate, (ii) to develop new achievement to understand the ionization mechanism, (iii) to understand the chemistry on the paper using different papers, (iv) to distinguish between solubility and micro-flow of the sample across PSI and liquid extraction surface analysis (LESA), (v) to demonstrate the potential of PSI as a metabolites fingerprinting technique (vi) to develop new approach for direct analysis of solid tissues without sample preparation, and (vii) to develop new achievement to understand the interaction between analyte and surface of the paper. Silica nanoparticles were used in this study. The orientation of the paper substrate and the optimal type of paper was determined. Interestingly, when the paper was dried in an oven to reduce a water content and non-polar solvents were used, the ionization was inhibited. When non-polar solvents were used on paper acclimatized to normal lab humidity, ionization was observed. These results indicate that residual water on the paper surface plays a role in the ionization mechanism. The results showed the hydrophilic properties of the paper leads PSI to be utilized as a separation/ionization technique at one-step. The sensitivity was compared over two instruments and two surface ionization techniques. The results showed better peak response in the MS/MS system. It was demonstrated that PSI has low accuracy to obtain a profile for complex samples such as saffron. Mechanical press extraction and paper substrate coated with silica nanoparticles were developed as potential improvements for the PSI method. Together, the data in this study demonstrate the potential of PSI as an alternative technique for direct analysis.
CHARACTERIZATION AND APPLICATIONS OF PAPER SPRAY IONIZATION MASS SPECTROMETRY by Wejdan Taha Alsaggaf Ph.D., The University of British Columbia, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE COLLEGE OF GRADUATE STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) February 2015 © Wejdan Taha Alsaggaf, 2015 ii Abstract A survey of literature indicates an increased interest in developing direct analysis of samples with minimum preparation. Paper spray ionization mass spectrometry (PSI/MS) was developed for this purpose over the last four years. More than 50 manuscripts have been published describing this technique but several aspects remain to be characterized and optimized. The overall objectives of this work are as follows: (i) to construct a lab-made PSI/MS set up and optimize parameters for the orientation of the paper substrate, (ii) to develop new achievement to understand the ionization mechanism, (iii) to understand the chemistry on the paper using different papers, (iv) to distinguish between solubility and micro-flow of the sample across PSI and liquid extraction surface analysis (LESA), (v) to demonstrate the potential of PSI as a metabolites fingerprinting technique (vi) to develop new approach for direct analysis of solid tissues without sample preparation, and (vii) to develop new achievement to understand the interaction between analyte and surface of the paper. Silica nanoparticles were used in this study. The orientation of the paper substrate and the optimal type of paper was determined. Interestingly, when the paper was dried in an oven to reduce a water content and non-polar solvents were used, the ionization was inhibited. When non-polar solvents were used on paper acclimatized to normal lab humidity, ionization was observed. These results indicate that residual water on the paper surface plays a role in the ionization mechanism. The results showed the hydrophilic properties of the paper leads PSI to be utilized as a separation/ionization technique at one-step. The sensitivity was compared over two instruments and two surface ionization techniques. The results showed better peak response in the MS/MS system. It was demonstrated that PSI has low accuracy to obtain a profile for complex samples such as saffron. Mechanical press extraction and paper substrate coated with silica nanoparticles were developed as potential improvements for the PSI method. Together, the data in this study demonstrate the potential of PSI as an alternative technique for direct analysis. iii Preface The work contained in this thesis was designed, conducted, analyzed, and interpreted by Wejdan T. Alsaggaf. Wejdan T. Alsaggaf collected samples. Versions of several of the chapters were presented at conferences, submitted and are in preparation to submit to scientific journals for publication as below:  A version of Chapter 2 was presented as a poster by Wejdan T. Alsaggaf and it was published as Wejdan T. Alsaggaf and Rob O’Brien (2012) An Investigation of Mechanisms Occurring in Paper Spray Ionization Mass Spectrometry in American Society of Mass Spectrometry (ASMS) conference, Vancouver, May 20-24, 2012.  Chapter 6 was presented as a poster by Wejdan T. Alsaggaf and was published as Alsaggaf WT, Murch SJ (2013) Paper Spray Ionization for Mass Spectrometry of Saffron Crocus sativus Extracts in the 11th Annual Natural Health Product Research Society of Canada (NHPRS) Conference, Kelowna, May 13-16, 2014.  Chapter 1 has been prepared as a manuscript and submitted for publication. Alsaggaf WT, O’Brien R (2014) An Overview on Paper Spray Ionization Mass Spectrometry, submitted November 26th, 2014, Arabian Journal of Chemistry.  Chapter 3 has been prepared as a manuscript and submitted for publication. Alsaggaf WT, O’Brien R (2015) A Simultaneous Determination of Natural Compounds in 35 Foodstuffs, submitted January 9th, 2015, Food Chemistry.  A manuscript has been prepared and submitted for publication. Alsaggaf WT (2015) Paper Spray Profile of a model plant extract: Saffron Crocus sativus, submitted January 10th, 2015, Food Chemistry. iv Table of Contents Abstract ................................................................................................................................... ii Preface .................................................................................................................................... iii Table of Contents ................................................................................................................... iv List of Tables ............................................................................................................................ x List of Figures ....................................................................................................................... xii List of Abbreviations ........................................................................................................... xvi Acknowledgements ............................................................................................................ xviii Dedication ............................................................................................................................. xix Chapter 1 Introduction ........................................................................................................... 1 1.1 Mass Spectrometry ............................................................................................................... 1 1.2 Ionization Stratiges ............................................................................................................... 2 1.3 Atmosheric Pressure Ionization (API) .................................................................................. 2 1.3.1 Electrospray Ionization (ESI) ........................................................................................... 2 1.3.1.1 ESI Mechanism ........................................................................................................ 3 1.3.1.2 Importance of Water for ESI Signals ....................................................................... 4 1.3.1.3 Applications of ESI .................................................................................................. 6 1.3.1.3.1 Hormones ............................................................................................................. 6 1.3.1.3.2 Carotenoids in Food and Biological Samples ...................................................... 6 1.3.1.3.3 Metabolomics ...................................................................................................... 9 1.3.1.3.4 Crocus sativus (Saffron) ...................................................................................... 9 1.4 Ambient Ionization Mass Spectrometry ............................................................................. 11 1.4.1 Paper Spray Ionization Mass Spectrometry (PSI/MS) ................................................... 11 1.4.1.1 Mechanism of PSI/MS ........................................................................................... 14 1.4.1.2 Parameters Affecting PSI/MS ................................................................................ 15 1.4.1.2.1 Geometry of Paper ............................................................................................. 15 1.4.1.2.2 Type of Paper ..................................................................................................... 16 1.4.1.2.3 Non-Polar Solvents Influence on PSI/MS .......................................................... 16 1.4.1.3 New Advancement in PSI/MS ............................................................................... 17 v 1.4.1.3.1 Sampling Method ............................................................................................... 17 1.4.1.3.2 A High Throughput Device ................................................................................ 19 1.4.1.4 Applications of PSI ................................................................................................ 20 1.4.2 Liquid Extraction Surface Analysis (LESA) .................................................................. 25 1.5 Mass Analyzer .................................................................................................................... 25 1.5.1 Triple Quadrupole Mass Spectrometry (MS/MS) .......................................................... 25 1.5.2 Time of Flight Mass Spectrometry (TOF/MS) .............................................................. 26 1.6 Extraction Methodology ..................................................................................................... 26 1.7 Mechanical Press Technique .............................................................................................. 28 1.8 Silicon Dioxide Nanoparticles ............................................................................................ 28 1.9 Research Aims and Objectives ........................................................................................... 29 Chapter 2 The Role of Paper Surface on Paper Spray Ionization Mass Spectrometry ................................................................................................................................................. 31 2.1 Synopsis .............................................................................................................................. 31 2.2 Experimental ....................................................................................................................... 32 2.2.1 Chemicals and Materials ................................................................................................ 32 2.2.2 Apparatus........................................................................................................................ 34 2.2.3 Paper Substrate Imaging ................................................................................................. 35 2.2.4 Characteristic of Instrumental Parameters ...................................................................... 35 2.2.5 Characteristic of Paper Substrate.................................................................................... 35 2.2.6 Repeatability ................................................................................................................... 37 2.2.7 Figure of Merits of the Developed PSI/MS Technique .................................................. 37 2.3 Results................................................................................................................................. 37 2.3.1 PSI/MS Parameters Optimization and Spectra Characterisitics ..................................... 37 2.3.2 Influence of Solvent ....................................................................................................... 41 2.3.3 Characteristics of the Paper Substrate ............................................................................ 43 2.3.4 The Influence of Water Molecules ................................................................................. 47 2.3.5 Intra- and Inter-Day Repeatability .................................................................................. 48 2.3.6 Figure of Merits of the Developed PSI/MS Technique .................................................. 49 2.4 Discussion ........................................................................................................................... 53 2.4.1 The Role of Water Clusters ............................................................................................ 53 2.4.2 A Cone Shaped Distribution of Sprayed Jets ................................................................. 55 2.4.3 Properties of Paper ......................................................................................................... 56 vi 2.4.3.1 Solubility and Polarity ........................................................................................... 56 2.4.3.2 Size of Analyte and Pores ...................................................................................... 58 2.4.3.3 Trace Element and Analyte/Surface Interaction .................................................... 59 2.4.4 Paper Tip Constancy ...................................................................................................... 60 Chapter 3 Separation of Complex Samples on Paper ........................................................ 61 3.1 Synopsis .............................................................................................................................. 61 3.2 Experimental ....................................................................................................................... 61 3.2.1 Chemicals and Reagents ................................................................................................. 61 3.2.2 Apparatus........................................................................................................................ 64 3.2.3 Analytical Performance .................................................................................................. 64 3.2.4 Solvent Effects ............................................................................................................... 65 3.2.5 Quantitative Analysis of Model Compounds ................................................................. 65 3.2.6 Vegetable and Fruit Samples .......................................................................................... 66 3.2.7 Saffron Samples ............................................................................................................. 67 3.2.8 Commercial Samples ...................................................................................................... 68 3.3 Results................................................................................................................................. 68 3.3.1 Comparison Between PSI and Two Standard Separation Methods ............................... 68 3.3.2 Characteristics of PSI/MS Spectra ................................................................................. 71 3.3.3 Analytical Performance .................................................................................................. 71 3.3.4 Paper/Solvent Interaction ............................................................................................... 73 3.3.4.1 Trace Elements ...................................................................................................... 73 3.3.4.2 Solvents ................................................................................................................. 73 3.3.5 Quantitative Experiments ............................................................................................... 74 3.3.5.1 Quantification Analysis of Natural Carotenoids in Fruit and Vegetables ............. 74 3.3.5.2 Quantification Analysis of Natural Carotenoids in Commercial and Saffron ....... 76 3.3.5.3 Quantification Analysis of Caffeine in Samples .................................................... 77 3.3.5.4 Recovery ................................................................................................................ 78 3.4 Discussion ........................................................................................................................... 78 3.4.1 PSI/MS as Separation/Ionization Technique .................................................................. 78 3.4.2 PSI Efficiency for Quantitative Analysis ....................................................................... 79 Chapter 4 Comparison of Three Ionization Techniques: Corona Discharge .................. 81 4.1 Synopsis .............................................................................................................................. 81 4.2 Experimental ....................................................................................................................... 81 vii 4.2.1 Chemicals and Reagents ................................................................................................. 81 4.2.2 Apparatus........................................................................................................................ 81 4.2.3 Analytical Performance .................................................................................................. 82 4.2.4 F-test ............................................................................................................................... 82 4.3 Results................................................................................................................................. 82 4.3.1 MS Spectra Characterization .......................................................................................... 82 4.3.2 Analytical Performance Using Three Ionization Techniques ......................................... 84 4.3.3 F-test ............................................................................................................................... 86 4.4 Discussion ........................................................................................................................... 87 Chapter 5 Comparison of Two Mass Analyzers and Two Surface Ionization Sources ................................................................................................................................................. 89 5.1 Synopsis .............................................................................................................................. 89 5.2 Experimental ....................................................................................................................... 89 5.2.1 Chemicals and Reagents ................................................................................................. 89 5.2.2 Triple Quadrupole Mass Spectrometer Conditions ........................................................ 90 5.2.3 Tim of Flight Mass Spectrometer Conditions ................................................................ 90 5.2.4 Surface Ionization Sources Instrumentation ................................................................... 90 5.2.5 Analysis of Samples ....................................................................................................... 91 5.2.5.1 Recommended Procedures for the Quantitative Analysis of Each Compound ..... 91 5.2.5.2 Analysis of Thyroid Hormones.............................................................................. 91 5.3 Results................................................................................................................................. 92 5.3.1 Comparison Between Two Mass Analyzers ................................................................... 92 5.3.1.1 Comparison of Mass Spectra Obtained by TQ/MS and TOF with PSI… ............. 92 5.3.1.2 Comparison of Analytical Performance ................................................................ 95 5.3.1.3 Analysis of Thyroid Hormones.............................................................................. 95 5.3.2 Comparison Between Two Surface Ionization Sources ................................................. 98 5.3.2.1 Comparison of Mass Spectra Obtained by Two Surface Ionization Sources ........ 98 5.3.2.2 Comparison of Analytical Performance .............................................................. 101 5.4 Discussion ......................................................................................................................... 102 5.4.1 Sensitivity of PSI Lab-Made Apparatus via Different Instruments.............................. 102 5.4.2 Mobility and Analyte Extraction Influence with Two Surface Ionization Lab-Made Apparatus ................................................................................................................................... 104 Chapter 6 PSI Profile of Saffron Crocus sativus Extracts ............................................... 106 viii 6.1 Synopsis ............................................................................................................................ 106 6.2 Experimental ..................................................................................................................... 106 6.2.1 Chemicals and Reagents ............................................................................................... 106 6.2.2 Sample Preparation ....................................................................................................... 106 6.2.3 Apparatus...................................................................................................................... 107 6.2.4 PSI/MS Profile ............................................................................................................. 107 6.2.5 Analytical Performance of the Developed PSI/MS ...................................................... 107 6.2.6 Quantification Analysis of Known Metabolites ........................................................... 107 6.3 Results............................................................................................................................... 108 6.3.1 Profiles of Two Different Saffron Samples Using PSI/MS .......................................... 108 6.3.2 The Repeatability of PSI Profile ................................................................................... 115 6.3.3 PSI/MS Profile of the Blanks ....................................................................................... 116 6.3.4 PSI/MS Spectra of Known Metabolites in Saffron ...................................................... 118 6.3.5 Analytical Performance for the Selected Compounds .................................................. 119 6.3.6 Characteristics of the Solvent Extraction ..................................................................... 122 6.3.7 Quantitative Analysis of Known Metabolites in Saffron by Regions .......................... 123 6.3.8 Quantitative Analysis of Known Metabolites in Different Saffron Tissues.. ............... 125 6.3.9 Accuracy ....................................................................................................................... 127 6.3.10 Study the Quality of Various Saffron Samples ............................................................ 127 6.3 Discussion ......................................................................................................................... 128 Chapter 7 New Approach for Rapid Analysis of Solid Sample ....................................... 132 7.1 Synopsis ............................................................................................................................ 132 7.2 Experimental ..................................................................................................................... 132 7.2.1 Chemicals and Reagents ............................................................................................... 132 7.2.2 Apparatus...................................................................................................................... 132 7.2.3 Sample Preparation ....................................................................................................... 133 7.2.4 Precision and Recovery ................................................................................................ 133 7.3 Results............................................................................................................................... 135 7.3.1 Parameters Optimization .............................................................................................. 135 7.3.2 Quantitative Analysis of Thyroid Hormones................................................................ 136 7.3.3 Method Comparison ..................................................................................................... 138 7.4 Discussion ......................................................................................................................... 139 7.4.1 Extraction Efficiency, Accuracy and Precision of MPE .............................................. 139 7.4.2 Stability of Carotenoids in MPE Paper ........................................................................ 142 ix Chapter 8 Silica Nanoparticles-Paper Spray Ionization Mass Spectrometry ................ 144 8.1 Synopsis ............................................................................................................................ 144 8.2 Experimental ..................................................................................................................... 145 8.2.1 Chemicals and Reagents ............................................................................................... 145 8.2.2 Apparatus...................................................................................................................... 145 8.2.3 Silica Nanoparticles Film ............................................................................................. 145 8.2.4 Imaging of Paper Substrate .......................................................................................... 145 8.3 Results............................................................................................................................... 145 8.3.1 Paper Substrate Characterization .................................................................................. 145 8.3.2 Method Comparison ..................................................................................................... 149 8.3.3 Accuracy ....................................................................................................................... 151 8.4 Discussion ......................................................................................................................... 151 8.4.1 Ionization Efficiency .................................................................................................... 151 8.4.2 Ionization Mechanism .................................................................................................. 153 Chapter 9 Conclusion .......................................................................................................... 155 9.1 The Role of Paper Surface on Paper Spray Ionization Mass Spectrometry ...................... 155 9.2 Separation of Compounds from Complex Sample Matrices by Paper Substrate ............. 156 9.3 Comparison of Three Ionization Techniques/Corona Discharge ...................................... 156 9.4 Comparison of Two Mass Analyzers and Two Surface Ionization Sources ..................... 157 9.5 PSI Profile of a Model Plant Extract: Saffron Crocus sativus .......................................... 158 9.6 New Approach for Rapid Analysis of Solid Sample ........................................................ 158 9.7 Silica Nanoparticles-Paper Spray Ionization Mass Spectrometry .................................... 158 9.8 Strengths and Limitations of the Thesis Research ............................................................ 159 9.9 Future Research on Paper Spray Ionization Mass Spectrometry ...................................... 159 References ............................................................................................................................. 160 x List of Tables Table 1.1 Medical benefits of six natural compounds ............................................................ 8 Table 1.2 The chemical formula and the class of several metabolites in saffron ................. 10 Table 1.3 Key advancements on paper spray ionization mass spectrometry ....................... 13 Table 1.4 Analysis of contaminants in various foods using paper spray ionization mass spectrometry*…….………………………………………………….…..22 Table 1.5 Applications of paper spray ionization mass spectrometry (PSI/MS) ................. 24 Table 2.1 Preparation of standard solutions ........................................................................ 33 Table 2.2 Mass spectrometry optimized parameters in both ion modes.............................. 36 Table 2.3 Characteristic of five types of paper .................................................................... 37 Table 2.4 Trace elements that detected in paper substrate using elemental distribution spectra ................................................................................................................... 46 Table 2.5 Linear equation of the selected compounds ......................................................... 51 Table 2.6 Analytical features of the developed paper spray ionization mass spectrometry and other reported ionization methods ................................................................. 52 Table 3.1 The extracted part of different fruit and vegetables ............................................. 67 Table 3.2 Comparison between paper spray ionization and two standard methods, including (a) paper chromatography and (b) thin layer chromatography ............................. 70 Table 3.3 Optimized parameters and analytical performance for paper spray ionization .... 72 Table 3.4 Concentrations of each carotenoid over 34 samples, and calculated % recovery .............................................................................................................................. 75 Table 4.1 Optimized parameters for paper spray ionization, electrospray ionization and electrospray chemical ionization ......................................................................... 83 Table 4.2 Analytical performance for paper spray ionization, electrospray ionization and electrospray chemical ionization .......................................................................... 86 Table 5.1 Parameters and linear regression obtained by either triple quadrupole or time of flight mass spectrometry with paper spray ionization .......................................... 93 Table 5.2 Analytical performance obtained by either triple quadrupole or time of flight mass spectrometry with paper spray ionization .................................................... 96 Table 5.3 Levels of the tested hormones (T3&T4) in different samples determined by standard addition using the developed paper spray ionization technique............. 97 xi Table 5.4 Recovery and inter-day repeatability by using triple quadrupole and time of flight mass spectrometry with paper spray ionization .................................................... 98 Table 5.5 Parameters and linear regression obtained by either paper spray ionization or liquid extraction surface analysis with triple quadrupole mass spectrometry .... 100 Table 5.6 Analytical performance obtained by either paper spray ionization or liquid extraction surface analysis with triple quadrupole mass spectrometry .............. 102 Table 6.1 Paper spray ionization mass spectrometry profiles of Egyptian (S1) and Indian (S2) saffron ......................................................................................................... 109 Table 6.2 Identification of several investigated metabolites .............................................. 111 Table 6.3 Summary of the total number of the observed compounds over six paper substrates............................................................................................................. 118 Table 6.4 Linear equations for the selected compounds..................................................... 121 Table 6.5 Analytical performance of seven known metabolites using methanol as a mobile phase ................................................................................................................... 122 Table 7.1 The concentrations (µg/mL) of several organic compounds in eleven solid samples that pressed on the paper substrate using mechanical press extraction technique ............................................................................................................. 137 Table 7.2 The concentrations (µg/mL) of several organic compounds using mechanical press extraction and standard extraction method (sonication) ............................ 139 Table 8.1 Applied voltages and major ions using standard and coated paper .................... 150 xii List of Figures Figure 1.1 Schematic diagram of mass spectrometry regions ................................................ 1 Figure 1.2 Diagram of paper spray ionization mass spectrometry ....................................... 12 Figure 1.3 Number of published works on paper spray ionization mass spectrometry per year ...................................................................................................................... 12 Figure 1.4 (A) A capillary sampler prepared by coating it with an internal standard (IS) solution through capillary action and drying in air to form an IS coating on its inner surface; (B) Capillary sampler to deposit the blood sample onto a paper substrate to prepare a dried blood spot containing internal standard ................... 18 Figure 2.1 Triple quadrupole mass spectrometry in the left side, and (A) lab-made paper spray ionization apparatus. (B) Solvent will pass thruogh the paper by using capillary tube. (C) Infused injection method ...................................................... 34 Figure 2.2 The optimized geometric parameters for paper spray ionization: (A) the distance between the tip of the paper substrate and MS inlet and (B) the position at angle 60º, 75º, 90º, 120º, and 140º from left to right..................................................... 38 Figure 2.3 The effect of flow rate. (A) The plot show the variety in peak responses using different flow rate, and (B) the time of analysis was obtained within several minutes at different flow rate ............................................................................... 40 Figure 2.4 The paper spray ionization mass spectrometry spectra for (A) caffeine, (B) astaxanthin, (C) 17α-hydroxyprogesterone, (D) T4 using 10 µg/mL in positive ion mode ............................................................................................................. 41 Figure 2.5 (A) Scanning electron microscope image of the analyte that adsorbed on the paper substrate (thin fiber) where it was not obtained on the paper substrate without the addition of the analyte, and (B) the optimal solvent for each compound, where methanol and acetone was obtained as optimal solvent for progesterone and carotenoids, respectively. A mixture of acetone and acetonitrile (ACN) was the optimal solvent for thyroid hormones. For steroid hormones, the optimal solvent was a mixture of methanol and acetonitrile ............................... 43 Figure 2.6 The paper effect (Whatman filter paper grade 1 (G1), grade 4 (G4), grade 5 (G5), glass microfiber filter G6 (GFP), and 1PS phase separator (SP) were compared. Whatman filter paper grade 1 showed the highest peak intensity while xiii glass microfiber showed the lowest peak intensity.............................................. 45 Figure 2.7 Different adduct peaks formation using Whatman filter paper grade 1 at 10 µg/mL of each analyte. ....................................................................................... 47 Figure 2.8 The relative standard deviation values within 21 replicates using either T3 or T4. The values ranged between 3-5 % within replicates lower than six; and it reached 25 % within 21 replicates ...................................................................... 49 Figure 2.9 Calibration curves of progesterone and T4. A proportional calibration curve was obtained for β-carotene, where this compound showed low peak response ....... 50 Figure 2.10 The ions sprayed in a cone shape with different intense .................................... 56 Figure 3.1 Chemical structures of the tested compounds .................................................... 63 Figure 3.2 The paper spray ionization time of flight mass spectrometry; paper spray ionization apparatus and syringe pump on the right side ................................... 65 Figure 3.3 The natural pigments solution (astaxanthin, zeaxanthin, and crocin) that was separated using (A) paper spray ionization, (B) paper chromatography and (C) thin layer chromatography (C)............................................................................. 69 Figure 3.4 Solvent effect on mobility/extraction of the tested compounds. Acetone was selected as a mobile phase for quantitative analysis of these compounds ........... 74 Figure 3.5 Standard addition curve of (A) astaxanthin, (B) kaempferol, (C) β-carotene, and (D) zeaxanthin in mango ..................................................................................... 76 Figure 3.6 The concentrations of caffeine in different samples ........................................... 77 Figure 4.1 Calibration curve of astaxanthin using (A) electrospray chemical ionization, (B) electrospray ionization, and (C) paper spray ionization ...................................... 85 Figure 5.1 Image of lab-made liquid extraction surface analysis (LESA) setup with triple quadrupole mass spectrometry. The right side of the probe was responsible for making the liquid micro junction on the paper substrate and the extracted analyte flow to electrospray probe via the left side .......................................................... 91 Figure 5.2 The peak responses obtained by (A) triple quadrupole and (B) time of flight mass spectrometry with paper spray ionization .................................................. 94 Figure 5.3 The peak responses obtained by paper spray ionization and liquid extraction surface analysis with triple quadrupole mass spectrometry .............................. 101 Figure 6.1 The number of investigated metabolites within 30 minutes where X refer to time xiv of analysis .......................................................................................................... 110 Figure 6.2 Chemical structures of identified metabolites ................................................... 114 Figure 6.3 The total number of the found metabolites within a wide range of relative standard deviation for (A) Indian and (B) Egyptian saffron .............................. 115 Figure 6.4 The total number of compounds observed over six paper substrates using methanol as a mobile phase ............................................................................... 117 Figure 6.5 Calibration curve of (A) isophorone, (B) β-ionone, (C) safranal, and (D) quercetin using methanol as a mobile phase ..................................................... 120 Figure 6.6 Extractive solvent optimization using methanol, acetone, acetonitrile, toluene and hexane. Methanol gave the highest extraction yield ................................... 123 Figure 6.7 Concentrations of the selected compounds over five saffron samples. Crocin was obtained with the highest level .................................................................. 125 Figure 6.8 Concentrations of the selected compounds in saffron Crocus sativus flower (A) stigma, (B) petals, and (C) corm using standard addition method. The results show that corm contains all the selected compounds with different levels ....... 126 Figure 6.9 Concentrations of safranal and crocin in saffron Crocus sativus collected from different regions. Spanish saffron (S4) showed the highest quality of all saffron samples, where the highest level 24 and 32 µg/mL were observed in Spanish saffron for safranal and crocin, respectively ....................................................... 128 Figure 7.1 (A) Mechanical press extraction technique. The right images show different food samples, which was pressed on the paper, including spinach, lettuce, saffron, dill, and broccoli. The below images show the proposed apparatus of the technique. (B) The lab-made MPE apparatus, where the sample was placed between the two pieces of metal during the process. (C) The paper and the pressed sample (lettuce) after cut the paper into triangle ............................................................. 134 Figure 7.2 The recovery percentage within several days. The recovery decreased with increasing the time after MPE was applied ....................................................... 136 Figure 7.3 Standard addition curve of (a) proline, (b) isophorone, (c) quercetin, (d) gallic acid, (e) β-ionone, (f) kaempferol in zucchini ................................................... 138 Figure 7.4 (A) Few drops of solvent is added on the solid sample, the sample is pressed on the paper under high pressure (about 103 psi). The paper is cut into triangle xv shape for the next analysis. (B) The solvent is pass through the layers (outer and inner) of the solid sample, which affects the extraction efficiency ................... 141 Figure 8.1 Scanning electron microscopy images of paper substrate (whatman filter paper grade 1) after it was coated with silica nanoparticles film. The lower image shows the silica nanoparticles coated the surface of cellulose fiber .................. 146 Figure 8.2 Scanning electron microscopy image of paper substrate (whatman filter paper grade 1) after immersion in silica nanoparticles solution (15 minutes). The spectra showed the existence of silica nanoparticles within range of 2.2 - 36 %wt. Many pores on the paper were not filled by silica nanoparticles ............. 147 Figure 8.3 Scanning electron microscopy image of paper substrate (whatman filter paper grade 1) after immersion in silica nanoparticles solution (72 h). The spectra showed the existence of silica nanoparticles within range of 30 - 36 %wt. Most pores on the paper were filled by silica nanoparticles ....................................... 148 Figure 8.4 Comparison of peak responses with standard and coated papers. The plot show peak responses with coated paper higher than standard paper ......................... 151 Figure 8.5 The schematic of the proposed interaction between silicon dioxide nanoparticles and cellulose molecules ..................................................................................... 154 xvi List of Abbreviations CAN Acetonitrile APCI Atmospheric Pressure Chemical Ionization API Atmospheric Pressure Ionization CI Chemical Ionization CRM Charge Residue Model DART Direct Analysis in Real Time DESI Desorption Electrospray Ionization EI Electron Ionization ESCI Electrospray Chemical Ionization ESI Electrospray Ionization FT-ICR/MS Fourier Transform Ion Cyclotron Resonance Mass Spectrometry FT-IR Fourier Transform Infrared GC Gas Chromatography HPLC High Performance Liquid Chromatography IEM Ion Evaporation Model IS Internal Standard LDI Laser Desorption Ionization LESA Liquid Extraction Surface Analysis LLE Liquid-Liquid Extraction MALDI Matrix Assisted Laser Desorption Ionization MPE Mechanical Press Extraction MS Mass Spectrometry xvii MS/MS Triple Quadrupole Mass Spectrometry NSAIDs Nonsteroidal Antiflammatory Drugs PAHs Polysyclic Aromatic Hydrocarbons PC Paper Chromatography PCBs Poly Chlorinated Biphenyls PD/MS Plasma Desorption Mass Spectrometry PEG Polyethylene Glycol PFE Pressurized Fluid Extraction PPG Polypropylene Glycol PSI Paper Spray Ionization RSD Relative Standard Deviation S/N Signal-to-Noise SALDI Surface Assisted Laser Desorption Ionization SE Standard Extraction SEM Scanning Electron Microscopy TLC Thin Layer Chromatography TOF/MS Time of Flight Mass Spectrometry tp Tip Position tp Tip Distance TQ/MS Triple Quadrupole Mass Spectrometry UV Ultraviolet xviii Acknowledgements I would like to thank the faculty and staff at the UBC, who have supported me to continue my work in this field. I would like to thank my supervisor Dr. Susan Murch and Dr. Rob O’Brien for valuable and expert supervision, advice, and encouragement during the experimental work and preparation of the thesis. Her door is always open for advices and support. I would like to thank my committee Dr. Kirsten Wolthers and Dr. Edward Neeland for encouragement and supports during the preparation the thesis. I would like to thank David Arkinstall for his excellent contribution on the SEM imaging for paper substrate. I would like to thank King Abdulaziz University-Jeddah, Kingdom of Saudi Arabia and Saudi Cultural Bureau-Ottawa, Canada for financial support and helpful critiques during my education. Special thanks are owed to my parents, who have advised and supported me throughout my years of education and who have tolerated, provided much over the years including love and support. My deepest gratitude to them who have supported me all the way to the point where now I am, no words can sufficiently express my gratitude towards my parents. I am thankful to my brothers and my sisters for their love and support over the years. xix Dedication To my parents for the love, being my all time support, big help in so many ways and encouragement… Wejdan T. Alsaggaf 1 Chapter 1 Introduction 1.1 Mass Spectrometry Mass spectrometry (MS) is used for analysis of a wide range of molecules including natural products, drugs, and environmental pollutants. Both qualitative identification as well as sensitive quantification can be provided by MS (Luftman et al., 2004; Rouessac and Roussac, 2007; Poole, 1999; Sherma, 2002). MS technique requires analytes to be converted to gas phase and this is accomplished in a part referred to as the ionization source. Ions can be identified based on their mass-to-charge ratio using various types of magnetic and electric fields, in a region called the mass analyzer. Ions are collected as responses (intensity of the ion formed) and the signals are translated to digital data (Figure 1.1) (Luftman et al., 2004; Rouessac and Rouessac, 2007; Poole, 1999; Sherma, 2002; Hoffman and Stroobant, 2007; Banerjee and Mazumbdar, 2012). Figure 1.1 Schematic diagram of mass spectrometry Detectore Mass analyzer Ionization source 2 1.2 Ionization Strategies MS analysis requires the analyte to be converted into a gas-phase ion (Luftman, 2004). This process can be accomplished using different ionization strategies that are optimized for individual compounds and sample matrices (Luftman, 2004). The earliest commercially available ion sources operated in the vacuum region, and were called electron ionization (EI) and chemical ionization (CI) sources. The first chromatographic systems coupled to a mass spectrometer were gas chromatography (GC) but it has limited analysis due to the variability of responses among volatile and thermally stable molecules (Luftman, 2004; Dole et al., 1968; Fenn, 2002). MacFarlane and his co-workers discovered Plasma Desorption ionization MS (PDMS) technique (Dole et al., 1968; Fenn, 2002; Hilf, 1993). In 1984, electrospray ionization (ESI) was used for biomolecule analysis (Dole et al., 1968; Fenn, 1996; Hilf, 1993; Luftman, 2004; Dole et al., 1968; Fenn, 1996; Hilf, 1993). ESI is considered as one of atmospheric pressure ionization techniques. 1.3 Atmospheric Pressure Ionization (API) Atmospheric pressure ionization (API) is a term describing chemical ionization that occurs at atmospheric pressure. API techniques may provide more sensitivity and accuracy than the classical techniques (Niessen, 1998; Bruins, 1994). Several techniques are developed to be used under atmospheric pressure such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photo ionization (APPI). 1.3.1 Electrospray Ionization (ESI) Electrospray ionization mass spectrometry (ESI/MS) is the most commonly used ionization method because it provides a sensitive, robust, and reliable tool for studying both non-volatile and thermally labile biomolecules that could not be analyzed by other techniques (Sherma, 2004; Chai et al., 2003). ESI uses electrical potential to assist the transfer of ions from the solution to the gas phase before the ions are passed into the MS (Sherma, 2004). In this technique, small fine charged droplets are produced by applying a high voltage between a capillary and counter electrodes; ions are produced in different mechanisms (Chai et al., 2003; Konermann et al., 2013). Neutral species can also be converted to ionic form in the solution or the gas phase by protonation or cationisation (e.g. association with Na+ or K+) (York et al., 1990; Konermann et al., 2013). This method is especially useful in producing ions from molecules with a large size because it overcomes the propensity of these molecules 3 to fragment when ionized (Sherma, 2004; Chai, 2003;York et al., 1990; Konermann et al., 2013). 1.3.1.1 ESI Mechanism The mechanism of ESI can be consider to consist of three distinct process (i) production of charged droplets, (ii) the evolution of the charged droplets, and (iii) the formation of the final ions in gas phase (Cook et al., 2011; Dole et al., 1968). Several factors influence the initial production of charged droplets including; solvent evaporation, acid-base reactions, droplet fission, properties of the solvents, sample matrix, concentration of ionic analyte and/or solvent flow rate (Dole et al., 1968; Iribarne & Thomson, 1976; Konermann et al., 2013). Evaluation of the charged droplets is influenced by droplet size, charge, surface activity and ion-pairing (Cook et al., 2011; Dole et al., 1968; Iribarne & Thomson, 1976; Konermann et al., 2013). A Taylor cone is formed at the capillary electrode where a fine charged droplet is produced (Konermann et al., 2013). The formation of ions is influenced by adduct association with common cations such as H+, NH4+, Na+, and/or K+ (Konermann et al., 2012). The droplet carries the charges until the surface tension reached the Rayleigh limit, causing the droplets reduce in size (Konermann et al., 2013). This phenomenon is called Columbic repulsion (Konermann et al., 2013; Wu et al., 2013). The main mechanisms were suggested for this process are Charge Residue Model (CRM) and the Ion Evaporation Model (IEM) (Konermann et al., 2013; Wu et al., 2013). CRM was proposed in 2000 (Konermann et al., 2013). In this mechanism, a series of droplets evaporated, leading to production of fine droplets, which bring one or more charges (Konermann et al., 2013; Dole et al., 1968; Iribarne & Thomson, 1976). In 1979, Thomson and Iribarne proposed IEM. After the droplets have reached a critical size (droplet diameter ≤ 20 nm); ion evaporation mechanisms may occur (Iribarne & Thomson, 1976). This leads to production of multiple gas-phase ions (Konermann et al., 2013; Dole et al., 1968; Iribarne & Thomson, 1976). Other mechanisms occur that may affect ion formation including electrochemical oxidation and reduction the analyte (Konermann et al., 2013; Dole et al., 1968; Iribarne & Thomson, 1976). 4 1.3.1.2 Importance of Water for ESI Signals It is well established that in order to obtain signals in MS while using an ESI source that one needs to add at least some water to the eluent flow. A more careful analysis reveals that the addition of water is simply the easiest way to make the solution conductive, as water is a polar ionizable solvent. This observation is explained in the following discussion. The sensitivity of ESI method is directly correlated to the ionization efficiency, or in other words, the effectiveness of producing gas-phase ions from analyte molecules in solution. The capacity to transfer the charged species from atmospheric pressure to the low-pressure region of the mass analyzer (transmission efficiency) is another factor that influences the sensitivity (Kebarle and Tang, 1993; Schneider et al., 2006; Cole, 2000; Cech and Enke, 2001). Increasing ionization efficiency leads to better response, limits of detection (LOD) and limit of quantification (LOQ) (Wilm and Mann, 1994; Wahl et al., 1993; Goodlett et al., 1993; Henderson and Mcindoe, 2006). Ionic solution chemistry plays an important role in the ionization efficiency, which is influenced by a number of factors including analyte properties (Sjoberg et al., 2001; Page et al., 2007). Electrolyte concentration and pH will all influence the analytical ion signal. However, some of the most important factors are related to properties of the eluents, including volatility, surface tension, conductivity, ionic strength, flow rate, and/or solvent polarity. As suggested earlier, no ions are observed when using non-polar solvents without the addition of another ionic or polar solvent. The use of water as one of the eluent solvents is one of the easiest ways to enable ionization and thus high ionization efficiency (Sjoberg et al., 2001; Page et al., 2007). Although, the water fraction of eluent composition can strongly influence the response with ESI (Sjoberg et al., 2001; Page et al., 2007). The chemical composition of the electrosprayed ionic solution might be altered during the spray process due to electrochemical redox reactions (Sjoberg et al., 2001; Page et al., 2007; Blades et al., 1991; Zhou and Cook, 2001), and this reactivity may be another role that water impacts (Blades et al., 1991; Zhou and Cook, 2001). In any case, the overall ionization efficiency tends to increase as the mole fraction of water in the eluent increases. (Wilm and Mann, 1994; Wahl et al., 1993; Goodlett et al., 1993; Henderson and Mcindoe, 2006). In most cases, polar compounds are the most effectively ionized in mass spectrometry using ESI methods, and many of these can be ionized as solutes in polar solvents (Sannino et 5 al., 2004; Henderson and Mcindoe, 2006). However, despite the role of charged non-polar droplets, Sannino has demonstrated that non-polar solvents are not successful in ESI without the addition of water (Sannino et al., 2004; Henderson and Mcindoe, 2006). The electrosprayed ionic solution in ESI containing the analytes of interest is scattered into a fine aerosol. Because the ion formation involves extensive solvent evaporation (desolvation), the typical solvents for electrospray ionization are prepared by mixing water with organic compounds such as methanol, and acetonitrile (Sannino et al., 2004; Henderson and Mcindoe, 2006; Sjoberg et al, 2001). As previously discussed, the formation of a sprayed solution at the tip of ESI capillary electrode (Taylor cone) is critical in the formation of a stable ESI spray. It is believed that it is the presence of water, or other polar solvent, that enables Taylor cone formation (Henderson and Mcindoe, 2006). To date, most studies have been focused on structurally similar compounds in order to better grasp the factors that affect ionization efficiency. Henriksen et al. determined the response of thirty-one acidic analytes in pure acetonitrile, neat methanol, and mixtures of each organic solvent with water (1:1) in order to indicate the relative influence of acidity and polarity on negative-ion response. The results indicated that compounds ionized better in methanol than acetonitrile in the presence of water rather than pure solvent where the number of protons increased with the addition of water molecules. Schug and McNair (2002) compared the ionization responses of six pharmaceutical compounds with various solvent conditions (Schug and McNair, 2002). The tendency to form cluster ions was found to be related to the structure and properties, including polarity and nature of substituents group, of each compound (Schug and McNair, 2002). More recently, these researchers examined the negative ion mode of a number of analytes, which have similarity in the structure and related it to analyte acidity (Schug and McNair, 2003). Both studies were carried out in mixtures of acetonitrile and water using a series of substituted benzoic acids and pharmaceutical compounds (Schug and McNair, 2002 & 2003). The results of both studies indicated that compounds ionized better in the present of water rather than pure solvent. In another study, it was indicated that the sodium ion is known to form clusters with solvents to lower the energy hindrance for ion evaporation (Kebarle and Tang, 1993). This cluster has a high tendency to occur in the presence of water molecules (Schug and 6 McNair, 2002; Kebarle and Tang, 1993). The probability to form clusters increases with mole fraction of water in the eluent. It was indicated that the gas-phase ions carrying the analytes to the MS inlet consists of clusters containing water and other polar solvents present in the eluent (Schug and McNair, 2002; Kebarle and Tang, 1993). In summary, all the previous studies show that the ionization efficiency improves with the presence of water molecules in the eluent and furthermore ESI sprays are not obtainable in non-polar solvents. 1.3.1.3 Applications of ESI 1.3.1.3.1 Hormones ESI has been used for hormonal analyses in numerous types of samples (Gounden et al., 2014, Kunisue et al., 2010, Szeitz et al., 2014; McWhinney et al., 2010; Staufenbiel et al., 2013). Over 6491 published works have described analysis of thyroid hormones with mass spectrometry, with over 950 publications using ESI (Science direct, Jun 9th, 2014). For cortisone and hydrocortisone hormones, approximately 65 publications reported using ESI, while over than 526 manuscripts were published using MS techniques. These hormones is consider as one of the most important hormones that control several functions in human body. 1.3.1.3.2 Carotenoids in Food and Biological Samples Carotenoids that are biologically important to humans have been observed in various plants, including vegetables, fruits, algae and some bacteria. The carotenoids can be divided into two main groups; the first group contains oxygen, and includes such compounds as astaxanthin, lutein, kaempferol, and zeaxanthin. A second class of carotenoid, contains a polyene hydrocarbon but are non-oxygenated, e.g. β-carotene, and lycopene (Rivera et al., 2011; Frassanito et al., 2008; Li et al., 2005; Breemen, 1995; Careri; 1999). Several recent studies of the role of carotenoids in human health provided evidence that many diseases such as heart disease, night blindness, cancer, rough, scaly skin, and disorders in tooth and bone growth can be prevented by using different carotenoid compounds (Table 1.1) (Schwartz & Patroni-Killam, 1985; Amorim-Carrilho et al., 2014; Valduga et al., 2014; Shen et al., 2014). Approximately 700 carotenoids have been investigated in different plants and animals (Li et al., 2010; Breemen, 1995; Li et al., 2005). 7 Several techniques have been used for quantification and qualification of carotenoids such as ESI/MS and APCI/MS (Careri et al., 1999; Ghlissi, et al., 2014; Zheng et al., 2014; Mattei et al., 2011; Zhang et al., 2014; Sigurgisladottir et al., 1994). 8 Table 1.1 Medical benefits of six natural compounds Compound Chemical structure Benefits Ref Astaxanthin OOO HH O Strong antioxidant; anti-inflammatory; beneficial against diabetes. Ghlissi, et al., 2014; Zheng et al., 2014; Mattei et al., 2011; Zhang et al., 2014; Sigurgisladottir et al., 1994. β-carotene Antioxidant; have lower risks of cardiovascular disease, strokes, and some cancers. McGill et al., 2003; Wawrzyniak et al., 2013; Karppi et al., 2013; Paolini et al., 2003; Zhang & Omaye, 2001. β-ionone O Antioxidant Hanaura et al., 2014; Lobo et al., 2012. Kaempferol OO HH OO HO HO Antioxidant Gancel et al, 2008. Caffeine NNNNOOCH3CH3H3C Antihistamine, a mild stimulant and vasodilator Li et al, 2010 Gallic acid OHHOOHOHO Antioxidant Gancel et al, 2008 9 1.3.1.3.3 Metabolomics Metabolomics study focuses on the analysis of metabolites with different size (small and large) in biological systems (Dunn & Ellis, 2005; Ibanez, 2013; Peng and Li, 2013). This developing approach enhances characterization of metabolites (over hundreds) from complex samples, including plant, human and animal tissues, in a single measurement (Dunn & Ellis, 2005; Ibanez et al., 2013; Putri et al., 2013). Recent years have seen an upsurge of interest in identifying a metabolomics profile of various plants. 1.3.1.3.4 Crocus sativus (Saffron) Saffron is an autumn flower and one of the most expensive spices cultivated in many countries; including Egypt, Morocco, Pakistan, Spain, Greece, Azerbaijan, New Zealand, Australia, and India. About 160,000 flowers of saffron are used to produce only 1 kg of saffron (Karimi et al., 2010; Haghighi et al., 2007; Caballero-Ortega et al., 2007). The total amount of saffron produced all over the world is 190 tons per year (Karimi et al., 2010; Haghighi et al., 2007; Caballero-Ortega et al., 2007; Gonda et al., 2012). Saffron is the dried stigma of the flower Crocus sativus L. (Iridaceae) (Karimi et al., 2010; Haghighi et al., 2007; Caballero-Ortega et al., 2007; Gonda et al., 2012). This spice is used for several medication purposes such as bronchospasm, pain relief, and cardiovascular diseases. Also, saffron is used for other purposes such as a food coloring. Saffron is comprised of many metabolites such as carotenoid, flavonoid and phenolic acid (Table 1.2) (Zhang et al., 2013; Amin and Hosseinzadeh, 2012; Kanakis et al., 2009; Verma and Middha, 2010). 10 Table 1.2 The chemical formula and the class of several metabolites in saffron Compound Chemical formula m/z Class Astaxanthin C40H52O4 596.84 Carotenoid β-carotene C40H56 536.87 Carotenoid β-ionone C13H20O 192.3 Carotenoid Zexanthin C40H56O2 568.8 Carotenoid Kaempferol C15H10O6 286.23 Flavonoid Crocin C44H64O24 976.96 Carotenoid Safranal C10H14O 150.21 Carotenoid Caffiene C8H10N4O2 194.19 Alkaloid Gallic acid C7H6O5 170.12 Phenolic acid Isophorone C9H14O 138.21 Carotenoid Quercetin C15H10O7 302.236 Flavonoid Proline C5H9NO2 115.13 Amino acid 11 1.4 Ambient Ionization Mass Spectrometry The term Ambient Ionization refers to any ionization source in mass spectrometry where an analyte signal is generated outside the mass spectrometry directly from a sample with little or no sample preparation or separation. This approach can be classified into four types depending on how the analyte is extracted from the substrate (Monge et al., 2013; Huang et al., 2011; Benninghoven et al., 1993; Todd et al., 2004) (i) liquid and gas jet desorption/ionization, (ii) laser desorption/ionization (LDI), (iii) thermal desorption with secondary ionization, and (iv) liquid extraction ionization (Monge et al., 2013; Huang et al., 2011). All these techniques can analyze a solid sample with minimum sample preparation. However, in all cases, solid samples that are by their very nature heterogeneous can have analytes deeply sequestered within substructures such as cellular organelles. It is quite challenging to observe stable analyte signals and repeated measurements on the same systems, such as plant tissues can generate responses with high variance. These high variances affect analytical performance to an extent that these results may not be considered quantitative. Two important ambient surface ionization techniques, paper spray ionization (PSI) and liquid extraction surface analysis (LESA), are the focus of this thesis. 1.4.1 Paper Spray Ionization Mass Spectrometry (PSI/MS) In PSI/MS, a triangular filter paper substrate is used to collect a sample by loaded the paper with few microliter of a sample. Then analyte ions are produced by applying a high electric field and a flush of solvent (Figure 1.2) (Liu et al., 2010; Espy et al., 2012). Typically, samples are preloaded on the paper substrate in a solution by injecting sample on substrate using syringe (Liu et al., 2010; Espy et al., 2012). In total, over 50 research publications have appeared describing PSI/MS with an increasing number of reports each year (Figure 1.3) (web of science, August 24th, 2014). Some studies have described the effect of the geometry of the paper such as the shape of the paper tip (Wang et al., 2011, Liu et al., 2010). PSI has been used in various areas for analyzing several samples (Table 1.3). 12 Figure 1.2 Diagram of paper spray ionization mass spectrometry. Figure 1.3 Number of published works on paper spray ionization mass spectrometry per year. 13 Table 1.3 Key advancements on paper spray ionization mass spectrometry Year Advancements References 2010 Characterization and application of PSI/MS Liu et al., 2010 Analysis of dried blood spot on paper Wang et al., 2010 2011 Paper spray ionization has been demonstrated with ion mobility spectrometer Sukumar et al., 2011 Quantitative analysis of therapeutic drugs in DBS Manicke et al., 2011 Non-polar solvent generated ions Li et al., 2011 Quantitative analysis of pharmaceutical in blood spot Manicke et al., 2011 2012 Characterization and biomedical application of PSI/MS Yang et al., 2012 PSI coupled with FTICR/MS Quinn et al., 2012 Analysis of several contaminants in different foodstuffs Zhang et al., 2012 Characterization of polar lipid in green microalgae Oradu and Cooks, 2012 Spray mechanism in PSI/MS Espy et al., 2012 2013 Chemical reactivity on reactive paper Yan et al., 2013 Chemical fingerprint of Bansha herbal tea Deng and Yang, 2013 Identification and assay of underivatized urinary acylcarnitines Naccarato et al., 2013 Coated capillary tube was used for sampling in PSI/MS Liu et al., 2013 2014 Understanding chemical reactivity Bain et al., 2014 Paper spray ionization for inorganic analysis using ICP/MS Cody and Dan, 2014 14 1.4.1.1 Mechanism of PSI/MS In PSI/MS, an electric field is generated when applied to the high electric potential between the paper substrate and the MS inlet (Liu et al., 2010). It is hypothesized that this phenomenon is similar to electrospray ionization (ESI), where charged droplets are formed when the electric field becomes higher than the surface tension of the liquid at the paper tip (Liu et al., 2010; Espy et al., 2012). It is demonstrated that charged droplets are produced from a Taylor cone that forms at the tip of the paper. The charged droplets undergo a series of desolvation processes leading to the formation of the analyte ions (Liu et al., 2010; Espy et al., 2012). Droplet size may be a factor that affects paper spray ionization depending on the solvent system (Liu et al., 2010; Espy et al., 2012). When a large volume of solvent (methanol and water (80:20)) was used with relatively low current, a range of droplet sizes were produced due to the asymmetrical electric field at the paper tip and inequality of the flow rate across the jets (Liu et al., 2010; Espy et al., 2012). When a low solvent flow was provided with higher current (~ 1 µA) applied, the droplet size was less than one micron. Five spray jets were observed at or near the paper tip with the presence of Taylor cones (Liu et al., 2010; Espy et al., 2012). Some electron-transfer species such as radical cations or anions, and typical Bronsted acid-base species and/or salt adducts were observed (Liu et al., 2010; Espy et al., 2012). Wang and his co-worker compared PSI/MS with nano spray for analysis of cocaine (Wang et al., 2010). Data from this study indicated that differences in the effective size of the capillary channels on the paper substrate required a much higher spray voltage (>2.8 kV) than nano spray (>0.8 kV) (Wang et al., 2010). In addition, the peak response and the corresponding spray currents were also compared and the variation in signal intensity with a range of voltages was obtained (Wang et al., 2010). These data indicated a need to control and optimize the current for effective PSI methods. In addition, Li and his co-workers (2011) found that non-polar solvents could produce ions without addition of any polar solvent or water (Li et al., 2011). This is important because non-polar solvents can not produce ions with ESI without addition of water or other polar solvents. The fact of this investigation has not been demonstrated. 15 1.4.1.2 Parameters Affecting PSI/MS 1.4.1.2.1 Geometry of Paper Several factors are optimized to improve the analytical performance of PSI/MS. In one case, Yang et.al, (2012) found that the shape of the paper tip affects the ionization efficiency, sensitivity and quantitation accuracy. The researchers cut a paper substrate with different angle (30º, 60º, 90º, 120º, and 150º) which altered the total spray current and signal intensity (Yang et al., 2012). The spray plume was decreased as the angle increased; and no spray could be observed for the tip with an angle 150o (Yang et al., 2012). In addition, as the angle was increased, the applied voltage on paper substrate increased (Yang et al., 2012). Sukumar et al. (2011) tested the effect of the amount of solvent. They studied the changes of the tip of paper substrate on the variations in quantitative analysis. Different amounts of solvent (0, 5, 10, and 15 μl) were added with and without the application of electric fields. There was no change on the paper tip related to either the addition of solvent or the wetting/drying cycle with the substrate (Sukumar et al., 2011). Further study revealed that the sharpness of the portion of the triangular paper tip affected the ionization efficiency (Espy et al., 2012). All of these parameters are needed to be optimized before the analysis in order to get a good accuracy. Other geometric parameters such as the distance between the paper tip and MS inlet is not demonstrated to date. In this thesis, it is suggested that short distance might affect the ionization efficiency because the probability of production of corona discharge. The production of corona discharge leads to decrease the ionization efficiency. Ions might be neutralized by electron transfer reaction (Munson et al., 2000). 16 1.4.1.2.2 Type of Paper A number of studies investigated the impact of the type of different papers used as a substrate for paper spray ionization, including a series of filter papers of different pore sizes (Zhang et al., 2012; and Ren et al., 2013). Filter papers and the chromatography paper are made from cellulose, while the glass fiber paper was made from glass microfiber. In comparative studies, all paper substrates were cut into triangles with the same dimensions (Jhang et al., 2012). Small amounts of sample were loaded on the paper substrate (Jhang et al., 2012). Multiple non-analyte signals were observed and the authors hypothesized that these were due to chemical solvents. The highest spectrum performance with the highest single to noise ratio (S/N) value was obtained with chromatography paper (Jhang et al., 2012). This approach is important to understand the chemistry on the paper that has not been discussed to date. One motivations of this thesis is to develop new achievement to show the activity of the paper surface depending on the type of the paper. 1.4.1.2.3 Non-polar Solvents Influence on PSI/MS The ionic strength of applied solvent is a significant factor that affects the ionization efficiency of any technique that based on electrospray phenomena (Page et al., 2007).The ions in that solution leads to increase the electrical field on the solution. Recently, it was reported PSI/MS could produce ions using completely non-polar solvent without the addition of any aqueous solution (Li et al., 2011). Pure non-polar solvents like hexane were used in order to identify insoluble drugs, peptides, nucleotides, and phospholipids as solids from paper substrate. This solvent allows ionization of these compounds without addition of any water or other polar solvents (Li et al., 2011). Pentane and toluene were used in this study. (Li et al., 2011). These observations are confusing when compared to ESI results because some fraction of water would be required to see these types of signals with ESI. Zhang et al., 2012 investigated the effect of solvent properties on the movement of analytes across the paper substrate and the effects on ion formation. The researchers used several solvents with various polarities in pure form, including hexane, dichloromethane, methanol, ethanol, isopropanol, and water (Zhang et al., 2012). The previous studies indicated that pure non-polar solvents could induce ionization with PSI/MS. No explanation was given as to what mechanism could enable such results. 17 It was further determined that polar compounds such as peptide and drugs are largely insoluble in these solvents. Nevertheless, polar or ionic analytes deposited on the paper are transported by the non-polar solvent, released into field-evaporated non-polar droplets and enter the mass spectrometer (Li et al., 2011). This approach was unexpected and is difficult to explain as in comparison to ESI where production of analyte ions is not possible using pure non-polar solvents. In both cases, a sustainable spray is required to produce ions. The critical question as to why pure non-polar solvents induce ions with PSI while it is not with ESI remains unanswered in the literature. Finding an answer to this question was one of the motivations of this thesis. 1.4.1.3 New Advancements in PSI/MS 1.4.1.3.1 Sampling Method Jain et.al. (2011) used a large surface area-sampling tool coupled with PSI/MS. It allowed for a much larger area of substrate to be sampled compared with traditional swabbing method. In this method, the paper is swap the area where analyte is needed to be analyzed. Droplets of sample solution were loaded on an aluminum surface within an area of 2 cm by 1 cm. Results showed that this method is more accurate than swabbing as a method of surface sampling by two to three orders of magnitude (Jain et al., 2011). Liu and his co-worker (2013) used the glass capillary sampling method for quantitative analysis of complex mixtures, which were present in trace levels in a small volume (~ 1µL). Liquid samples can be analyzed in a convenient way by using this new sampling method. A small amount of a standard solution of analyte was added to a capillary tube and left until dried. Then a small amount of real sample was taken with equal volume of the standard solution. Both the standard solution and the real sample will be mixed automatically. Then, the paper substrate was cut into a triangle for paper spray ionization mass spectrometry analysis. A diagram of this process is given in Figure 1.4 (Liu et al., 2013). Sub-microliter droplets were generated by using the capillary tube because of the gravity and the electrostatic attraction of the capillary tube. All parameters that affect the droplet size and frequency were investigated. Solvent was delivered to the surface of the paper substrate by using a capillary tube. Both rhodamine 6G and l-phenylalanine were 18 detected. This method was used for the determination of the amine-aldehyde condensation reaction of butylamine and benzaldehyde (Liu et al., 2013). This development will increase the capability of PSI/MS for liquid samples analysis. Figure 1.4 (A) A capillary sampler prepared by coating it with an internal standard (IS) solution through capillary action and drying in air to form an IS coating on its inner surface; (B) Capillary sampler to deposit the blood sample onto a paper substrate to prepare a dried blood spot containing internal standard. 19 1.4.1.3.2 A High Throughput Device A high throughput device based on PSI/MS was developed for fast analysis of complex mixtures (Shen et al., 2013). In this technique, the samples were loaded onto triangles on the paper substrate and dried blood spots were formed after they left the sample to dry at room temperature (Shen et al., 2013). Several experimental parameters were optimized, including the amount of solvent added on the paper substrate, the analysis time, and the sample amount loaded for each measurement (Shen et al., 2013). In the experimental optimization, five samples were used for testing. One tested parameter was the effect of sample load on the substrate. This experiment was done with spray solvent within a small amount of sample (12 μL). Results showed that little change in both the absolute ion response of the analyte and the ratio of the analyte to the internal standard once the sample amount was more than 1 μL (Shen et al., 2013). The ion response did not have direct proportion with the sample amount. Solvent wetting is another parameter that was optimized. The results showed variety in peak response with different amount of solvents. The results in this study demonstrate that the most important factor, which has an effect on eluting the analyte on paper substrate for the high throughput analysis is the solvent wetting (Shen et al., 2013). The results showed that spray solvent with amounts equal to 12 μL, and 1 μL of each blood sample for the preparation of dried blood samples were selected as the optimized set of experimental parameters for high throughput analysis of dried blood spots using paper spray ionization mass spectrometry technique (Shen et al., 2013). The performance for quantitation analysis using the high throughput device was then characterized with a series of bovine blood samples (Shen et al., 2013). This development added more advantages on PSI/MS where multiple analyses can be obtained within few seconds. Several triangular papers are loaded with few microliters of a sample first followed by PSI analysis. 20 1.4.1.4 Applications of PSI Quantitative and qualitative analyses of several compounds within different areas have been determined (Cook et al., 2011; Manicke et al., 2011). For example, atenolol in bovine blood (Manicke et al., 2011) and the interaction between the protein and the drug was studied by using PSI/MS (Manicke et al., 2011; Wang et al., 2011). Fifteen therapeutic drugs including hydrophobic and weakly basic drugs such as sunitinib, citalopram, and verapamil, were detected in another study at levels about 1 ng/mL (Manicke et al., 2011). While direct analysis of several molecules, such as hormones, lipids, and therapeutic drugs in tissue samples with minimum sample pre-treatment were analyzed using PSI/MS (Wange et al., 2011). In this study, a needle was used to prepare a tissue sample on the paper substrate (Manicke et al., 2011; Wang et al., 2011). The analysis of the solid sample (tissue) in this study was obtained for a portion of the sample where the solvent moved through the outer layer only. Thus, the analyte, which exist in the inner layer, might not be detected. Therefore, a new extraction technique is developed in this thesis for direct analysis of solid sample. Direct analysis of several compounds in fruits was accomplished using PSI/MS (Wang et al., 2011; Zhang et al., 2012; Naccarato, 2013). In another study, PSI/MS was used for characterizing lipids in two crude green microalga samples (Oradu and Cooks, 2012) and results agreed with previous reports using desorption electrospray ionization (DESI) (Oradu and Cooks, 2012). In addition, qualitative and quantitative analysis of 4-methylimidazole (4-MEI) in different samples, including caramel and beverage was obtained using PSI/MS (Li et al., 2013). The lowest concentration of pure 4-MEI detectable was 3 pg/μL in neat solvent and 5 pg/μL in a matrix. Eleven caramel samples for 4-MEI were analyzed; the average of RSD approached 15% (Li et al., 2013). PSI/MS was used for the assay of acylcarnitines and carnitine analysis in urine. Internal standard solutions of propionylcarnitine, octanoylcarnitine, and palmitoylcarnitine were used for quantitative analysis using calibration curves (Wange et al., 2011; Zhange et al., 2012; Naccarato, 2013). A range of linearity was obtained with good correlation coefficients within a range 0.96-0.99 (Wange et al., 2011; Zhange et al., 2012; Naccarato, 2013). Several contaminants, such as clenbuterol, melamine, plasticizer, and Sudan red were 21 quantified in foodstuffs, including meat, milk, sports drinks, and chili powder (Table 1.4). Relative standard deviation (RSD %) values were obtained within a range 3.7-21.8% with four replicates. These published works showed that the RSD percentage reached to 21%. This makes us ask why these high values were obtained where all samples were extracted using the same method. There is a certain question in this section is PSI a good technique for quantitative analysis. To date all quantitative analyses were obtained without taking into account substances that might be adsorbed on the paper. This thesis discuss the effect of these adsorbed molecules on the accuracy of PSI/MS for quantitative analysis. 22 Table 1.4 Analysis of contaminants in various foods using paper spray ionization mass spectrometry * Analyte Matrix Linear range(ng/g) LOD (ng/g) Solvent Clenbuterol Pork 1.0-1000 1.0 MeOH:H2O (9:1) Beef 5.0-1000 5.0 Terbutaline Pork 1.0-1000 1.0 MeOH:H2O (9:1) Beef 5.0-1000 5.0 Salbutamol Pork 5.0-1000 5.0 MeOH:H2O (9:1) Beef 5.0-1000 5.0 Ractopamine Pork 1.0-1000 1.0 MeOH:H2O (9:1) Beef 1.0-1000 1.0 Melamine Milk 20-1000** 20** Acetonitrile:H2O containing 26µM citric acid and 5.0 mM ammonium acetate (99:1) Sudan Red II Formula powder 50-1000 50 Dichloromethane:isopropanol containing 0.5% H2O (9:1) Chili powder 50-1000 50 Sudan Red III Chili powder 50-1000 50 Dichloromethane:isopropanol containing 0.5% H2O (9:1) Sudan Red IV Chili powder 50-1000 50 Dichloromethane:isopropanol containing 0.5% H2O (9:1) Sudan Red G Chili powder 100-1000 100 Dichloromethane:isopropanol containing 0.5% H2O (9:1) Bis(2-ethylhexyl) phthalate (DEHP) Sports juice 200-10000** 200** Dichloromethane:isopropanol containing 0.2% formic acid (9:1) Bis(2-ethylhexyl) adipate (DEHA) Sports juice 50-10000** 50** Dichloromethane:isopropanol containing 0.2% formic acid (9:1) * Zhang et al., 2012 **Concentration in ng/mL 23 A novel approach for the identification and evaluation of nonsteroidal anti-inflammatory drugs (NSAIDs) in olive oil was demonstrated using PSI/MS (Yan et al., 2013). Also, polypropylene glycol (PPG) and polyethylene glycol (PEG) were identified in urine and calf lung extract by using PSI coupled with fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS) (Quinn et al., 2012). The chemical profile of Bansha herbal tea was obtained by using PSI/MS (Deng and Yang, 2013). Approximately 38 active compounds were obtained in different samples (Deng and Yang, 2013). The previous works provided evidence that PSI is a suitable technique for a wide range of applications, including environmental pollution, natural product research and/or others. This would show the necessity of a commercial apparatus of PSI/MS. However, the effect of the adsorbed molecules on the paper was not discussed in the previous studies. These molecules might affects the real number of compounds for a metabolites profile. One motivations of this thesis is to determine whether PSI is a good fingerprinting technique. PSI has been used for several applications (Table 1.5). 24 Table 1.5 Applications of paper spray ionization mass spectrometry (PSI/MS) Area of Analysis Matrix Target Solvent Ref. Pharmaceutical Bovine and human blood, dried blood spot, saliva. Sunitinib, citalopram, verapamil, amitriptyline, paclitaxel, acetaminophen, sitamaquine, proguanil, benzethonium chloride, pazopanib, tamoxifen, Imatinib, cyclophosphamide, paclitaxel, irinotecan, docetaxel, topotecan, Propranolol, atenolol, neurotensin (NT), maltoheptaose, 4-chloroamphetamine, o-, m-, p-chloroamphetamine and o-, m-, p-fluoroamphetamine MeOH:H2O Wang et al., 2010; Sukumar et al., 2011; Espy et al., 2012; Jhang et al., 2012; Manicke et al., 2011; Lee et al., 2012. Biological Urine, Human serum, Porcine adrenal gland, mouse liver tissue, human prostate tumor and normal tissue sections Heroin, atenolol, cholesterol, hydralazine, Vitamin K1, 16 alpha, 17-beta-estriol, 17-beta-D-glucuronide, tetrahydroaldosterone-3-glucuronide, 25-hydroxyvitamin, D2-25-glucuronide, acylcarnitines, acetycarnitine, propionylcarnitine, isovalerylcarnitine, hexanoylcarnitine, octanoylcarnitine, decanoylcarnitine, lauroylcarnitine, myristoylcarnitine, palmitoylcarnitine and stearoylcarnitine. MeOH:H2O Liu et al., 2014; Wang et al., 2011; Quinn et al., 2012; Yang et al., 2012. Hormones Animal tissue, rat kidney Epinephrine, norepinephrine and hydralazine - Wang et al., 2011. Environmental pollution Beef, milk, sport juice Salbutamol, ractopamine, terbutaline, melamine, Bis(2-ethylhexyl) phthalate and Bis(2-ethylhexyl) adipate. - Zhang et al., 2012. Food Chili pepper, chili powder, lemon peel, beef. Sudan I, II, III, IV, para red, Sudan red II, Sudan red III, Sudan red IV, Sudan red G, thiabendazole, nicotine and nicotine-d3, clenbuterol, terbutaline and ractopamine. - Liu et al., 2010; Shen et al., 2013; Zhang et al., 2012. 25 1.4.2 Liquid Extraction Surface Analysis (LESA) A liquid extraction surface analysis (LESA) with a micro junction probe was designed by Wachs and Henion (Wachs and Henion, 2001). LESA operates in the same ionization process as electrospray ionization (ESI), where both techniques produce ions from the mobile phase at the tip of capillary electrode. However, the sampling area is physically sealed off and extracted with high flow rate. Both PSI and LESA are generally classified as liquid surface extraction techniques where the targeted analyte is loaded on the substrate and it is extracted from the substrate with varying amounts of solvent. The sensitivity of both techniques is monitored by the mobilization and/or extraction efficiency of the sample into spray solutions (Cheng et al., 2011; Wachs and Henion, 2001). Thus, they are included in the genre of liquid extraction based surface sampling techniques for MS. 1.5 Mass Analyzer Mass spectrometry uses a wide range of mass analyzers, with different sensitivities. This section is responsible to separate ions according to their mass to charge ratio (Hernandez et al., 2005; Liu et al., 2014). Several mass analyzers have been developed. This study will focus on two mass analyzers including, triple quadrupole and time of flight. 1.5.1 Triple Quadrupole Mass Spectrometry (MS/MS) 1326378 articles describe the use of this mass analyzer (Science direct, June 10th, 2014). In fact, two symbols have been used to define triple quadrupole mass spectrometry, including TQ/MS and MS/MS. TQ refers to triple quadrupole with ion scan mode while MS/MS refers to daughter ion mode (Hernandez et al., 2005). Most researchers prefer to use MS/MS rather than TQ/MS, where the total number of articles that have been published using MS/MS is over 1,313,623. In contrast, about 12,755 articles have been published using TQ/MS. This technique has several advantages such as high selectivity with less interference of matrix components, good signal-to-noise, and low level of target analyte can be detected (Hernandez et al., 2005). 26 1.5.2 Time of Flight Mass Spectrometry (TOF/MS) 45978 articles describe the use of this technique (Science direct, June 10th, 2014). Several advantages associated with TOF/MS include the fastest MS analyzer, and high resolution. It is good for pulsed ionization method such as matrix assisted laser desorption ionization (MALDI). However, TOF/MS has limitations such as low selectivity and requires pulsed ionization sources to produce high accuracy (Bings et al., 2000). 1.6 Extraction Methodology Traditional analysis of solid or heterogeneous samples containing solids requires an extraction step to bring sequestered analytes into a homogenous solution. There have been various approaches used for this, but sonication, soxhlet extraction and Liquid-Liquid Extraction (LLE) are the most common (Thurbide and Hughes, 2000; Leon et al., 2000; Peng and Li, 2013). In cases such as soxhlet extraction, the time required for efficient extraction can approach 24 hours. In others such as sonication, extraction efficiencies can be as low as 10-20 % (Ferguson et al., 2001). However, new extraction techniques have been developed to save time and to reduce the amount of solvent and sample consumption without losing extraction efficiency (Lara-Martin et al., 2008). Pressurized fluid extraction (PFE) is one of these techniques (Eichhorn et al., 2005; Petrovic et al., 2002; Lara-Martin et al., 2006; Croce et al., 2003). This technique has similar extraction efficiencies to soxhlet extraction, but can achieve these in a fraction of the time. In some cases, extractions that take day can be completed in minutes. Many common solvents can be used effectively with PFE. In short, extractions are achieved at temperatures above ambient, typically over 60ºC. In order to ensure solvents stay in their liquid state, solvents are typically pressurized (less than 500 psi). The elevated temperature improves the diffusion of the solvent into the sample matrix, mobilizes the sample within the matrix to further facilitate extraction (Eichhorn et al., 2005; Petrovic et al., 2002; Lara-Martin et al., 2006; Croce et al., 2003). The pressure (approximately less than 500 psi) allows solvents to maintain liquid state (Eichhorn et al., 2005; Petrovic et al., 2002; Lara-Martin et al., 2006; Croce et al., 2003; Lara-Martin et al., 2008). 27 Analyses on persistent organic pollutants (e.g., polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs)) performed using PFE in less than 20 minutes with a low solvent volume (less than 50 mL) (Croce et al., 2003). A good extract yield and the total polycyclic aromatic hydrocarbon content in different samples were investigated in another study using this technique (Croce et al., 2003). This technique improved the extraction process without sacrificing high recovery values (Lara-Martin et al., 2008). Other previous work used the pressurized fluid extraction method for the extraction of aflatoxins B1 and B2 from contaminated pistachio samples (Sheibani and Ghaziaskar, 2009). All these studies show that PFE reduces the amount of solvent required for extraction and the time for analysis. There is an effort to try to produce rapid real time analysis from a variety of sample types including those that are composed of heterogeneous materials (plants) where analytes can be sequestered into structural compartments within the material. More recently, ambient ionization techniques have been developed for direct analyses of solid samples such as PSI, desorption electrospray ionization (DESI) and direct analysis in real time (DART) (Zhang et al., 2012; Wang et al., 2011; Cooks et al., 2006; Takats et al., 2004; Mulligan et al., 2006; Fouyer et al., 2012). Previous published works on PSI/MS the researchers used liquid sample in their analysis. Few manuscripts described the analysis of solid samples with PSI/MS. Several contaminants, such as clenbuterol, melamine, plasticizer, and Sudan red were quantified in foodstuffs in powder form such as milk and chili powder (Wang et al., 2011; Zhang et al., 2012; Naccarato, 2013). Fouyer and his co-worker (2012) analyzed industrial polyethylene pellets without any sampling step using DART (Fouyer et al., 2012). DESI was used to examine solids samples (Cooks et al., 2006). DESI allows analytes to be desorbed directly from the solid sample such as pharmaceutical tablets (Takats et al., 2004), alkaloids from plant tissue or pesticides from corn leaves (Mulligan et al., 2006). All these technique extracted the analyte from the surface of the solid samples. The RSD values were high in many published works (more than 15 %) (Mattarozzi et al., 2012; Zhang et al., 2012). These data raise the question whether DESI and MALDI can be applied as a rapid screening method for chemicals in solid samples analysis. There is that in commercial samples or complex samples, such as homogenized food or plant, difficult to relate the quantified data obtained after direct detection from the surface of sample especially with contaminants. Contaminants are often not evenly distributed on the surface of the 28 sample. Some contaminants such as pesticides may be located inside certain parts of the solid sample, such as crop tissue, rather than on the outer surface (Garcia-Reyes et al., 2009). This direct detection of these compounds from the surface without consider the inner surface will affect the recovery values. Rapid measurement of the contaminant from the outer and inner surfaces will increase accuracy; sensitivity and eliminate non-homogeneity issues. Thus, there is upsurge to develop new extraction methods for direct sample analysis with less solvent and sample without losing the extraction efficiency and/or recovery. In this thesis, I hypothesized that we could develop a rapid approach that uses the scientific basis of PFE to extract solid samples onto a surface suitable for analysis by ambient ionization approaches. The RSD values associated with ambient ionization will drop dramatically, enabling routine analysis by this approach. 1.7 Mechanical Press Technique The previous works pressed solid powder into pellets using a mechanical press. The applied pressure and solvent affect the strength of the pellet. Many published works used mechanical press techniques to produce a pellet of the synthesized compound for IR spectroscopy analysis (Bell et al., 1991; Braga et al., 2002). 1.8 Silicon Dioxide Nanoparticles Nanoparticles materials have been used successfully in various MS experiments such as graphite, carbon nanotubes, Au, Sn, Si, and TiO2 nanoparticles (Latorre et al., 2012; Okoturo-Evans et al., 2013; Lewis et al., 2003; Liu and He, 2008). Silicon dioxide nanoparticles, also known as silica nanoparticles or nanosilica, are the basis for a great deal of biomedical research due to their stability, low toxicity, and ability to be functionalized with a range of molecules and polymers (Liu and He, 2008; Tsao et al., 2012; Okoturo-Evans et al., 2013). Silicon nanoparticles have been used with different surface ionization techniques such as matrix assisted laser desorption ionization (MALDI), and desorption electrospray ionization (Liu and He, 2008; Tsao et al., 2012; Okoturo-Evans et al., 2013; Lewis et al., 2003). Silicon nanoparticles has been successfully used in several applications, including determination of the metabolites profile, indicate organic synthetic products, indicate active drug components, and determine small molecules in biological samples (Liu and He, 2008; Tsao et al., 2012; Okoturo-Evans et al., 2013; Lewis et al., 2003; Latorre et al., 29 2012). These nanoparticles were used to modify the paper in this thesis in order to improve the ionization efficiency on the paper. 1.9 Research Aims and Objectives The overall objective of this thesis was to characterize the potential of PSI. The objective of this study to understand the ionization mechanism and to develop potential applications of PSI/MS. To achieve this overall objective, several specific objectives were designed including: 1- To design and construct an apparatus for PSI. 2- To understand the chemistry of the paper and the impact of paper on ionization. 3- To determine the potential of PSI as a quantitative technique. 4- To utilize PSI as separating tool for complex samples. 5- To determine the variability of the lab-made PSI apparatus between different mass spectrometers. 6- To compare solubility and micro-flow of the sample across PSI and LESA. 7- To determine the capacity of PSI as a metabolite fingerprinting technique using Crocus sativus as a model system. 8- To develop novel method for introduction a solid sample onto the paper substrate, which is defined as mechanical press extraction. 9- To determine the potential of silicon dioxide nanoparticles to improve the ionization efficiency. Together these studies are a comprehensive evaluation of the PSI/MS and provide insights into ionization mechanism of surface analysis techniques based on electrospray, the challenges and opportunities afford by the chemistry and complexity of the paper. The saffron metabolomics profile described identify the degree of accuracy for PSI to be utilized as a metabolite fingerprinting technique. A new approach is described to improve the direct analysis of solid samples in their native shape. The approach developed for experimental 30 design can be applied to a range of other ambient ionization techniques. A new achievement is described to boost the ionization efficiency of cellulosic paper using silica nanoparticles. 31 Chapter 2 The Role of Paper Surface in Paper Spray Ionization Mass Spectrometry 2.1 Synopsis For PSI it was perplexing as to why a spray could be obtained using even completely non-polar solvents. It was further intriguing that very polar analytes could be analysed with completely non-polar solvents with PSI/MS while it is not possible with ESI (Sjoberg et al., 2001; Page et al., 2007; Henderson and Mclndoe, 2006; Sannino et al., 2004). To date, several published works used non-polar solvent with PSI/MS but no one indicated the reason of ionisation (Li et al., 2011; Zhang et al., 2012). There are two main working hypotheses for the research described herein, the theoretical bases of which are discussed in detail in the theory section in Chapter 1. The first hypothesis is water molecules are in the paper. It was suggested that this water is essential for the technique. The second hypothesis is that response should be directly affected to motion of water and analyte through the paper. Therefore, the goal of the research is to independently test the two hypotheses in order to develop new achievement to understand the ionization mechanism. The current work encompasses the following: i) geometric parameters of the paper substrate on the ionization efficiency; ii) the chemistry of paper on the ionization efficiency, iii) changes to the paper substrate tip after multiple measurements. In addition, it is hypothesized that the spray is emitted from the paper tip to form a cone shaped distribution of ions. Experiments involved testing optimal distance and position of the tip of the paper substrate and five types of paper. The parameters were tested with nine compounds, which had a range of polarities and solubilities in methanol, acetone, acetonitrile, hexane, toluene, and pentane. It is hypothesized that the surface of the paper is not inert, which affects the accuracy of PSI. The trace elements that adsorbed in the paper surface was demonstrated using different type of papers. The results of these studies significantly improved our understanding of the PSI fundamental aspects. 32 2.2 Experimental 2.2.1 Chemicals and Materials 17α-hydroxyprogesterone, 3, 3, 5-triiodo-L-thyronine (T3), L-thyroxin (T4), cortisone, hydrocortisone, β-carotene, astaxanthin, caffeine, estriol, and estrone were obtained from Sigma-Aldrich (St. Louis, MO). Several polar and non-polar solvents were used of analytical grade including methanol, acetone, acetonitrile, hexane, toluene, and pentane, and they were obtained from Fisher Scientific (Mississauga, ON). A series of dilute solutions were prepared (Table 2.1). Whatman filter paper (grade 1, grade 4, and grade 5), glass microfiber filter G6, which is manufactured from 100% borosilicate glass, ashless and 1PS phase separators filter paper provided the paper interface (Fisher Scientific, Mississauga, ON). 33 Table 2.1 Preparation of standard solutions Compounds Solvent Caffeine Acetone Astaxanthin Dichloromethane Estrone MeOH Estriol MeOH Cortisone MeOH Hydrocortisone MeOH α-Hydroxy progesterone MeOH T3 MeOH + NH4OH (95:5) T4 MeOH + NH4OH (95:5) 34 2.2.2 Apparatus All experiments were performed using a Waters Quattro Premier liquid chromatography-tandem mass spectrometer (Waters Inc., Mississauga, ONVAAQ68) equipped with a custom-built assembly for PSI/MS (Figure 2.1). Mass spectra were collected in a full scan mode. Parameters of applied voltage, cone voltage, extractor voltage, RF lens voltages, and flow rate (µl/min) were optimized based on the most intense peak for each compound. Paper was cut into a triangle with approximate dimensions: height 10 mm and base 5 mm. A steel metal clip was used to hold the paper section. A small amount of solvent containing analyte was loaded to the paper before eluting with solvent that was applied via infused injection method ( Figure 2.1). The PSI source was operated in the positive and negative ion modes. Ultrapure water was obtained from Pure lab Plus water purification system (18 mΩ; US filter). Figure 2.1 Triple quadrupole mass spectrometry in the left side, and (A) lab-made paper spray ionization apparatus where the paper should adjacent to MS inlet cone. (B) Solvent will pass through the paper by using capillary tube. (C) Infused injection method. 35 2.2.3 Paper Substrate Imaging The image of paper substrate was obtained using the Tescan Mira3 XMU Field Emission Scanning Electron microscope in the UBC Okanagan Facility for Scanning-Electron Microscopy (SEM). SEM image was used to show analyte binds to cellulosic fiber in paper. SEM analysis was used to determine the trace metals that associated in the paper. 2.2.4 Characteristic of Instrumental Parameters In order to improve the peak responses, both PSI and MS instrumental parameters were optimized (Table 2.2). For PSI, geometric parameters of the paper substrate were studied, including distance from the paper substrate to MS inlet (tip distance= td) and paper’s tip position (tp) toward MS inlet. This study was studied over wide ranges of 1-10 mm and 60-140º for td and tp, respectively. In addition, the influence of applied potential, cone voltage, and RF lens were optimized at ranges of 1-4.5 KV, 10-50 V, and 1-4 V, respectively. Flow rate is another factor that was optimized; a wide range of 1-30 µL/min was used for this study. 2.2.5 Characteristic of Paper Substrate Six types of paper were used in this study, including Whatman filter paper (grade 1, grade 4, and grade 5), glass microfiber filter G6, Whatman 1PS phase separator and ashless filter paper (Table 2.3). Standard filter paper (Whatman filter paper grade 1) was used either as received or after being dried in an oven (Isotemp oven) at 85 ºC for 24 hours. 36 Table 2.2 Mass spectrometry optimized parameters in both ion modes Compounds Positive mode Negative mode Capillary volt (KV) Cone volt (V) RF lens (V) Capillary volt (KV) Cone volt (V) Rf lens 17α-hydroxyprogesterone 3.66 35 1.3 3.17 35 1 T3 3.5 41 1.3 3 30 1.3 T4 3 45 1.3 3 41 1.3 Astaxanthin 3 35 1.3 3 41 1 β-Carotene 3.30 35 1.3 3 41 1 Cortisone 3.5 45 1.3 3.63 45 1.3 Hydrocortisone 3.63 45 1.1 3.1 45 1.3 Estoil 3.23 35 1.4 2.32 41 0.9 Estrone 3.31 31 1 3.11 41 1.3 Caffeine 3.50 31 1.1 3 35 1.1 37 Table 2.3 Characteristic of five types of paper 2.2.6 Repeatability In order to study the tip shape degradation of the paper substrate, intra- and inter-day repeatability were used for this purpose. Both tests were expressed as RSD % and rsd %, respectively. The RSD % was carried out within 21 replicates, while rsd % was carried out within three replicates during six weeks. 2.2.7 Figure of Merits of the Developed PSI/MS Technique Stock solutions for each compound were prepared in different solvents. A series of standard solutions of each compound (0.05-250 µg/mL) were prepared by sequential dilution with several solvents. Both stock and working solutions were stored at 4 °C and kept in dark vials until further use. The analytical performance (dynamic linear range, lower limit of detection and quantification) were investigated by analyzing pure standard solutions. The limit of detection (LOD) and limit of quantification (LOQ) were determined according to the standard IUPAC approach (Miller & Miller., 1994). 2.3 Results 2.3.1 PSI/MS Parameters Optimization and Spectra Characteristics Geometric parameters such as paper tip position (tp) and distance from paper tip to MS inlet (tip distance= td) were studied for signal intensity and stability. The influence of the Types Porosity Flow rate Particle retention (µm) Whatman Grade 1 Medium Medium 11 Whatman Grade 4 Coarse Fast 20-25 Whatman Grade5 Fine Slow 2.5 Glass fiber microfiber Fine Fast 1.6 1PS phase separator Medium Medium 11 Ashless filter paper Coarse Fast 20-25 38 distance between paper substrate and MS inlet on the PSI/MS peaks of each compound was tested over a wide range (1-12 mm). The results show that the peak intensity increased steadily on increasing the td until 5 mm (Figure 2.2). At higher values (td > 5 mm), the intensity decreased. Consequently, a td of 5 mm was selected in the remaining experiments work. At 5 mm, excellent background, and protonated peaks were observed in positive ion mode (Figure 2.2). The tip position toward MS inlet is one of the most important parameters in the PSI/MS method. It has a pronounced effect on the sensitivity, and analytical performance. This parameter was optimized over a wide range (60º - 140º). The highest protonated peak responses, showing well defined and sharp peaks, were investigated for each compound and were found to be achieved at tp=90º. At further positions, peak response began to decrease (Figure 2.2). Figure 2.2 The optimized geometric parameters for paper spray ionization: (A) the distance between the tip of the paper substrate and MS inlet and (B) tip position at angle 60°, 75°, 90°, 120°, and 140° from left to right. B 39 The influence of the applied potential (1.5-4.5 KV) on the peak response of the selected compounds was then established using the optimal paper tip position and distance from paper to MS inlet. Good sensitivity and sharp and symmetric peaks were found at 3-3.66 KV. Thus, in the next work the applied potential values of 3.50, 3.66, 3.23, 3.31, 3.50, 3.63, 3.30, 3, 3.5, and 3 KV were adopted for caffeine, progesterone, estriol, estrone, cortisone, hydrocortisone, β-carotene, astaxanthin, T3, and T4, respectively. Other MS parameters, including cone voltage and RF lens were optimized. The studies were carried out with ranges of 10-50 V for optimal cone voltage, and 1-4 V for RF lens. Different protonated peaks were shown variability in the responses. Cone voltage values at 35, 35, 41, 45, 35, 35, 45, 45, 35, and 31 V were found for caffeine, progesterone, T3, T4, astaxanthin, β-carotene, cortisone, hydrocortisone, estroil, and estrone. For RF the values were 1.1 V, and 1.4 V for hydrocortisone and estroil. The value 1.3 V was optimized for T3, T4, astaxanthin, β-carotene, and cortisone. All instrumental parameters are given in Table 2.2. Solvent flow rate may also be expected to have an effect on the PSI efficiency. It is indicated that low flow rate (≈ 3 µL/min), will inhibit transfer of analytes to the tip of the paper, while a high flow rate (20 µL/min) can dilute the analytes and oversaturate the paper, which will result in a restricted current and no observed peaks. A low flow rate took more time than high flow rate to extract the analyte from the surface (Figure 2.3). These potential complications with solvent addition were overcome by using an infused injection method ( Figure 2.1). A continuous flow of solvent using the infused injection method will make the evaporation process more stable and it will keep the consistency of analyte mobility over the paper substrate. A flow rate of 10 µL/min was found to be optimal for this study. 40 Figure 2.3 The effect of low rate. (A) The plot show the variety in peak responses using different flow rate, and (B) the time of analysis was obtained within several secondes at different flow rate. Optimization of all these instrumental parameters revealed that the conventional positive ion mode of the paper spray was compatible with the tested analytes. In the PSI/MS technique, spectra for each analyte in positive ion mode showed protonated ion peaks at m/z 195, 289, 271, 331, 361, 363, 537, 597, 651, and 777 for caffeine (m/z194), estriol (m/z 288), estrone (m/z 270), progesterone (m/z 330), cortisone (m/z 360), hydrocortisone (m/z 362), β-carotene (m/z 536), astaxanthin (m/z 596), T3 (m/z 650), and T4 (m/z 776), respectively in positive ion mode (Figure 2.4). In addition, PSI/MS spectra showed an ion peak at 731 which potentially corresponds to [M- COOH] for T4, while lower relative intensities within the mass range of interest were observed in negative mode. Deprotonated ion peaks at m/z 193, 287, 269, 329, 359, 361, 535, 595, 649, and 775 were observed for these analytes in negative mode, respectively. According to the results positive ion mode was selected for this study because the selected analytes showed low tendency to lose proton in negative ion mode. A B Flow rate (µL/min) Time of analysis (s) % 41 Figure 2.4 The paper spray ionization mass spectrometry spectra for (A) caffeine, (B) astaxanthin, (C) 17α-hydroxyprogesterone, (D) T4 using 10 µg/mL in positive ion mode. 2.3.2 Influence of Solvent In determining the optimal solvents, peak responses for several compounds were obtained using methanol, acetone, acetonitrile, and mixtures of them (1:1). A small amount of sample was loaded on the paper substrate and left until dry at room temperature for few seconds. The adsorbed analyte on the paper substrate was indicated by using SEM (Figure 2.5).The best solvent to mobilize the analyte from the surface was investigated for each compound. The responses were compared to determine the effect of solvent on the target analytes’ signals. Methanol extracted 17αhydroxyprogesterone with extraction yield equal to 20 %, while the other solvents detected this compound with values ranged 12-18 %. In contrast, acetone shows the highest percentage of extraction for both β-carotene and astaxanthine. Other solvents including acetonitrile, methanol:acetone, methanol:acetonitrile, acetone:acetonitrile gave a range of extraction percentages. For β-carotene, it was equal to A D C B 42 16, 15, 16, 15, 14 %, respectively, while this percentage was within range of 7-23 % for astaxanthin (Figure 2.5). Based on these results, methanol and acetone were selected to be the mobile phase for progesterone and the carotenoid compounds, respectively. For thyroid hormones (T3 &T4), a mixture of acetone and acetonitrile (1:1) gave the highest responses with the highest extraction efficiency (30%). The range of extraction for thyroid hormones was 0.7-30 %, where the mixture of methanol and acetonitrile gave the lowest percentage equal to 0.7% and 7% for T3 and T4, respectively. In addition, the ranges of the extraction percentage 8-12 % and 7-29 % were established for hydrocortisone and cortisone, respectively. This result was obtained by using methanol, acetone, acetonitrile, and mixtures of them. The mixture of methanol and acetonitrile showed the highest extraction percentage for the previous steroid hormones. The values were equal to 37 % and 33% for hydrocortisone and cortisone, respectively. 43 Figure 2.5 (A) Scanning electron microscope image of the analyte that adsorbed on the paper substrate (thin fiber) where it was not obtained on the paper substrate without the addition of the analyte, and (B) the optimal solvent for each compound, where methanol and acetone was obtained as optimal solvent for progesterone and carotenoids, respectively. A mixture of acetone and acetonitrile (ACN) was the optimal solvent for thyroid hormones. For steroid hormones, the optimal solvent was a mixture of methanol and acetonitrile. 2.3.3 Characteristics of the Paper Substrate To understand the chemical and physical effects of the paper, five types of paper were investigated including glass microfiber, 1PS phase separator, and Whatman filter paper with different grade numbers. Different elements such as Na+, K+, Ca2+, Al3+, B3+ and other inherently are associated with the paper. Protonated peaks were observed for each analyte using the five types of paper. A range of peak responses was observed for these papers. Of all A B 44 types, glass fiber paper showed the lowest peak response, while 1PS phase separators filter paper gave responses higher than the glass fiber paper but lower than the Whatman filter paper grad 1 (Figure 2.6). In addition, Whatman filter paper with three different grades (G1, G4, and G5) was compared in order to study the effect of the chemical and physical properties of the paper on the ionization efficiency. The results showed that grade 5 produced the lowest peak responses with ratios within range of 23-27 %. In contrast, grade 1 produced the highest PSI/MS peak response for all the tested compounds within ranges of 41-48 %. Whatman filter paper grade1 was selected for further study. 45 Figure 2.6 The paper effect (Whatman filter paper grade 1 (G1), grade 4 (G4), grade 5 (G5), glass microfiber filter G6 (GF), and 1PS phase separator (SP) were compared. Whatman filter paper grade 1 showed the highest peak intensity while glass microfiber showed the lowest peak intensity. In order to identify the distribution and/or the level of the elements that are associated within the paper substrate scanning electron microscope was used. The spectra were investigated at different positions with resolution equal to 10 µm. The results in the current study provided evidence that several elements were observed on the paper, including carbon, oxygen, sulfur, chlorine, potassium, and calcium. These elements were obtained at levels 0.04-75 % wt (Table 2.4). 0102030G1 G4 G5 1PS GFProgesteroneCortisoneHydrocortisoneT3T4Peak response (103) %Type of paper 46 Table 2.4 Trace elements that detected in paper substrate using elemental distribution spectra Many adduct peaks beside protonated peaks were observed. Of all adduct peaks, a peak that is related to [M+Na]+ was the predominant adduct formation peak. For example, the peaks at m/z 619, 311, 383, 385, and 799 for astaxanthin, estriol, cortisone, hydrocortisone, and T4 gave evidence of sodium adduct formation. This adduct produced within values 60, 30, 12, 17, 16, 19 % for progesterone, astaxanthin, hydrocortisone, cortisone, T3, and T4, respectively. Other adducts peaks such as [M+K], [M+O], [M+2H+K] were observed for astaxanthin, β-carotene, and hydrocortisone, respectively. All adduct peaks which were observed are given in Figure 2.7. Most adduct peaks were obtained using Whatman filter paper grade 1 and show no signal with the other types of paper. The analysis time is an important factor that must be considered especially in quantitative analysis. The variability between the signal responses amongst analyte peaks was dependent on the time since the initiation of the ionization. The results revealed that the relative signal intensities are typically more stable between 30-90 seconds. It was hypothesized that porosity and/or polarity issues can increase the time of the analysis. Elements Level (% wt) C 63-65 O 34-37 K 0.1 Cl 0.1 47 Figure 2.7 Different adduct peaks formation using Whatman filter paper grade 1 at 10 µg/mL of each analyte. 2.3.4 The Influence of Water Molecules Six solvents with a range of polarity were used in order to add new novel insight in PSI/MS. Methanol, acetone, acetonitrile, hexane, pentane, and toluene were used for this purpose. Both standard and oven-dried paper were used with each selected solvent. Cluster water peaks were observed with analyte and solvent using standard paper in different form such as [M+H2O]+. and [M+H+(H2O)2]+. In addition, a water cluster peak, which is related to the [M+H+H2O]+, was obtained with all polar solvents. The most significant water cluster peak was obtained at m/z 41 which is related to [Na+H2O]+. Another water cluster was shown at m/z 57, which associated to [K+H2O]+. These clusters were observed with either polar or non-polar solvents. All these water clusters were indicated with standard paper while oven-dried paper show no existence of the water cluster even with polar solvent (acetone, acetonitrile, and methanol). Many adduct peaks beside protonated peaks were observed with the standard paper. Of all adduct peaks, a peak that is related to [M+Na]+ was the predominant adduct formation peak. For example, the peaks at m/z 619, 311, 383, 385, and 799 for astaxanthin, estriol, cortisone, hydrocortisone, and T4 gave evidence of sodium adduct formation. Other adducts 48 peaks such as [M+K]+ was observed for astaxanthin, β-carotene, and hydrocortisone, respectively. All adduct peaks which were observed are given in Figure 2.8. 2.3.5 Intra- and Inter-Day Repeatability A final parameter that needed to be determined was the repeatability. The inter-day repeatability (within different days) was obtained by analyzing the analytes within three replicates. The value of inter-day (rsd %) was 23 and 10 % at the low concentration level tested using T3 and T4, respectively. The rsd % test was done within six weeks. The values of rsd % were expected to be high where the accurate shape of the tip was changed. Even though the paper was cut with standard dimensions (approximately 5 x 10 mm), it is hard to keep the consistency of the accurate shape for all measurements within different days because the sharpness of the paper might be change by cutting the paper with the scissors. Other factors, such as impurities that might be associated with the paper from the lab, could affect the reproducibility. However, the chemistry of the paper is discussed in this chapter. Furthermore, the intra-day (RSD %) repeatability within 21 replicates was used to study the stability of the tip shape for the paper substrate. The values of the RSD % were investigated with an acceptable values range of 3-5 % within five replicates. At higher number of replicates, the RSD % values increased within range of 9-25 % (Figure 2.8). 49 Figure 2.8 The relative standard deviation values within 21 replicates using either T3 or T4. The values ranged between 3-5 % within replicates lower than six; and it reached 25 % within 21 replicates. 2.3.6 Figure of Merits of the Developed PSI/MS Technique Once the optimum experimental conditions of instrumental, solvent, and paper substrate parameters were determined, the IUPAC figure of merits (dynamic linear range, lower limit of detection and lower limit of quantification) were indicated. The peak response at various concentrations (0.05-250 µg/mL) of each analyte were recorded individually. The linear range was determined in each plot of the peak intensity versus the concentrations (Figure 2.9). The regression equation for the linear range for each analyte were accomplished with a range of slope from 1344 x 10+6 to 3 x 10+6 (Table 2.5). For β-carotene, a proportional calibration curve was obtained where the compound showed low peak response. The LOD values were found equal to 20, 5, 1.5, 3.8, 30, 28, 25, and 22 (µg/L) for caffeine, progesterone, cortisone, hydrocortisone, β-carotene, astaxanthin, T3, and T4, respectively. The LOQ was equal to 66.6, 17, 4.8, 12.6, 99.9, 93.2, 83.3, and 73.3 (µg/L) for the same compounds, respectively. The main analytical performance and analytical 50 040801200 30 60 90Peak response (106)Concentration of progesterone (g m/-1)parameters of the PSI/MS method were compared successfully with many of the previously published ionization methods (Table 2.6). Figure 2.9 Calibration curves of progesterone and T4. A proportional calibration curve was obtained for β-carotene, where this compound showed low peak response. 51 Table 2.5 Linear equation of the selected compounds Compound Y= aX + b R2 Astaxanthin Y= 150274X + 25695 0.9996 β-carotene Y= 0.0349X + 0.0058 0.9834 Caffeine Y= 1344.1X + 1820 0.9180 Cortisone Y= 1E+06X + 3E+06 0.9906 Hydrocortisone Y= 304142X +270936 0.9904 17α-hydroxyprogesterone Y= 1e+06X + 3E+06 0.9906 T3 Y= 82679X + 51888 0.9810 T4 Y= 25874X + 127967 0.9936 52 Table 2.6 Analytical features of the developed paper spray ionization mass spectrometry and other reported ionization methods Compounds Ion mode Base ion peak LOD (µg/L) LOQ Linearity (mg/L) Flow rate (µL/min) Injected sample (µL) Applied voltage (KV) Ref. Caffeine [M+H]+ 110 ESI + 380 400 - 4 Sorensen, 2011 0.1 0.333 (0.1-1000)x10-3 40 20 3 Baker and Kasprzyk-Hordern, 2011 242.5 807.5 0.5-78 600 0.7 3 Ptolemy et al., 2010 11.9 39.6 (12-36)x10-3 6200 5 - Perrone et al., 2008 nanoESI + [M+H]+ - - - 10 100 5 Hu et al., 2010 HTLC/DESI/IM + [M+H]+ 16 2384 - 250 - 3.5 Harrey et al., 2009 TLC/ESI + [M+H]+ 2230 4450 (20-102)x10-3 100 - 3.5 Aranda & Morlock, 2006 PSI + [M+H]+ 20 66.6 0.5–100 10 1.5 3.5 present work Progesterone ESI + [M+H]+ 0.24x10-3 0.81x10-3 (0.5-50)x10-6 500 20 - Muz et al., 2012 PSI + [M+H]+ 5 17 0.05 - 80 10 1.5 3.6 present work β-carotene APCI + [M+H]+ 25 100 0.1 - 1 400 100 Frenich et al., 2005 1.01 2.19 (4.8-402)x10-3 20 Rivera et al., 2011 ESI + [M].+ - - - - - 3.5 Rivera et al., 2011 PSI + [M+H]+ 30 99.9 0.5 - 20 10 1.5 3.3 present work T3 ESI + [M+H]+ 1.63 3.25 – 300 10 5 Piehl et al., 2008 PSI + [M+H]+ 25 83.3 0.05 - 50 10 1.5 3.5 present work T4 ESI + [M+H]+ 1.94 3.88 - 300 10 5 Piehl et al.,2008 PSI + [M+H]+ 22 73.3 0.05-100 10 1.5 3 Present work 53 2.4 Discussion 2.4.1 The Role of Water Clusters The mass spectra generated from PSI with standard filter paper contained clear evidence of water adduct formation. The results showed no signals with non-polar solvents when the paper is dried, as all signal is lost. This strongly implies that the water content within the paper matrix is essential for the spraying process and the formation of a Taylor cone. It could be argued that drying the paper changes the structural characteristics and micro-channels within the paper and this could explain the observation. Thus, few drops of water were added on the dried paper. The results showed that the ions were detected with dried paper after few drops of water were added. The water cluster peaks were observed with all type of cellulosic papers. These results showed that the water molecules affects the ionization efficiency. However, the large complexes containing water ligands are observed when even using completely non-polar eluent. This could help to explain why sprays are observable under these conditions. We deduce that in traditional ESI, the water content enables current to be conducted within the fluid and with the high-applied voltage. This leads to a Taylor cone. It is well established that when voltages are applied to conductive surfaces, the field strength is maximized at sharp points, in this case the tip of the Taylor cone. It is my assertion that once this field strength reaches some threshold that a sustained electrospray can be obtained and this electrospray leads to the effective generation of analyte ions. In paper spray, the water content associated with the paper enables the conductive process and once the paper is cut into an appropriate shape and solvent applied, the correct conditions exist for a sustained spray to be achieved. In the absence of the water, this condition is no longer satisfied. The fact that the fluid can be completely non-polar has been confusing to other researchers, however, our explanation and supporting experimental data adequately explains the phenomenon. The previous works on PSI/MS discovered that polar compounds were analyzed using a non-polar solvent (Li et al., 2011, Zhang et al., 2012). In the model proposed here, the residual water molecules that are naturally associated the hydroxyl groups of the cellulose paper substrate creates a conductive surface and can assist with the flux of ionized analytes by forming large complexes that contain water as ligands. Evidence of these complexes are 54 evident in the observed mass spectra and all signals are lost once the paper has been dried in an oven prior to application of analyte or eluent. This result was obtained with non-polar solvent. No water cluster peaks were observed with dried paper using either polar or non-polar solvents. A range of non-polar solvents such as toluene, pentane, and hexane generated the same type of response. Water cluster peaks at different mass were observed with either solvent or analyte. These peaks are related to water cluster formation (Figure 2.8). In addition, a water cluster peak which is related to the [M+H2O]+. formation was obtained with all non-polar solvents. These results reproduce previous work by Li and his co-worker (Li et al., 2011), but go further to provide a valid explanation as to the mechanism involved. In addition to water clusters, a sodium adduct peak [M+Na]+ was observed for most analytes. Other adduct peaks were also observed such as [M+K]+ and [K+H2O]+ʘ. These peaks usually occur in ESI when water and/or salts are present (Schug and McNair, 2002; Kebarle and Tang, 1993). This indicates that this is further evidence that the water molecules move through paper substrate and draw the observed metals (cations) to the electrosprayed solution, leading to adduct formation. It could be argued that the trace elements within the paper induce the ionization using non-polar solvent. Ashless filter paper, which made of cellulose but the trace elements were removed by washing the paper with HCl, was used. This paper produce ions with non-polar solvent. Water cluster peak was also obtained with this paper. This is another evidence supports the hypothesis that water molecules on the paper is the main reason to induce the ionization with completely non-polar solvents. In conclusion, although a spray can be sustained using non-polar solvent without adding any water into the eluent, there is still water present and associated with the paper substrate. Further, if the paper is dehydrated, a spray can no longer be sustained with non-polar solvents. This is an important finding, as there has been no explanation of this phenomenon to date. 55 2.4.2 A Cone Shaped Distribution of Sprayed Jets Geometric parameters were demonstrated in this thesis in order to determine the shape distribution of the sprayed ions. Geometric parameter such as the size and the shape of the tip of paper substrate was investigated (Yang et al., 2012; Espy et al., 2012; Liu et al., 2010). The researchers discovered that the angle of the shape of the tip during cut the paper affects the ionization efficiency. In the current study, two geometric parameters were demonstrated. First, the distance between the paper tip and MS inlet cone. The result showed that at distance lower than 5 mm the peak response was decreased. A shorter distances (lower than 5 mm) a corona discharge might be produced, which decreased the peak responses. Espy and his co-worker suggested that corona discharge might produce with PSI because the change of the solvent uniformity on the paper (Espy et al., 2012). The production of corona discharge is discussed in Chapter 4. However, the peak response generally related to three factors, (i) the amount of ions that introduce to MS inlet, (ii) the ionization efficiency, and (iii) the ion suppression of the system. Second, the position of the tip toward the MS inlet also affects the ionization efficiency. To test this hypothesis the paper substrate was placed at different directions to MS inlet. The results showed the highest peak response when the paper tip was placed at 90° to MS inlet cone, which means the most intense sprayed jet produced at the paper tip since Espy and his co-worker demonstrated that several jets with different intensity could be sprayed at the paper tip. Based on these results, further information on the ions distribution was demonstrated in this thesis. It is hypothesized that the sprayed jets are emitted from the paper tip to form a cone shaped distribution of ions with various intensity. To investigate this hypothesis, the shape of the electrospray at the tip was determined on three dimensions. A paper substrate was placed at 5 mm and was moved on the y, x, and z directions at voltage (3.6 KV). Peak response was observed in all directions. Based on these observations, I hypothesize that the ions sprayed in a cone shape (Figure 2.10). I predict that ions spread in approximately 164.85 mm3. This value was calculated according to the follow equations: Region = SA + V 9 Where SA and V were calculated as follow: SA = π r s + π r2 10 V = 0.33 π r2 h 11 56 Where r, s, and h were obtained as shown in Figure 2.10. Figure 2.10 The ions sprayed in a cone shape with different intense. 2.4.3 Properties of Paper Four factors were demonstrated in order to study the chemistry of the paper; including (i) solubility and polarity of analyte, (ii) size of analyte and pores on paper, (iii) the presence of trace elements on the paper, and (iv) the possibility of analyte/surface interaction. In previous works, a wide range of molecules have been analyzed using PSI/MS (Liu et al., 2010; Cooks et al., 2011; Wang et al., 2011; Liu et al., 2014; Zhang et al., 2014; Bain et al., 2014; Espy et al., 2012 & 2014). 2.4.3.1 Solubility and Polarity The solution of each analyte was loaded first on the paper substrate, and left until dry at room temperature. It was hypothesized that the analyte adsorbed to the paper by two ways. First, it adsorbed in the pores within the paper. Second, it adsorbed on the paper surface by analyte/surface interaction. Both affect the ionization efficiency, where a portion of analyte’s molecules lost. Different solvents have been used in order to study the effect of solubility and polarity of analyte on the ionization efficiency. It is well known that individual molecules of 57 an analyte separate from their neighbors and move between spaces of solvent molecules when the analyte dissolves in the solvent (Pape et al., 2014; Rouessac and Rouessac, 2007). The solvent molecules collide with the analyte molecules and the intermolecular forces of attraction between the analyte and the solvent molecules hold the analyte molecules in the spaces (Pape et al., 2014; Rouessac and Rouessac, 2007). There are two types of energy required for the extracting process: (i) the energy required to overcome the attraction between solvent molecules, where the solvent molecules must move apart to make room for analyte molecules, (ii) the analyte molecules must separate from the surface. This process also requires energy to overcome the forces of attraction between the analyte molecules and the surface. Then, the intermolecular forces of attraction between analyte and solvent take hold and the molecules move closer when the analyte molecules move between the solvent molecules. The attraction force is different from one analyte to another depending on the polarity of both the solvent and the analyte. Most of the previous works used a mixture of solvents (Pape et al., 2014; Liu et al., 2010; Li et al., 2011; Cooks et al., 2011; Zhang et al., 2012; Quinn et al., 2012; Yang et al., 2012; Liu et al., 2014). Each analyte is solubilized and extracted from the paper surface using an optimal solvent, which was selected depending on analyte/solvent interaction. In this thesis, both pure and mixture solvents have been used in order to mobilized or extract the analytes from the paper substrate, where in PSI/MS pure solvent can ionize the sample without the requiring the mixing of water (Liu et al., 2010; Li et al., 2011; Cooks et al., 2011). Several researchers have used a mixture of solvent for their analyses (Zhang et al., 2012; Quinn et al., 2012; Yang et al., 2012; Liu et al., 2014). The results in this thesis also show a good extraction percentage for the tested compounds using either pure or a mixture of solvents. The results showed that progesterone and the carotenoid compounds were extracted using pure methanol and acetone, respectively. The data showed that it is unlikely that a single solvent will effectively solubilize and facilitate the ionization of thyroid and steroid hormones. A series of solvents may be required to extract these hormones from the paper. However, it was demonstrated that water molecules play an important role in inducing the ionization. Pure solvents can extract the analyte from the paper surface depending on the polarity, and solubility of the analyte. 58 2.4.3.2 Size of Analyte and Pores The size of analyte and pores on the paper is another factor that might affect the analyte extraction from the surface. Five types of commercially available paper, which have different pore sizes, were selected and compared. The comparison accomplished according to the peak intensity and recovery percentage of the analyte in pure form. Recovery % was obtained for progesterone (10 µg/mL) using two cellulosic filter papers (Whatman filter paper grade 1 and grade 4). The results showed that paper with pores size 11 µm (Whatman filter paper grade 1) gave the highest recovery (96%). The paper with pores size approximately 20-25 µm (Whatman filter paper grade 4) gave recovery less than 94 %. In addition, the time of analysis was studied for each analyte using filter paper with pore size 11 µm. The result showed that the large molecules (T4) spend time (approximately 90 seconds) to be extracted from the surface more than small molecules (17α-hydroxyprogesterone, approximately 30 seconds). The interaction between the analyte and the surface might leads to this results. This hypothesis is discussed in the text further. However, it is suggested that a portion of both small and large molecules filled the pores but large molecules need more force to be extracted from these pores. Based on these results, not all molecules can be extracted from the surface, which affects the ionization efficiency. In addition, the peak intensity of the analytes were compared using the five types of commercial paper, cellulosic filter paper with different pores size (11, 20-25, 2.5 µm) and glass microfiber paper. The results showed that glass microfiber paper gave the lowest intensity. These results reproduce previous work by Liu and his co-worker, but go further to provide a valid explanation (Liu et al., 2010). Liu and his team used six types of paper including cellulosic, chromatography and glass fiber paper. They found that glass microfiber paper gave the lowest peak intensity (Liu et al., 2010). The major reason for this result was not discussed to date. In the current study, it is suggested that the poor conductivity of the borosilicate affects the ionization efficiency. It was demonstrated that the electric field on the surface of the solution affects the electrospray production using ESI (Sherma, 2004; Chai, 2003; York et al., 1990; Konermann et al., 2013). Under the effect of surface tension, the liquid form a semi-spherical shape at the tip of the emitter. It is crucial that the electric field on the solution surface is higher than the surface tension. This field leads to liquid polarization. The negative/positive ions migrate away from the electrode where the voltage is 59 applied. At this point, the solution sprayed forming Taylor cone at the tip (Sherma, 2004; Chai, 2003; York et al., 1990; Konermann et al., 2013). Since the glass microfiber made of pure borosilicate, the current on the surface is very low. Thus, the electric field at the paper tip is not enough to be higher than the surface tension of the solution. Thus, the poor conductivity of the glass microfiber surface decrease the ionization efficiency. Based on this result, the substrate with a good conductive material boost the efficiency of Taylor cone production. In this study, all cellulosic papers showed intensity higher than glass microfiber paper, where cellulose is a good conductive material. However, Whatman filter paper grade 1 was selected for the rest experiments in this thesis. 2.4.3.3 Trace Element and Analyte/Surface Interaction Whatman filter paper grade 1 (pore size 11 µm) showed the highest peak intensity of all other papers. Interestingly, cellulosic papers with the same pore size (1 PS) and smaller pores size (Whatman filter paper grade 5) showed peak intensity lower than grade 1. It is hypothesized that either the trace elements on the paper or the analyte/surface interaction affects the ionization efficiency. For cellulosic paper, which has the same pore size (1PS), it is suggested that the type and the amount of the trace elements on the paper affects the ionization efficiency. 1PS is made for allowing organic solutions to pass through while water-repellent to retain aqueous solutions. This paper has been washed repeatedly during the manufacture process using organic solvents, which means the trace elements and water molecules that presence on the surface have been removed. In addition, ashless filter paper was used, low intensity and no adduct peak were observed with this paper. Based on this result, the trace elements might affects the ionization efficiency. It is suggested that these trace element on the surface induce the ionization by different reactions such as adduct formation, charge transfer, and/or electron transfer in case of oxidation and/or reduction reactions. For whatman filter paper grade 5, it is suggested to have greater peak intensity because it has small pore size (2.5 µm). Interestingly, the results showed that grade 5 has the lowest peak intensity of all other cellulosic papers. It might be because the analyte/surface interaction is different from grade 1. This interaction might be occurred by hydrogen and/or oxygen bond formation, where Ullah et al. (2013) demonstrated that the interaction between 60 cellulose molecules and inorganic molecules was accomplished through oxygen atom which arise from a combined of electrostatic and H-bound (Ullah et al., 2013). 2.4.4 Paper Tip Constancy It was hypothesized that the shape of the tip might be changed with a high number of replicates. To test this hypothesis thyroid hormones, including T3 and T4 were determined within 21 replicates. The results showed that the measurements should be ≤ five number of replicates. The lowest value of RSD % (≤ 5 %) was obtained with number of replicates below six. The results showed wide range of RSD %, which obtained at the same experimental conditions. It is hypothesized that the shape of the paper tip changed during the analysis leading to this variation, where Yang et al. (2012) investigated that the variation in the shape of the tip affects the peak response (Yang et al., 2012). Thus, the experiments in this thesis were made within less than six replicates in order to increase sensitivity and accuracy of PSI. 61 Chapter 3 Separation of Complex Samples on Paper 3.1 Synopsis In this chapter, two hypotheses were studied. First, it was hypothesized that the hydrophilic surface of the paper could serve as a separation/ionization substrate, simultaneously. Thus, PSI might be utilized for a complex sample separation and enable identification of unknowns on a TLC plate. To test this hypothesis the separation efficiency was compared using PSI/MS, paper chromatography and thin layer chromatography. Retention factor (Rf) values of three carotenoids were obtained for each method in order to study the separation efficiency of PSI/MS. The results showed a good separation using PSI/MS. Second hypothesis was the trace elements on the paper affect the separation and quantitative analysis. To test this hypothesis recovery was accomplished with and without washing the paper. A series of model food chemicals were analyzed. Selected natural compounds, including kaempferol, β-carotene, zeaxanthin, astaxanthin, and caffeine were determined in 34 food samples. 3.2 Experimental 3.2.1 Chemicals and Reagents All analytes including kaempferol, β-carotene, zeaxanthin, astaxanthin, crocin, and caffeine were received from Sigma-Aldrich (St.Louis, MO). The chemical structure of the selected compounds is given in Figure 3.1. Several analytical grade solvents were used, including methanol, acetone, ethanol, acetonitrile, and dichloromethane; all of them were obtained from Fisher Scientific (Mississauga, ON). The stock solutions of β-carotene and astaxanthin were prepared using dichloromethane, while a series of dilute solutions were prepared using acetone. Methanol was used for preparing the other compound solutions. Whatman filter paper grade 1 (Fisher Scientific, Mississauga, ON) was the paper substrate for ionization. 62 OOOHHO NNNNOOCH3CH3H3C Caffeine Kaempferol Astaxanthin β-carotene OOHHOOHOHO 63 CH3CH3H3CCH3 CH3CH3CH3H3CCH3H3C OHHO OOOHOOHOHHOHOHOOOOOOOHOHOHHOOHOHOHOOCH3CH3CH3CH3OH Figure 3.1 Chemical structures of the tested compounds.Zeaxanthin Crocin 64 3.2.2 Apparatus All experiments were performed using a PremierTM series TOF/MS (Waters Inc., Mississauga, ON) and equipped with a custom-built assembly for PSI/MS. Mass spectra were collected in a full scan mode. Paper was cut into a triangle with approximate dimensions as was described in Chapter 2. Continuous flow rate of a mobile phase was applied through the paper substrate by using a syringe pump (Figure 3.2). MS parameters of capillary voltage, cone voltage, and flow rate (µl/min) were optimized based on the most intense peak for each compound. Ultrapure water was obtained from Millipore-Q 3 UV with a pump (Millipore S.A.S. 6712 Molsheim, France) throughout the work. The analyses were obtained with standard paper and washed paper, which was rinsed in an organic solvent (methanol). 3.2.3 Analytical Performance Stock solutions for each compound were prepared in different solvents. A series of standard solutions of each compound (1-250 µg/mL) were prepared by sequential dilution with several solvents. All solutions were stored at 4 °C, and kept in dark vials until further use. The analytical performance (range of linearity, LOD and LOQ) were obtained by analyzing pure standards. Both LOD and LOQ were calculated according to the IUPAC equations (Miller & Miller, 1994). The accuracy was defined as recovery %. Each paper substrate was injected with three levels of each standard solution. Recovery of each compound was defined as: % recovery= X/(X0 + C) x 100 (3.1) where X0 is the concentration of the compound in the sample, X is the found concentration of the compound, and C is the added concentration. Both X0 and X were obtained from the standard addition curve. 65 Figure 3.2 The paper spray ionization time of flight mass spectrometry; paper spray ionization apparatus and syringe pump on the right side. 3.2.4 Solvent Effects For PSI/MS, the mobile phase for quantitative analysis was optimized by using four polar organic solvents including methanol, acetone, ethanol, and acetonitrile. Each solvent was used in pure form without addition of any reagent, where one advantage of this technique is pure solvent can ionize analytes without using a mixture of solvents as a mobile phase (Wang et al., 2010). 3.2.5 Quantitative Analysis of Model Compounds An accurate volume (3-4 µL) of each sample was loaded on the paper substrate (Whatman filter paper grade 1). In this study, standard addition method based on five replicates was used. Peak intensity was measured before and after addition of increasing concentration of the standard solution for each sample. The change in the peak intensity was recorded and used for quantification. 66 3.2.6 Vegetable and Fruit Samples According to the reported method of Gonzalez-Rodriguez et al., 2008, each sample was prepared as follows: 34 samples were extracted, including fruit (apple, orange, papaya, pomegranate, fig opuntia, muskmelon, strawberry, mango), and vegetables (spinach, cilantro, lettuce, carrot, tomato, fava bean, pea, green pepper, broccoli, zucchini, mint). The part of each fruit and vegetable that was extracted is given in Table 3.1. Each sample was cut into small pieces and ground in a mortar and pestle, freeze-dried and stored, followed by extraction procedures. An accurate weight of each stored sample was placed in a 50 mL glass bottle and extracted with an approximate volume (20 – 30 mL) of acetonitrile. The glass container was vigorously homogenized in an ultrasonic bath for 20-25 min in order to extract the analytes from the sample. After sonication, the extracted solutions were separated out by filtration through filter paper (Gonzalez-Rodriguez et al., 2008).The oil layer in orange was extracted by using the same procedure, and separated from aqueous layer using a separating funnel. Subsequently, 0.45 µm cellulose membrane filter was used to filter the extracted samples. 67 Table 3.1 The extracted part of different fruit and vegetables Fruit Genus species Extracted Part Symbol Apple Malus domestica Borkh Peel F1 Orange Citrus sinensis L essential oil from peel F2 Peel F3 Pulp F4 Papaya Carica papaya L Slice F5 Peel F6 Seed F7 Pomegranate Punica malus Linnaeus Peel F8 Fig Opuntia Opuntia ficus-indica Linnaeus Slice F9 Peel F10 Muskmelon Cucumis melo L Slice F10 Seed F11 Strawberry Fragaria ananassa Duchesne Slice F13 Mango Mangifera Indica Watkins Peel F14 Vegetable Spinach Spinacia oleracea L Leave V1 Cilantro Coriandrum Sativum L Leave V2 Lettuce Lactuca sativa L Leaves V3 Carrot Daucus carota subsp. Sativus Hoffm V4 Tomato Solanum lycopersicum L Mesocarp and epidermis V5 Fava bean Vicia faba L Whole piece V6 Pea Pisum sativum L Whole piece V7 Green pepper Capsicum Annum L Whole piece V8 Broccoli Brassica oleracea var. botrytis Whole piece V9 Zucchini Cucurbita pepo Peel V10 Mint Mentha spicata L Leaves V11 Stems V12 3.2.7 Saffron Samples Five saffron samples were collected from different locations, Egypt (S1), India (S2), Iran (S3), Spain (S4), and Canada (S5). Saffron samples were extracted according to the reported method of Lage et.al, 2009 as follow: first, saffron stigma of each sample was ground using mortar and pestle. An accurate weight (0.1-0.4 g) of each ground sample placed in 20 ml dark vial and extracted with an approximate volume (12-20 mL) of methanol, then it 68 was mixed for 1 min. The contents of the dark vials were vigorously homogenized in the ultrasonic bath for 60 min in order to extract the analytes from the sample. All bottles were kept at 4 ºC (overnight) and the sonication process was repeated. Each extracted sample was filtered through 0.45 mm cellulose membrane filter and stored in a dark bottle (Lage & Cantrell, 2009). Petals (S6) and root (S7) of saffron flower are also extracted by using the same procedure, where the flower grew in Kelowna-Canada. 3.2.8 Commercial Samples Commercial samples including two tea samples, coffee, and tomato paste were analyzed. An accurate weight of each sample was extracted according to the recommended procedure, which is described in the last section (Gonzalez-Rodriguez et al., 2008). 3.3 Results 3.3.1 Comparison Between PSI and Two Standard Separation Methods In order to estimate the efficiency of PSI for sample component separation, several paper substrates with a range of length (0.5-5 cm) were prepared. A mixture solution of natural pigments, including astaxanthin, zeaxanthin, and crocin was used for this study. The paper was placed at 5 mm, and 3.5 KV of voltage was applied. The flow has a constant value of 10 µL/min with all lengths of the paper substrate. It was provided that the total time of analysis increased with increasing the length of the substrate. Furthermore, RSD % was obtained in order to study the precision of this experiment. The values of RSD % were less than 8 %. In order to determine the capability of paper spray ionization for sample component separation Retention factor (Rf) values of three pigments (astaxanthin, zeaxanthin, and crocin) were obtained. Furthermore, the results were compared in Rf values that obtained by two standard methods, including paper chromatography and thin layer chromatography (Figure 3.3). Rf values were within a range of 0.80 – 0.83 for astaxanthin using the three methods. Rf values were within ranges of 0.77-0.78 and 0.95-0.98 for zeaxanthin and crocin, respectively. The data provided evidence that paper spray ionization can be used as separation/ionization technique for sample component analyses, simultaneously (Table 3.2). 69 Figure 3.3 The natural pigments solution (astaxanthin, zeaxanthin, and crocin) that was separated using (A) paper spray ionization, (B) paper chromatography and (C) thin layer chromatography. C A B 70 Table 3.2 Comparison between paper spray ionization and two standard methods, including (a) paper chromatography and (b) thin layer chromatography Length (cm) Flow rate (µL/min) Distance of analyte move through the paper (cm) Retention factor (Rf) RSD% Astaxanthin Zeaxanthin Crocin Astaxanthin Zeaxanthin Crocin 2 10 1 1.2 0.9 0.79 0.79 0.98 6 4 10 2.7 2.8 2.5 0.80 0.78 0.97 7 5 10 3.4 3.5 3 0.81 0.77 0.95 8 5 (PC) - 3.8 3.9 2.8 0.85 0.78 0.96 - 5 (TLC) - 3.7 4 2.9 0.83 0.77 0.95 - 71 3.3.2 Characteristics of PSI/MS Spectra In order to investigate the reliability of PSI/MS for model compounds analysis, both positive and negative ion modes were used. Parameters, including capillary volt and flow rate, were optimized at 3.5 KV and 10 µL/min using PSI, respectively. The highest responses were obtained for the tested analytes in the positive ion mode. The peak response was low for β-carotene because this compound is non-polar. The spectra in positive ion mode showed protonated ion peaks at m/z 195, 287, 537, 569, and 597 for caffeine (m/z 194), kaempferol (m/z 286), β-carotene (m/z 536), zeaxanthin (m/z 568), and astaxanthine (m/z 596), respectively. The analyses in this chapter is based on the protonated peak for each analyte. 3.3.3 Analytical Performance The peak response of each analyte was determined at various concentrations (1-200 µg/mL). The values of linear regression were typically higher than 0.997 and showed good linearity in the range 1-30 µg/mL for caffeine, kaempferol, β-carotene and zeaxanthin, while the linearity with the range of 1-100 µg/mL was found for astaxanthin (Table 3.3). The value of LOD was equal to 0.53, 0.29, 0.22, 0.45, and 0.89 µg/mL for caffeine, kaempferol, β-carotene, zeaxanthin, and astaxanthin, respectively. The limit of quantification for each analyte was investigated within a range of 0.73-2.99 µg/mL. 72 Table 3.3 Optimized parameters and analytical performance for paper spray ionization Compound Time of analysis (sec.) Flow rate (µL/min) Mobile phase LOD (µg/mL) LOQ (µg/mL) Linearity (µg/mL) Caffeine 30 10 MeOH 0.53 1.79 1-30 Kaempferol 30 10 MeOH 0.29 0.97 1-30 β-carotene 60 10 MeOH 0.22 0.73 1-30 Zeaxanthin 60 10 MeOH 0.45 1.45 1-30 Astaxanthin 90 10 MeOH 0.89 2.99 1-100 73 3.3.4 Paper/Solvent Interaction 3.3.4.1 Trace Elements Both standard paper and paper that was washed were utilized in order to study the effect of the trace elements, such as K, C, O, and Cl, and/or the analyte/surface interaction on quantitative analysis. The paper was washed using organic solvent (methanol) prior the analysis. Recovery percentage was calculated with each paper using the selected analytes in their pure solution. The results showed that the recovery value with the standard paper (95 %) was less than the values with washed paper (99 %) for progesterone at the same conditions. Thus, the quantitative analyses in this thesis were obtained with washed paper. 3.3.4.2 Solvents In order to optimize the solvent with the highest efficiency, four polar solvents were investigated, including methanol, acetone, ethanol, and acetonitrile. The results revealed that acetone gave the highest signal response for all compounds. Approximately 39 % of zeaxanthin was detected by using acetone, while other solvents extracted this analyte with lower percent equal to methanol (21%), ethanol (6 %), and acetonitrile (34%). In addition, acetone extracted the remaining analytes with different extraction ratio, including kaempferol (37.5 %), β-carotene (36.4 %), and astaxanthin (37.5%) (Figure 3.4). Similarly, acetonitrile extracted the target analytes with percentage lower than acetone but higher than the rest solvents with an exception of kaempferol (Figure 3.4). In contrast, ethanol extracted these compounds from paper substrate with lowest ratios equal to 11.6 %, 9.1 %, 5.2 %, and 7.8 % for kaempferol, β-carotene, zeaxanthin and astaxanthin, respectively. For methanol, ranges of 21.4-27.6 % of the compounds were extracted. Given these results, acetone was selected as a mobile phase for quantitative analysis of these compounds. 74 061218Kaempferol β-carotene Zeaxanthin AstaxanthinMeOHEtOHAcetoneAcetonitrilePeak respons (103) Figure 3.4 Solvent effect on mobility/extraction of the tested compounds. Acetone was selected as a mobile phase for quantitative analysis of these compounds. 3.3.5 Quantitative Experiments 3.3.5.1 Quantification Analysis of Natural Carotenoids in Fruit and Vegetables The concentrations of the selected natural compounds in all food samples (fruit and vegetable) are shown in Table 3.4. In the present study, 34 food samples were analyzed. Kaempferol is observed in all fruit samples with the exception of F8, F11, and F12. This compound was detected in fruit samples within a range of concentrations (58-837 µM). For vegetables, zucchini was the only sample that did not have this compound. The symbol that refers to each sample is given in Table 3.1. Eleven samples (fruit and vegetable) did not contain either β-carotene or astaxanthin. Two samples (cilantro, lettuce) have neither astaxanthin nor β-carotene. A wide range of concentrations (12-347 µM) was investigated for both carotenoid compounds through different samples. β-carotene was found with the highest concentration in spinach, while astaxanthin was the predominant carotenoid in orange pulp. Similarly, zeaxanthin was found in most samples either fruit or vegetable. Four samples did not contain zeaxanthin; including F2, F8, V1, and V9. Figure 3.5 represents the standard addition curves of the carotenoids in mango. 75 Table 3.4 Concentrations of each carotenoid over 34 samples, and calculated % recovery Samples Kaempferol β-carotene Zeaxanthin Astaxanthin Fruit C (µM) Recovery% C (µM) Recovery% C (µM) Recovery% C (µm) Recovery% Apple 174 97 62 98 146 99 96 100 Orange Oil layer from peel 174 98 56 95 0 - 39 98 Peel 58 98 155 98 146 97 347 97 Pulp 150 98 0 - 185 98 247 99 Papaya Slice 233 99 186 98 88 99 134 100 Peel 290 95 0 - 105 97 28 - Seed 524 97 62 99 88 99 0 - Pomegranate 0 - 62 100 0 - 39 97 Fig opuntia Fig opuntia slice 465 99 124 96 176 96 129 98 Peel 465 97 248 96 100 95 112 95 Muskmelon Slice 0 - 62 99 29 98 39 99 Seed 0 - 112 98 116 96 156 97 Strawberry 837 96 104 98 105 97 73 99 Mango 349 96 104 97 205 98 163 97 Vegetable Spinach 233 96 250 96 0 - 0 - Cilantro 233 94.9 0 - 135 95 0 - Lettuce 349 96 0 - 439 98 0 - Carrot 233 97 93 97 158 98 0 - Tomato 116 99 62 96 47 95 73 97 Fava bean 58 97 0 - 12 98 12 98 Pea 233 96 80 97 58 95 28 99 Green pepper 233 98 124 99 135 95 112 99 Broccoli 150 95 31 95 0 - 39 100 Zucchini 0 - 19 97 40 94.9 50 97 Mint Leaves 233 97 155 99 88 96 39 98 Stems 408 96 0 - 70 97 70 99 Saffron S1 465 95 248 97 193 99 0 - S2 185 96 112 99 100 97 79 98 S3 233 96 62 98 105 98 67 99 S4 129 96 0 - 216 97 101 98 S5 147 98 104 99 135 99 39 99 S6 66 99 43 99 29 99 11 96 S7 147 99 0 - 234 98 0 - Artificial sample Tomato paste 174 96 0 - 58 99 0 - 76 Figure 3.5 Standard addition curve of (A) astaxanthin, (B) kaempferol, (C) β-carotene, and (D) zeaxanthin in mango. 3.3.5.2 Quantification Analysis of Natural Carotenoids in Commercial and Saffron For commercial sample (tomato paste), only kaempferol and zeaxanthin were observed in this sample. These compounds were obtained at level 174 µM (kaempferol) and 58 µM (zeaxanthin). For saffron, kaempferol was the prevalent natural compound within 75% of the total concentration. Furthermore, this analyte was obtained with the highest ratio in all saffron samples. The exception was Spanish saffron, where zeaxanthin was found with the highest level (216 µM). Both β-carotene and astaxanthin were obtained in all saffron samples within ranges of 11-248 µM. In contrast, Spanish and Egyptian saffron samples provided no evidence of the presence of β-carotene and astaxanthin, respectively. Neither β-A B D C 77 carotene nor astaxanthin were present in saffron root. Zeaxanthin was observed at level ranged between 29-234 µM. 3.3.5.3 Quantification Analysis of Caffeine in Samples Over recent years, caffeine has been considered to be one of the most useful natural compounds. Caffeine affects our body in different ways. It is act as tranquilizer, myocardial stimulant, and smooth-muscle relaxant. Cold medications contain caffeine as it activates immune system (Aranda and Morlock, 2006; Perron et al., 2008; Li et al., 2010). This compound is found in different samples at various concentrations (Aranda and Morlock, 2006; Perron et al., 2008; Li et al., 2010). In the present chapter, caffeine was investigated at different concentration in three commercial samples, including two tea samples, and coffee. Recommended standard addition method was used in this session as described in experimental part, as well. Caffeine was found in the commercial samples within range of 86.1-171.2 µM in all samples (teas and coffee) (Figure 3.6). Figure 3.6 The concentrations of caffeine in different samples 78 3.3.5.4 Recovery In order to investigate the accuracy of PSI after the paper was washed, each sample was spiked within three levels of standard solutions. The result values for carotenoid compounds were within ranges of 95-100% in fruit samples, while they had ranges equal to 94-100 %, 95-99% and 96-99% in vegetables, saffron, and tomato paste samples. For caffeine, the range of 98-99.5 % of recovery was obtained. 3.4 Discussion 3.4.1 PSI/MS as Separation/Ionization Technique Recently, complex mixtures were ionized without prior sample preparation using several ambient ionization methods such as DESI (Wiliams et al., 2006), MALDI (Tyan et al., 2013; Chen et al., 2012), PSI (Wang et al., 2011; Espy et al., 2012), and LESA (Cheng et al., 2011). These studies focused on how we can analyze real samples directly. One limitation to previous work is the incomplete chromatographic separations of a complex sample. It was hypothesized that PSI/MS is useful for efficient separation of a complex sample. To test this hypothesis Rf values of three carotenoids (astaxanthin, zeaxanthin, and crocin) were compared with two standard separation methods in order to identify the separation efficiency of PSI/MS. The values were close to each other by using PSI/MS, paper chromatography and thin layer chromatography for the same analyte. RSD % values of this experiment were obtained with good values. For further addition, this experiment has been repeated at three different lengths of the paper substrate with flow rate 10 µL/min. Rf values were similar with the different lengths. For paper chromatography and TLC, the separation took 15 minutes to be finished while it took less than 5 minutes with PSI depending on the length of the paper. Based on these results, PSI/MS can separate a mixture of carotenoids with good separation efficiency within short time. This approach increase the capacity of PSI/MS to be utilized for a complex sample analysis within few seconds. In a previous work, liquid chromatography was used for carotenoids analysis. A high-pressure pump was required with that method (Verma and Middha, 2010). The total analysis required few minutes to be finished. Delpino-Rius et al determined 27 carotenoids in different fruit samples. The chromatographic analysis took less than 17 minutes, while it took only few seconds to finish the whole analysis in the current study without the requirement of 79 high pressure pump. In addition, it is well known that TLC has a weak detector (Cheng et al., 2011).When dealing with unknown samples, there is a need for a separation step in order to determine what is being analyzed (Cheng et al., 2011; Verma and Middha, 2010). The result in this thesis showed that PSI/MS is a good technique for sample separation where unknown compounds on TLC can be identified with PSI/MS. Thus, the novel approach in the current study increased the capacity of PSI/MS for a mixture sample separation with less time, less work potential and a good separation efficiency. 3.4.2 PSI Efficiency for Quantitative Analysis Washed papers were used in order to determine the accuracy of PSI for quantitative analysis. It is suggested that the trace elements or molecules adsorbed on the paper either during manufacturing process or from the lab environment affects the quantitative analysis. These elements and/or molecules might be removed from the paper by washing the paper with organic solvent prior the analysis. The results showed that the recovery percentage of the analyte (in pure solution) with standard paper was lower than washed paper. Thus, 34 food samples were analyzed using washed paper to test the effect of the washed (using methanol) paper by calculating the recovery percentage. The quality of fruit and vegetables is determined by several parameters; among these are color, stability and the presence of nutrients with health benefits. Carotenoids are one of these nutrients, which were used in various medication purposes (Schwartz & Patroni-Killam, 1985; Amorim-Carrilho et al., 2014; Valduga et al., 2014; Shen et al., 2014). It is discussed in Chapter 2 of this thesis how the solvent could extract the analyte from the paper surface. Based on the polarity and solubility of selected natural compounds, the optimal solvent in this study was acetone. Five natural compounds were determined in various foodstuffs, including fruits, vegetables, saffron, teas, coffee, and tomato paste. A previous work determined 27 carotenoids in apple, peach, and pear product (Delpino-Rius et al., 2014). The LOD was 0.022 and 0.157 µg/mL for zeaxanthin and β-carotene, respectively. It was 0.45 µg/mL and 0.22 µg/mL for the same carotenoids in the current study. The analytical performance was in agreement with the previous work. The concentrations of β-carotene ranged between 5.41 x 10-4-0.057 µM in different food samples (Delpino-Rius et al., 2014), while it ranged between 19 - 250 µM in the current study. For zeaxnthin, it ranged between 2.8 x 10-4 – 0.019 µM (Delpino-Rius et al., 2014), 80 while it ranged between 12 – 439 µM in the current study. The recoveries were 104 % (β-carotene) and 96 % (zeaxanthin) (Delpino-Rius et al., 2014). In another previous work, the recovery was obtained by adding standard solution to extracted solution (Dong et al., 2013). The recovery ranged between 99.79-102.13 % (Dong et al., 2013). In the current study, to evaluate the effect of the washed paper on the recoveries of the selected carotenoids, samples were spiked just prior the analysis. The recoveries ranged 95 – 100 % for both β-carotene and zeaxnthin over 15 fruit samples. For vegetable samples, a previous work determined kaempferol in broccoli with recovery ranged between 92-93 % (Koh et al., 2009). These low values might be obtained because the losing of the analyte during the extraction process. The recovery ranged between 95 – 99 % in the current study for kaempferol. Wang et al (2011) determined caffeine in different tea sample using high performance liquid chromatography (HPLC). The recovery ranged between 88.2 – 99.3 % (Wang et al., 2011). Another previous work determined this analyte in tea, and coffee. The recovery ranged between 96.2 – 101.2 % (Sereshti and Samadi, 2014). It reached to 100 % in the current study. Approximately all recovery values in the current study were more than 95 % using washed paper. Based on these results, it is indicated that the washed paper makes PSI more adequate for quantitative analysis. However, it is demonstrated that the porosity and analyte/surface interaction lead to adsorb the analyte on the paper (Chapter 2), which might affects the quantitative analysis. Thus, we need to modify the surface of the paper to improve the capacity of PSI for quantitative analysis. A new achievement on paper surface modification using silica nanoparticles is discussed in this thesis. 81 Chapter 4 Comparison of Three Ionization Techniques: Corona Discharge 4.1 Synopsis In Chapter 2, it was observed that the peak intensity decreased at short distance between the paper tip and MS inlet. It was hypothesized that corona discharge might be formed with a short distance between the paper tip and MS inlet. To test this hypothesis, the data was compared using three ionization techniques, based on electrospray. Paper spray ionization (PSI), electrospray ionization (ESI), and electrospray chemical ionization (ESCI) were used in this study. At distance 5 mm, PSI/MS provided results consistent with ESI (in the absence of corona discharge) more than ESCI (in the presence of corona discharge). In contrast, the results showed that PSI is more consistent with ESCI more than ESI in ion formation at distance lower than 5 mm. However, it is recommended to inhibit corona discharge; otherwise, it is hard to analyze the selected natural compounds using PSI/MS. Furthermore, Espy and his co-worker demonstrated that the corona discharge might be formed at low solvent flow rates because the distribution of the solvent through the paper was not uniform (Espy et al., 2012). In the current study, it is hypothesized that continuous flow of the solvent makes the distribution of the solvent constant through the paper, which inhibits the possibility to produce corona discharge. The continuous flow of solvent and selected distance (5 mm) between the paper tip and MS inlet showed inhibition in the production of corona discharge. 4.2 Experimental 4.2.1 Chemicals and Reagents All analytes were received from Sigma-Aldrich (St.Louis, MO). A series of dilute solutions were prepared as described in Chapter 3. 4.2.2 Apparatus All experiments were performed using a PremierTM series TOF/MS (Waters Inc., Mississauga, ON) as described in Chapter 3. Mass spectra were collected in a full scan mode. Three techniques including PSI, electrospray ionization (ESI), and electrospray chemical ionization (ESCI) were operated in the positive and negative ion modes. 82 4.2.3 Analytical Performance The values of analytical performance for each analyte was obtained as described in Chapter 3. These values were investigated with the three ionization techniques. Intra and inter-day test were obtained. 4.2.4 F-test In order to estimate the consistency of PSI with standard methods, F-test was used. The standard deviation for each technique was obtained within five replicates. F-test was calculated according to equation 4.1: F-test = S22/S12 (4.1) Where S2 and S1 are standard deviation of proposed method and standard method. The standard deviation was calculated according to equation 4.2: S2= (∑(xi-x)2/n-1) (4.2) Where x is the mean value for n replicates and xi is the value for each measurement. 4.3 Results 4.3.1 MS Spectra Characterization Both positive and negative ion modes were used in this study. Parameters, including capillary voltage and flow rate, were optimized at 3.5 KV and 10 µL/min using PSI, respectively, while 3.3 KV and over 30 µL/min were selected for ESI and ESCI. The highest responses were obtained for the tested analytes in the positive ion mode for all ionization sources. The spectra in positive ion mode showed protonated ion peaks at m/z 195, 287, 537, 569, and 597 for caffeine (m/z 194), kaempferol (m/z 286), β-carotene (m/z 536), zeaxanthin (m/z 568), and astaxanthine (m/z 596), respectively. The optimized parameters are given in Table 4.1. These spectra were observed using the three ionization techniques with an exception of caffeine and kaempferol. These compounds showed no detectable ion formation with ESCI. Other peaks were observed. A deoxygenated peak was observed for astaxanthin with both PSI and ESI, while ESCI show no existence of this peak. In addition, the protonated peak was the predominant peak for all tested compounds with an exception for astaxanthin where sodium adduct peak was the predominant peak of all other peaks. Both caffeine and kampferol showed no ions formation using ESCI because the presence of corona discharge. The results revealed that PSI is consistent with ESI when the distance between the paper tip 83 and MS inlet is 5 mm. At shorter distance, the results showed that PSI is more consistent with ESCI. However, the paper tip was placed at 5 mm to MS inlet for the rest study of this thesis. Furthermore, lower relative intensities within the mass range of interest were observed in negative mode. In this ion mode, deprotonated ion peaks at m/z 193, 285, 535, 567, and 595 were observed for caffeine (m/z 194), kaempferol (m/z 286), β-carotene (m/z 536), zeaxanthin (m/z 568), and astaxanthine (m/z 596), respectively. The results showed peak responses for the tested compounds with positive ion mode higher than negative ion mode using the three techniques. Based on this result, positive ion mode was selected for this study. Table 4.1 Optimized parameters for paper spray ionization, electrospray ionization and electrospray chemical ionization Compound PSI ESI ESCI Time of analysis (sec.) Flow rate Mobile phase Time of analysis (sec) Flow rate Mobile phase Time of analysis (sec.) Flow rate Mobile phase Caffeine 30 10 MeOH 180 35 MeOH+ 0.1% formic acid - - - Kaempferol 30 10 MeOH 180 35 MeOH+ 0.1% formic acid - - - β-carotene 60 10 MeOH 180 35 MeOH+ 0.1% formic acid 180 40 MeOH+ 0.1% formic acid Zeaxanthin 60 10 MeOH 180 35 MeOH+ 0.1% formic acid 180 40 MeOH+ 0.1% formic acid Astaxanthin 90 10 MeOH 180 35 MeOH+ 0.1% formic acid 180 40 MeOH+ 0.1% formic acid 84 4.3.2 Analytical Performance Using Three Ionization Techniques The peak response of each analyte was determined at various concentrations (1-200 µg/mL). The values of linear regression were typically higher than 0.997 and showed good linearity in the range 1-30 µg/mL for caffeine, kaempferol, β-carotene and zeaxanthin, while the linearity with the range of 1-100 µg/mL was found for astaxanthin. Figure 4.1 represents calibration curves of astaxanthin using the three ionization techniques. Similarly, ESI/MS gave calibration curves with the mean correlations within the range 0.992-0.999. Good linearity was shown in the ranges 1-30 µg/mL for caffeine, kaempferol, β-carotene, and zeaxanthin, whereas astaxanthin had linearity ranged between 1-50 µg/mL. For ESCI the range of linearity was within range of 1-10 µg/mL for β-carotene and zeaxanthin while it was within range of 1-30 µg/mL for astaxanthin. The correlation coefficients for each technique are given in Table 4.2. The value of LOD ranged between 0.22 – 0.89 µg/mL using the PSI/MS technique. It ranged between 0.03 – 0.71 µg/mL for the same compounds, respectively, using the ESI/MS. The limit of quantification for each analyte was investigated within a range of 0.73-2.99 µg/mL and 0.10-2.38 µg/mL, using PSI/MS and ESI/MS, respectively. For ESCI, the LOD ranged between 0.62-0.91 µg/mL. 85 Figure 4.1 Calibration curve of astaxanthin using (A) electrospray chemical ionization, (B) electrospray ionization, and (C) paper spray ionization. B C A Peak response Peak response Peak response (102) 86 Table 4.2 Analytical performance for paper spray ionization, electrospray ionization and electrospray chemical ionization 4.3.3 F-test In order to determine if there is significance difference between PSI and ESI, F-test value was obtained using several compounds. The results provided evidence that F-test was within range of 0.07- 1. According to this result, there is no significant difference between the new developed technique PSI and the standard ionization method ESI when the paper tip was placed at 5 mm from the MS inlet. Furthermore, the correlation curve between the two ionization sources was obtained. The results provided good correlation between PSI and ESI within level of confidence range of 90-99%. Compound PSI ESI ESCI LOD LOQ R2 Linearity LOD LOQ R2 Linearity LOD LOQ R2 Linearity Caffeine 0.53 1.79 0.995 1 – 30 0.03 0.1 0.997 1- 30 - - - - Kaempferol 0.29 0.97 0.996 1 – 30 0.25 0.83 0.995 1 – 30 - - - - β-carotene 0.22 0.73 0.997 1 – 30 0.1 0.34 0.997 1 – 30 0.87 2.89 0.9563 1-10 Zeaxanthin 0.45 1.45 0.999 1 – 30 0.71 2.38 0.999 1 – 30 0.62 2.04 0.9998 1-10 Astaxanthin 0.89 2.99 0.997 1 – 100 0.49 1.62 0.992 1 – 50 0.91 3.05 0.9849 1-30 87 4.4 Discussion Recently, direct ionization sources were developed, where a spray is produced directly from the substrate (Liu et al., 2010; Wang et al., 2010). Paper spray ionization is one of these techniques, where the first experiment on PSI/MS developed in 2010 by Cooks team (Liu et al., 2010; Wang et al., 2010). The mechanism of this technique was demonstrated by Espy and his co-worker (Espy et al., 2012). They indicated that corona discharge could be produced with the change in the uniformity and the amount of mobile phase on the paper substrate. They did their experiments with a small amount of solvent (Espy et al., 2012). In this thesis, it was hypothesized that a continuous injection of a solvent by using a pump syringe might inhibit the presence of corona discharge by keeping the solvent motion through the paper substrate more stable, making PSI/MS more consistent with ESI/MS. A pump syringe was used with the three techniques. The results showed consistency between PSI/MS and ESI/MS techniques under the optimal experimental conditions with continuous flow of solvent. MS spectra showed the same ion peaks for all the tested compounds using the PSI and ESI techniques. The results showed ions that obtained by ESCI different from the ions that obtained by the previous two ionization techniques either in the peak response or ion formation. Radical cations were observed with both PSI and ESI (for kaempferol, zeaxanthin and β-carotene), while it was not observed with ESCI. Based on these results the presence of corona discharge affects ion formation. Corona discharge might neutralize the ions formed by electron transfer reaction. This reaction is possible with the presence of corona discharge (Liang et al., 2006; Munson, 2000). By using a small amount of solvent (10 µL), variability in peak responses were observed, supporting the published work (Espy et al., 2012). The results of the current study showed no consistency between PSI and ESCI (presence of corona discharge) with a stable amount of solvent through the paper substrate. Thus, continuous flow of solvent inhibit the possibility to production of corona discharge. In addition, it was demonstrated that the peak responses decrease with a shorter distance between the paper tip and MS inlet cone (Chapter 2). It is hypothesized that the possibility of the production of corona discharge increase at short distance (< 5 mm). Corona discharge is a process by which a current flows from an electrode with a high-applied voltage. It is usually forms at highly sharp section on electrodes, such as sharp corners (Motret et al., 2002; Abdel-Salam et al., 2003; Skalny and Mason, 2002; Simek and Clupek, 88 2002; Ono and Oda, 2003). The ions generated eventually pass charge to nearby areas of lower potential (MS inlet), or recombine to form neutral molecules. This corona is produce when the applied potential is large enough (Motret et al., 2002; Abdel-Salam et al., 2003; Skalny and Mason, 2002; Simek and Clupek, 2002; Ono and Oda, 2003). Thus, the solution at the tip ionizes and it becomes conductive. If a tip of the paper has a sharp point, the electric field strength around that point will be higher than other regions. It was demonstrated that the area near the sharp point can become ionized (partially conductive), while regions more distant do not (Motret et al., 2002; Abdel-Salam et al., 2003; Skalny and Mason, 2002; Simek and Clupek, 2002; Ono and Oda, 2003). At a sufficiently high voltage between the electrodes, the discharge starts at the tip. It is suggested that there are two ways to create the corona discharge on the paper tip. First, an electric layer can cover the paper substrate. At a sufficiently high voltage, the discharge starts at the tip of the paper. It spreads out until it reaches the MS inlet. Second, the discharge develops when the voltage difference is high between the electrodes (paper substrate and MS inlet cone). It spreads out towards the MS inlet. There are two type of corona discharge depending on charge of applied voltage (Motret et al., 2002; Abdel-Salam et al., 2003; Skalny and Mason, 2002; Simek and Clupek, 2002; Ono and Oda, 2003). It is a positive corona when the paper substrate is connected to a positive output of power supply. A negative corona discharge produce when the paper is connected to the negative of the power supply. The results revealed that the peak response decrease in the presence of corona discharge (at short distance), which affects the ionization efficiency. Therefore, we need to avoid the production of corona discharge. There are two ways to inhibit the production of corona discharge at the paper tip. First, the voltage can be made low enough on the paper substrate to stop the spreading of the discharge somewhere before the MS inlet is reached. Second, decrease the field strength between the paper tip and MS inlet cone by increasing the distance. At 5 mm with continuous flow rate of solvent PSI was consistence with ESI more than ESCI. This novel achievements added more insight on the fundamental concept of the ionization mechanism. 89 Chapter 5 Comparison of Two Mass Analyzers and Two Surface Ionization Sources 5.1 Synopsis In this chapter, it is demonstrated that the PSI has different sensitivity across two MS instruments using the same lab-made apparatus. In order to further investigate the robustness of PSI as an introduction and ionization technique, the lab-made PSI system was interfaced to both a TQ/MS and TOF/MS system. The system were compared for analysis using caffeine, progesterone, cortisone, hydrocortisone, triiodothyronine (T3) and thyroxin (T4). Aspects such as sensitivity, peak response, and recoveries of paper spray ionization over the two mass analyzer were determined. Analytical performance including limit of detection, limit of quantification and range of linearity for each technique were indicated. It is hypothesized that the way of detection ions across two mass spectrometry affects the sensitivity of the PSI. Further, it is hypothesized that analyte mobilization or extraction from the paper is different between surface ionization techniques, which affects the ionization efficiency. To test this hypothesis a comparison between two surface ionization techniques, including PSI and liquid extraction surface analysis (LESA) were determined with triple quadrupole mass spectrometry. Recoveries were obtained for each method. These studies provide insight into distinguishing between solubility and micro-flow dynamics with surface/ionization techniques. 5.2 Experimental 5.2.1 Chemicals and Reagents All compounds including caffeine, progesterone, cortisone, hydrocortisone, T3, and T4 were purchased from Sigma-Aldrich Canada. Individual stock solutions were prepared by using methanol, and storage in dark container at 4 ̊C. Dilute solutions, at different concentration, were daily prepared. The solvents including methanol and acetone were obtained from Fisher Scientific (Mississauga, ON). Whatman filter paper grade 1 were also obtained from Sigma-Aldrich, Canada. 90 5.2.2 Triple Quadrupole Mass Spectrometer Conditions The Waters Quattro equipped with a PSI source as it was described in Chapter 2. Parameters were optimized for each analyte by continuous injection of a standard solution with flow rate 10 µL/min. The PSI source values were RF lens (1.5V), extractor (2 V), and cone temperature (120 ̊C). 5.2.3 Time of Flight Mass Spectrometer Conditions The Time of Flight mass spectrometer equipped with PSI source (Chapter 3). The standard solution of each analyte was injected to ionization source in order to optimize the instrumental parameters. The compounds were injected by using infusion method via syringe pump at flow rate 10 µL/min. The cone temperature was set to 120 ̊C. 5.2.4 Surface Ionization Sources Instrumentation For PSI, ~3 µL was loaded on the paper substrate. Each sample was injected at flow rate 10 µL/min in order to optimize the parameters. For LESA, the sample was loaded on the substrate with small amount (~10 µL). The parameters were optimized by injecting the standard solution of each compound continuously via a syringe pump at a high flow rate over than 100 µL/min of the mobile phase. LESA was designed and built in our lab (Figure 5.1). Electrospray probe was used for LESA to ionize the tested compound from the extracted solution. The mass spectrometry was run in full scan using both ionization sources. 91 Figure 5.1 Image of lab-made liquid extraction surface analysis (LESA) setup with triple quadrupole mass spectrometry. The right side of the probe was responsible for making the liquid micro junction on the paper substrate and the extracted analyte flow to electrospray probe via the left side. 5.2.5 Analysis of Samples 5.2.5.1 Recommended Procedures for the Quantitative Analysis of Each compound For PSI, ~3 µL of each sample was loaded on paper (Whatman filter paper grade 1). The standard addition method based on six replicate measurements was used. Peak intensity was measured before and after addition of increasing concentration of the standard solution for each compound. The change in the peak intensity was then recorded and used for quantification. 5.2.5.2 Analysis of Thyroid Hormones Larvae of Strongylocentrotus purpuratus urchin, sea water, eggs spawned as well as guts and mesenteries harvested from adult female sample were prepared by the authors Ashley E. M. Miller and Andreas Heyland in Department of Integrative Biology, College of Biological Science, The University of Guelph, Guelph, ON, Canada. The samples were prepared according to previously reported methods (Miller and Heyland, 2010). 92 5.3 Results 5.3.1 Comparison of Two Mass Analyzers 5.3.1.1 Comparison of Mass Spectra Obtained by TQ/MS and TOF with PSI A wide range of applied potential (1.5-4.5 KV) was used in order to optimize mass spectrometry parameters. The results show that the peak responses increased within a range of 3-3.6 KV and 3.5-3.7 KV using triple quadrupole and time of flight, respectively. The optimized parameters for each analyte are given in Table 5.1. The results in this thesis show that the ions formed were similar by TQ and TOF. Both ion modes were investigated using the two instruments. The results show that both techniques gave ions with responses in the positive ion mode higher than the negative ion mode. Thus, the positive ion mode was found to be optimal for this study. The ions for each analyte were obtained in full scan mode. The protonated ion peak was obtained as a predominant ion peak for all tested compounds using both mass analyzers. The peak response that was obtained by TQ was higher than the peak responses obtained by TOF by two to three orders of magnitude for the same tested compounds (Figure 5.2). 93 Table 5.1 Parameters and linear regression obtained by either triple quadrupole or time of flight mass spectrometry with paper spray ionization Compound Triple quadruple TOF Predominate ion Applied volt (KV) R2 Predominate ion Applied volt (KV) R2 Caffeine [M+H]+ 3.50 0.9180 [M+H]+ 3.50 0.9963 Progesterone [M+H]+ 3.66 0.9996 [M+H]+ 3.50 0.9946 Cortisone [M+H]+ 3.50 0.9987 [M+H]+ 3.70 0.9984 Hydrocortisone [M+H]+ 3.63 0.9904 [M+H]+ 3.50 0.9836 T3 [M+H]+ 3.50 0.9810 [M+H]+ 3.50 0.9753 T4 [M+H]+ 3.00 0.9936 [M+H]+ 3.50 0.9863 94 Figure 5.2 The peak responses obtained by (A) triple quadrupole and (B) time of flight mass spectrometry with paper spray ionization. A B 95 5.3.1.2 Comparison of Analytical Performance By plotting the peak responses via a wide range of concentrations, a calibration curve with linear regression was obtained for each compound. The values of linear regression are given in Table 5.1. Thereafter, the analytical performance was evaluated according to IUPAC guidelines (Miller& Miller, 1994). The limit of detection was established at the lowest level detection of the compound. The repeatability was obtained within-day repeatability with an acceptable value (<8%) by either PSI-TQ/MS or PSI-TOF/MS. Analytical performance for each analyte by using both mass analyzer are given in Table 5.2. 5.3.1.3 Analysis of Thyroid Hormones PSI/MS technique was used for the analysis of T3 and T4 in different samples. The detected levels for each hormone are presented in Table 5.3. The recovery was successfully detected with acceptable values (more than 85%). The recovery was obtained with an acceptable inter-day repeatability. The values of recovery and repeatability for each sample are given in Table 5.4. 96 Table 5.2 Analytical performance obtained by either triple quadrupole or time of flight mass spectrometry with paper spray ionization Compound Quadrupole TOF LOD (µg/mL) LOQ (µg/mL) Linearity (µg/mL) LOD (µg/mL) LOQ (µg/mL) Linearity (µg/mL) Caffeine 20x10-3 16.6x10-3 1-30 0.53 1.79 1-30 17α-hydroxyprogesterone 5x10-3 17x10-3 0.05-80 0.45 1.49 1-100 Cortisone 1.5x10-3 17x10-3 1-80 0.58 1.92 1-100 Hydrocortisone 3.8x10-3 4.8x10-3 1-100 0.59 1.99 1-100 β-carotene 30x10-3 99.9x10-3 0.5-20 0.22 0.74 1-30 T3 25x10-3 83.3x10-3 0.05-50 0.26 0.87 1-50 T4 22x10-3 73.3x10-3 0.05-100 0.68 2.27 1-100 97 Table 5.3 Levels of the tested hormones (T3&T4) in different samples determined by standard addition using the developed paper spray ionization technique Sample Quadrupole TOF T3 T4 T3 T4 0.4 um filtered sea water 3.78 1.96 2.98 1.75 Eggs spawned - 0.15 - 0.10 Guts and mesenteries harvested from adult female - 0.22 - 0.113 Strongylocentrotus purpuratus 3.91 2.35 3.73 2.01 98 Table 5.4 Recovery and inter-day repeatability by using triple quadrupole and time of flight mass spectrometry with paper spray ionization 5.3.2 Comparison Between Two Surface Ionization Sources 5.3.2.1 Comparison of Mass Spectra Obtained by Two Surface Ionization Sources In order to compare the applied potential using two surface ionization sources with TQ/MS a wide range of 1.5-4.5 KV was used. The results show that the peak responses increased within range of 3-3.6 KV and 3-3.5 KV using the paper spray ionization and liquid extraction surface analysis technique, respectively. In order to optimize the flow rate of liquid extraction surface analysis a wide range of 50-300 µL/min was used. The result show that flow rate at 200 µL/min was found to be optimal for LESA technique. The optimized parameters for each analyte are given in Table 5.5. Both ion modes were investigated using the two ionization sources. Our results show that both techniques gave ions with response in the positive mode higher than the negative ion mode. Thus, the positive ion mode found to be optimal for this study. The ions for each analyte were obtained in full scan mode. Protonated and sodium adduct peaks were observed Sample Quadrupole TOF Recovery % RSD % Recovery % RSD % T3 T4 T3 T4 T3 T4 T3 T4 0.4 um filtered sea water 97 93 2 5 94 96 5 5 Eggs spawned 89 99 6 6 96 87 4 7 Guts and mesenteries harvested from adult female 95 99 5 2 86 98 6 3 Strongylocentrotus purpuratus 96 99 4 3 92 95 7 4 99 for all the tested compounds. The results show that the major ion was the protonated peak for all the tested compounds using either PSI or LESA with an exception of cortisone and hydrocortisone where the sodium adduct peak was observed as a major ion with LESA. The peak response obtained by paper spray ionization was higher than the peak responses obtained by liquid extraction surface analysis for the same tested compounds (Figure 5.3). 100 Table 5.5 Parameters and linear regression obtained by either paper spray ionization or liquid extraction surface analysis with triple quadrupole mass spectrometry Compound PSI LESA Ions Precursor ion Applied potential Ions Precursor ion Applied potential Caffeine [M+H]+, [M+Na]+ [M+H]+ 3.5 [M+H]+ [M+H]+ 3.5 17α-hydroxyprogesterone [M+H]+, [M+Na]+ [M+H]+ 3.66 [M+H]+ [M+H]+ 3.45 Cortisone [M+H]+, [M+Na]+ [M+H]+ 3.5 [M+H]+, [M+Na]+ [M+Na]+ 3.45 Hydrocortisone [M+H]+, [M+Na]+ [M+H]+ 3.63 [M+H]+, [M+Na]+ [M+Na]+ 3.33 T3 [M+H]+, [M+Na]+ [M+H]+ 3.5 [M+H]+ [M+H]+ 3.5 T4 [M+H]+, [M+Na]+ [M+H]+ 3 [M+H]+ [M+H]+ 3 101 Figure 5.3 The peak responses obtained by paper spray ionization and liquid extraction surface analysis with triple quadrupole mass spectrometry. 5.3.2.2 Comparison of Analytical Performance The calibration curves with a good linear regression were created for each compound by plotting the peak responses for a wide range of concentrations (1-250 µg/mL). The values of linear regression were better than 0.98 and 0.92 for PSI and LESA, respectively. The values of LOD were within a range of 0.0015 – 0.003 µg/mL and 0.13 – 0.86 µg/mL using PSI and LESA, respectively. The linear range was obtained for each compound and it was approximately the same using either PSI or LESA, with the exception of progesterone and T4. The linear range was of 0.05-80 µg/mL and 1-100 µg/mL using PSI, while it was within a range of 1-30 µg/mL and 1-50 µg/mL for progesterone and T4; respectively, by LESA. The LOQ was detected as ten time of LOD for each tested compound. The analytical performances are given in Table 5.6. In addition, the repeatability was obtained with an acceptable value (<7%) by PSI while it was increased using LESA (over 24%). The recoveries were investigated at three levels for each analyte. Recovery was over 96% and 88% using PSI and LESA, respectively. 102 Table 5.6 Analytical performance obtained by either paper spray ionization or liquid extraction surface analysis with triple quadrupole mass spectrometry Compound PSI LESA LOD (µg/mL) LOQ (µg/mL) Linearity LOD (µg/mL) LOQ (µg/mL) Linearity Caffeine 20x10-3 16.6x10-3 1-30 0.51 1.71 1-30 Progesterone 5x10-3 17x10-3 0.05-80 0.54 1.79 1-30 Cortisone 1.5x10-3 17x10-3 1-80 0.86 2.85 1-80 Hydrocortisone 3.8x10-3 4.8x10-3 1-100 0.14 0.46 1-80 β-carotene 30x10-3 99.9x10-3 0.05-100 - - - T3 25x10-3 83.3x10-3 0.05-50 0.17 0.56 1-50 T4 22x10-3 73.3x10-3 0.05-100 0.13 0.42 1-50 5.4 Discussion 5.4.1 Sensitivity of PSI Lab-Made Apparatus via Different Instruments The previous published works on PSI/MS were obtained over several instruments. Ion trap (Bain et al., 2014), triple quadrupole (Yang et al., 2012; Manicke et al., 2011; Espy et al, 2014), linear trap quadrupole (Oradu and Cooks, 2012; Yang et al., 2012), and fourier transform ion cyclotron resonance (Quinn et al., 2012). The specific research questions that guided this section were: (i) can the variation in MS instruments influence the sensitivity of the same PSI lab-made apparatus, (ii) does the variation influence the accuracy of PSI lab-made apparatus for a certain applications. Two MS instruments were used, including triple quadrupole (TQ/MS) and time of flight mass spectrometry (TOF). Mass spectra provided 103 evidence that the tested compounds were ionized with the same mechanism by PSI independent of TQ/MS or TOF/MS. The most interesting findings in the current study was that the sensitivity and limit of detection varied for the selected MS instruments. In previous work, the limit of detection was calculated using the same formula that was used in the current study. The formula was 3 SB/m, where SB is the standard deviation of the blank, and m is the analytical sensitivity. The LOD was obtained at level in microgram per liter for all analytes examined with triple quadrupole mass spectrometry (Cooks et al., 2011). Another previous work was obtained LOD at the same level using linear trap quadrupole mass spectrometry (Liu et al., 2010). The results in this thesis showed the same level for the detection limit obtained by TQ/MS, while TOF/MS in our lab showed low sensitivity with PSI lab-made apparatus. These results show that our triple quadrupole has capacity to quantify the selected compounds at trace level (µg/L) more than TOF in our lab with the PSI lab-made apparatus. These results might be depending on the accuracy of each instrument. For TQ/MS, mass with only two decimal points could be detected, while TOF could detect mass with four decimal points. Thus, the ions that have the same integer mass with different decimal points gave the same peak with TQ/MS, while a unique peak can be observed for each ion using TOF. For example, CH3 and NH have the same integer mass (m/z= 15) and the difference in decimal points, TQ/MS give only one peak for both ions while TOF give two peaks for each ion. Based on these data, the peak response with TQ/MS related to all ions, which have the same integer mass, which might affects the intensity of the peak. TQ/MS gave select quantification of known compounds but the TOF allows us to look at all diversity of ions. However, further research is required to determine whether these results are applicable to the class of instruments or whether the results are unique to these individual instruments. The second section tested the level of T3 and T4 in several samples using two instruments in order to determine the accuracy of the PSI lab-made apparatus. T4 can itself degrade by losing iodine to form T3 (Eales et al., 2004; Fan et al., 2013). The researchers indicated that LOD for each hormone was at level microgram per litter. The results in this thesis showed the same level with TQ/MS. In addition, the results showed that T3 existed in the samples with concentration higher than T4. Both MS instruments showed the same fact that T3 exist in levels higher than T4 in filtered seawater and Strongylocentrotus purpuratus. 104 The repeatability (RSD %) was determined within three replicates. The values of RSD % indicated the validity of the method for each instrument. Furthermore, recovery was obtained in the current study in order to determine the potential of the PSI lab-made designed with MS instruments. In the current study, good recovery value was obtained for each thyroid hormone (T3 and T4) using either triple quadrupole or time of flight mass analyzer. Thyroid hormones were quantified in several matrices such as rat tissue by Vacek et al., 2010. The researcher used ion trap mass spectrometry. In addition, Higashi et al., 2011, quantified T4 in Saliva where they used MS/MS in their work (Vacek et al., 2010; Higashi et al.; 2011). Vacek et al 2010 determined a recovery of range 93-103.7% for T3 and a recovery of 95.5-101.7 for T4. Higashi et al.; 2011 indicated that the % recovery was 96.5 to 99.6 % for T4 (Vacek et al., 2010; Higashi et al.; 2011). In the current study, recovery percent ranged between 89-97 % for T3 and 93-104 % for T4 using PSI-TQ/MS; and the RSD % values were within range from 2 to 6 %. For PSI-TOF/MS, the recovery ranged between 86 - 96 % for T3 and 87-98% for T4 with RSD % ranged between 3 - 7 %. The recovery values were similar using both mass analyzers but the results in this thesis show that % recovery was lower than the other published works. I suggested that the procedure of the spiked sample might lead to this difference, where the sample was spiked with the standard solution before the extraction process. There are several factors affecting recovery; including the way of spiking the sample or injecting the sample, the period from spiking the sample to making the analysis, and the error that is possibly produced during standard solution preparation. However, % recovery values were close to each other using two MS instruments with the same PSI lab-made apparatus, indicating the recovery values using PSI lab-made designed. 5.4.2 Mobility and Analyte Extraction Influence with Two Surface Ionization Lab-Made Apparatus Many surface ionization techniques have been used that produce a protonated peak. For example, a protonated peak at m/z 195 was found for caffeine using PSI by Yang and his co-worker (Yang et al., 2012), while Williams used desorption electrospray ionization mass spectrometry (Williams et al., 2006). A protonated ion peak was found at m/z 651 and 777 for T3 and T4; respectively by Tyan et al., 2013 with matrix-assisted laser desorption (MALDI) (Tyan et al., 2013). In the current study, the protonated ions were found as a 105 predominant ion for all tested compounds using both ionization sources, which agree with the previous works with different surface ionization techniques (Williams et al., 2006; Tyan et al., 2013; Yang et al., 2012). The most significant finding of this section was the LOD values. The limit of detection ranged between 0.0015 - 0.003 µg/mL by PSI-TQ/MS while it ranged between 0.13-0.86 µg/mL by LESA-TQ/MS. These results indicated that the analyte at trace concentration (µg/L) could be detected by using paper spray ionization while it is not possible by using liquid extraction surface analysis technique. In addition, recovery was obtained using the standard solution of the selected analytes. The values were over 96% and 88% using PSI and LESA, respectively. Based on these results, the analyte was extracted and mobilized from the paper surface in PSI with extraction efficiency higher than LESA. However, more investigations with different apparatus, either PSI or LESA, are required to determine whether these results are unique to these individual apparatus. 106 Chapter 6 PSI Profile of a Model Plant Extract: Saffron Crocus sativus 6.1 Synopsis One of the key advantages of PSI is thought to be the capacity to determine a metabolites profile of complex sample matrices such as intact plant tissues. Saffron (Crocus sativus L.) was chosen as the model plant to investigate this idea because it is a complex tissue with polar, non-polar, small, and large metabolites. Seven major bioactive metabolites were quantified in different tissues of Crocus sativus plant; including anther, petals and corm (saffron flower’s root) with locally grown horticultural plants. The quality of saffron collected from five regions was investigated using known metabolites. 6.2 Experimental 6.2.1 Chemicals and Reagents Whatman filter paper grade 1 was purchased from Fisher Scientific (Mississauga, ON). Proline, isophorone, safranal, gallic acid, β-ionone, quercetin, and crocin were obtained from Sigma-Aldrich (St. Louis, MO). All polar and non-polar solvents were purchased from Fisher Scientific (Mississauga, ON). 6.2.2 Sample Preparation Five saffron samples (anthers) were collected from different locations; including Egypt (S1), India (S2), Iran (S3), Spain (S4), and Canada (S5). Saffron samples were extracted according to the reported method of Lage et.al (2009). Anthers of each sample were ground using mortar and pestle. An accurate weight (~1.5 g) of each ground sample was placed in 20 ml dark vial and extracted with an approximate volume (14-20 mL) of methanol, acetone, acetonitrile, hexane, and/or toluene. The dark vial was vigorously homogenized in an ultrasonic bath for 60 min. All bottles were kept in at 4 ºC (overnight) and the sonication process was repeated for 60 min. Each extracted sample was filtered through 0.45 µm cellulose membrane filter and stored in a dark bottle. The same, extraction procedure was repeated for different tissues of the saffron plant; including petals and corm with locally grown horticultural plants. 107 6.2.3 Apparatus All experiments were performed using a PremierTM series TOF/MS (Waters Inc., Mississauga, ON) and equipped with a custom-built assembly for PSI/MS. The mass spectra were collected in a full scan mode. The paper was prepared as described in Chapter 2. Mass spectrometry parameters of capillary voltage, cone voltage, and flow rate (µl/min) were investigated based on the most intense peak for each compound. Both positive and negative ion modes were used in this study. Ultra-pure water was obtained from Millipore-Q 3 UV (18 mΩ, Millipore S.A.S. 6712 Molsheim, France) throughout the work. 6.2.4 PSI/MS Profile PSI/MS profile was obtained using two saffron samples (S1 & S2). The blanks were summed and subtracted against profile data for each saffron sample. A series of algorithms equations were used in Excel (Microsoft). All masses which have significant responses are obtained. The blanks were subtracted from the sample spectra. 6.2.5 Analytical Performance of the Developed PSI/MS Stock solutions of each compound were prepared in different solvents. A series of standard solutions of each compound (1-250 µg/mL) were prepared by sequential dilution with several solvents. All solutions were stored at 4 °C and protected from light; kept in dark vials, until further use. The analytical performance (range of linearity, lower limit of detection (LOD) and lower limit of quantification (LOQ)) were obtained by analyzing pure standards. Both LOD and LOQ were calculated according to the IUPAC equations. The accuracy of each compound was defined as % recovery, where it was evaluated by the analysis of each sample at the same concentration within five replicates. Repeatability was expressed as RSD%. 6.2.6 Quantification Analysis of Known Metabolites An accurate volume (~ 3 µL) of each sample was loaded on the paper substrate. The standard addition method based on five replicates was used. Peak intensity was measured before and after addition of increasing concentration of the standard solution for each compound. The change in the peak intensity was then recorded and used for quantification. 108 6.3 Results 6.3.1 Profiles of Two Different Saffron Samples Using PSI/MS PSI/MS profiles of each saffron sample was obtained in positive ion mode. Each sample was analyzed in triplicates. A large amount of saffron samples were loaded on the paper substrate, and it left until dry. The total number of observed metabolites was 2261 in S1, and 2192 in S2. The total number observed in all samples equal to 4453 (Table 6.1). The total number of observed of distinct signals compounds common to both samples was 364. Approximately 51 % of the total detected metabolites was found in Egypt saffron. The total number of compounds that were found in S1 was significantly greater than the number of compounds in S2, with varying relative abundances. Most the observed metabolites were observed within less 5 minutes (Figure 6.1). 109 Table 6.1 Paper spray ionization mass spectrometry profiles of Egyptian (S1) and Indian (S2) saffron S 1 S2 Total number of signals observed 2261 2192 Average number of signals per treatment 1588 1075 Compounds common to all replicates of each treatment 915 124 Unique to each treatment 324 40 Total # unique to each treatment (at least one replicates) 1346 2068 Total # unique to each treatment (present in all replicates) 915 124 Total # unique to both treatment 364 Common to both treatments 22 Total # observed in all sample 4453 110 Figure 6.1 The number of investigated metabolites within 30 minutes where X refer to time of analysis. Several compounds were detected in our analysis phenolic acid, vanillic acid, gallic acid, and syringic acid. Other compounds including pyrogallol, β-ionone, quercetin, myricetin, isophorone, safranal, crocin and/or other were observed (Table 6.2). Some of them were identified by comparison with data from other published works (Verma & Middha, 2010; Lage & Cantrell, 2009; Moraga et al., 2009; Caballero-Ortega et al., 2007; Li et al., 1999; Straubinger et al., 1998; Tarantilis & Polissiou, 1997; Tarantilis et al., 1995). The chemical structures of the identified metabolites are given in Figure 6.2. 111 Table 6.2 Identification of several investigated metabolites 112 OHOOCH3OHOHOH O OOOHOHHOHOCH3H3CCH3O OHOOHOHOHOHOH OHOOHOHOCH3OHOHOHOOHOHOCH3OHOCH3 1 2 OOHHOOHOHO3 4 5 6 113 HOHOH OHHOOO HO OHNH2O O H3NNH3OO OHHOONH2 NHOHO OHOCH3OHO OHHOHOO 7 8 9 10 11 12 13 14 114 COOHH3COOHOCH3 OHOHHO OHOOHOHOHOHOOHOOHOHOHOHOHO H3C CH3CH3O O OHHOOHOHO O Figure 6.2 Chemical structures of identified metabolites. 15 16 17 18 19 21 22 23 115 6.3.2 The Repeatability of PSI Profile In order to test the validity of PSI/MS to be used as a metabolite fingerprinting, intra-day repeatability was obtained. Three paper substrates were used within three replicates for each sample. Two saffron samples were investigated, Egyptian saffron (S1) and Indian saffron (S2). A range of metabolites were obtained with different RSD ratios. In S1, 206 metabolites gave RSD% values within a range of 20-40 %, while only 68 metabolites gave RSD% less than ten percentages. For S2, the large number of metabolites was obtained at the range of 40-60%, while only 12 metabolites were obtained with RSD% value lower than ten percentages. Wide ranges of RSD% were obtained. The precision of the peak response of compounds is given in Figure 6.3. These results gave evidence that saffron samples have variability in metabolites quantity within intra-day repeatability. Thus, PSI/MS has low capacity to identify the fingerprint profile of a complex sample like saffron. Figure 6.3 The total number of the found metabolites within a wide range of relative standard deviation for (A) Indian and (B) Egyptian saffron. B 116 6.3.3 PSI/MS Profile of the Blanks In order to study the accuracy of PSI/MS to obtain the saffron profile, six paper substrates were used. The total number of compounds observed in the blank equal 31072, 29878, 14164, 22563, 15512, 25357, while 138546 compounds were observed for all replicates. Also, I found that there is no proportional relationship between the total number of compounds observed per replicate and the order of replicates (Figure 6.4). Thus, there are many sources of contamination with these compounds, including ionization source (PSI), contaminants from lab environment during preparation process of paper substrate and/or manufactory compounds present on a paper. Paper contributed to a sample matrix which might makes a metabolite fingerprinting more challenging. Thus, paper spray ionization mass spectrometry has a lower accuracy for characterizing the metabolomics profile of saffron. The summary of blank analysis is given in Table 6.3. 117 Figure 6.4 The total number of compounds observed over six paper substrates using methanol as a mobile phase. 118 Table 6.3 Summary of the total number of the observed compounds over six paper substrates 6.3.4 PSI/MS Spectra of Known Metabolites in Saffron In order to study the diversity of several compounds in saffron stigma, standard addition of the tested compounds were used. Protonated peaks were produced when a small amount of each sample was infused into the PSI/MS, which is operated in positive ion mode. Mass spectra were obtained within a range of voltages (3.3-3.5 KV). Direct infusion of a mobile phase to pass through the paper substrate was accomplished using a small volume of 10 µL/min by a syringe pump. Protonated ions including m/z 116, 139, 151, 171, 193, 303, and 977 were obtained for proline (m/z 115), isophorone (m/z 138), safranal (m/z 150), gallic acid (m/z 171), β-ionone (m/z 192), quercetin (m/z 302), and crocin (m/z 976), respectively. The deprotonated peaks at m/z 114, 137, 149, 169, 191, 301, and 975 were observed in the Total number of compounds observed 138546 Average number of compounds in the blank for SAFF1 25038 Average number of compounds in the blank for SAFF2 21144 Compounds common to all replicates 147 Compounds common to all replicates after first run 287 Unique to each substrate Blank 1 31072 Blank 2 29878 Blank 3 14164 Blank 4 22563 Blank 5 15512 Blank 6 25357 119 negative ion mode for the last compounds, respectively. The results showed that negative ion mode has less improvement in signal intensity than positive ion mode. As a result, positive ion mode was selected to be used for the quantitative analyses. 6.3.5 Analytical Performance for the Selected Compounds Under the optimum experimental conditions of the ionization sources and the mass spectrometry parameters, by plotting peak responses vs. concentrations, the calibration curve for each analyte was obtained at various concentrations 0.5-200 µg/mL. The mean correlation coefficients (R2) of these curves were typically higher than 0.97 and showed good linearity in the ranges of 1-30 µg/mL for crocin, and quercetin (Figure 6.5). The range of 1-100 µg/mL was obtained for β-ionone, gallic acid, and proline, while the linearity with the range of 0.5-30 µg/mL, and 1-50 µg/mL were found for safranal and isophorone, respectively. The plots of the change of the ionization peak intensities versus the concentrations were found linear with the good linear regression equations (Table 6.4). The figure of merits (lower limit of detection and quantification and repeatability) of the PSI/MS developed technique was determined according to IUPAC. The value of LOD was found equal to 0.7, 0.1, 0.3, 0.3, 0.3, 0.5 and 0.5 µg/mL for proline, isophorone, safranal, gallic acid, β-ionone, quercetin, and crocin, respectively. The limit of quantification for each analyte was investigated within range of 0.33-2.49 µg/mL. The analytical performance results are given in Table 6.5. Intra-day precision of each compound was obtained by analysing it within 5 replicates. RSD% was 6.5, 6.6, 6.4, 5.3, 6.7, 5.2, and 4.7% for proline, isophorone, safranal, gallic acid, β-ionone, quercetin, and crocin at the low concentration level tested. These data indicate that, the PSI/MS method can be used for quantitative analysis of the previous compounds. 120 Figure 6.5 Calibration curve of (A) isophorone, (B) β-ionone, (C) safranal, and (D) quercetin using methanol as a mobile phase. A C D B Peak response (103) Peak response (103) 121 Table 6.4 Linear equations for the selected compounds Compound Y= aX + b R2 β-ionone Y = 3217.2 X + 9661.2 0.9973 Crocin Y = 94.451 X + 20.317 0.9979 Gallic acid Y = 493.06 X + 3962.2 0.9728 Isophorone Y = 3283.6 X + 10782 0.9941 Proline Y = 81.66 X + 912.07 0.9926 Quercetin Y = 127.71 X + 51.57 0.9931 Safranal Y = 494.52 X + 2754.5 0.9980 122 Table 6.5 Analytical performance of seven known metabolites using methanol as a mobile phase 6.3.6 Characteristics of the Solvent Extraction In order to find the optimal solvent for sample extraction, different solvents were investigated. Polar and non-polar solvents were compared including methanol, acetone, acetonitrile, hexane, and toluene. In this study, the concentrations of each compound were obtained using methanol, acetone, and acetonitrile. Each extraction was repeated within three replicates. The results showed that the average concentrations were within range of 3-28 µg/mL. Of all solvents, methanol provided the highest concentrations of the tested compounds with yield of detection 59 %, whereas toluene gave lower values of extraction 2%. The range of the concentration 9-28 µg/mL was obtained using methanol. Approximately 14% of the extraction was established for both polar solvents acetone and acetonitrile. The ranges of 3-7 µg/mL and 1-6 µg/mL were obtained using acetone and acetonitrile, respectively. On the other hand, hexane gave range of 1-8 µg/mL for the tested compounds with yield of detection equal 12 %. According to these results, polar solvents extracted most of the tested compounds with yield of detection higher than non-polar Compounds LOD LOQ Linearity RSD% Isophorone 0.1 0.33 1-50 6.6 β-ionone 0.33 1.09 1-100 6.7 Gallic acid 0.3 0.99 1-50 5.3 Proline 0.7 2.49 1-100 6.4 Safranal 0.3 0.89 0.5-30 6.4 Crocin 0.5 1.81 1-30 4.7 Quercetin 0.52 1.75 1-30 5.2 123 solvents, meaning that polar solvents are more suitable for the saffron extraction (Figure 6.6). Figure 6.6 Extractive solvent optimization using methanol, acetone, acetonitrile, toluene and hexane. Methanol gave the highest extraction yield. 6.3.7 Quantitative Analysis of Known Metabolites in Saffron by Regions In order to study the diversity of the selected metabolites in saffron collected from different regions; a standard addition method was used to quantitate data. Compounds related to phenolic acid, flavonoid, carotenoid and amino acid were used in this study. First of all, tested compounds were determined in five saffron samples. Standard addition was used as the recommended procedure in experimental session. Each sample was loaded on the paper substrate followed by applying a small amount of a mobile phase. Methanol was used as the mobile phase (10 µL/min) for this study. Each sample provided the presence of the tested compounds over a wide range of concentrations. 124 The results showed a wide range of each analyte in Egyptian saffron (8-29 µg/mL) (Figure 6.7). Crocin was present at 26%, while safranal was obtained with the lowest level 8%. Other compounds proline, isophorone, gallic acid, β-ionone, and quercetin were obtained with ratios equal 20, 12, 9, 10, and 15%, respectively. For the Indian sample, crocin was also the major metabolite with level 28 µg/mL. Isophorone and proline were obtained at ratio 9% and 13 % of all compounds’ concentration, respectively. This sample shows no evidence of the presence of gallic acid. Similarly, crocin gave the highest concentration 29, 17 µg/mL in either S3 or S5; respectively, while isophorone was the predominant compound in S4. The other compounds were observed within ratios 7.5-72.5 %, 6-22 %, and 8-33 % in S3, S4, and S5, respectively. Iranian and Spanish saffron show no presence of proline. Gallic acid and quercetin are not detected in either S3 nor S5 samples. Of all saffron’s anther samples, Egyptian saffron showed the presence of all phenolic, flavonoid, and carotenoid compounds. 125 Figure 6.7 Concentrations of the selected compounds over five saffron samples. Crocin was obtained with the highest level. 6.3.8 Quantitative Analysis of Known Metabolites in Different Saffron Tissues In order to estimate the variety of each metabolite in different parts of saffron flower, three parts of the flower were investigated; petal, stigma, and corm. Each part was extracted using the recommended procedure in the experimental section. Proline, isophorone, safranal, gallic acid, β-ionone, quercetin, and crocin were also quantified using the same recommended procedure for the standard addition curves. According to the results, the flower’s petals showed the existence of crocin with the highest level 37%, while safranal was observed at 7%. On the other hand, gallic acid gave no evidence for the presence in the 02040S1 S2 S3 S4 S5L-prolineIsophoroneSafranalGalic acidβ-iononeQuercetinCrocinObtained concentrations (µg/mL) 126 petals. For the stigmas, various concentrations were obtained 9, 14, 4, 7, and 17 µg/mL for proline, isophorone, safranal, β-ionone, and crocin, respectively. The results showed no presence of gallic acid or quercetin. Of all flower parts, corm (saffron flower’s root) provided the presence of all tested compounds. The obtained concentration was 26, 8, 6, 7, 16, 24, and 69 µg/mL for proline, isophorone, safranal, gallic acid, β-ionone, quercetine, and crocin, respectively. Of all tested compounds, crocin showed the highest level (69 µg/mL) with percentage equal 44% (Figure 6.8). These results show that the diversity of the selected compounds in different saffron can be investigated using PSI/MS. Figure 6.8 Concentrations of the selected compounds in saffron Crocus sativus flower (A) stigma, (B) petals, and (C) corm using standard addition method. The results show that corm contains all the selected compounds with different levels. Found concentration (µg/mL) A B C 127 6.3.9 Accuracy In order to study the accuracy of PSI/MS to estimate the diversity of selected compounds in either saffron collected from different regions or different parts of saffron flower, each sample was injected with three levels of the standard solution. The accuracy is expressed in recovery %, and it was obtained according to recommended procedure in Chapter 3. The results provided evidence that PSI/MS showed a good accuracy for quantitative analysis of the selected compounds over eight different samples, including petal, stigmas and corm. The average of recovery percent ranged between 95-98.5 %. These results showed the accuracy of PSI/MS to estimate the diversity of the selected compounds over the samples. 6.3.10 Study the Quality of Various Saffron Samples The compounds were detected within range of over five saffron samples. Each measurement was obtained in triplicate. Methanol was used as a mobile phase for this study (Figure 6.9). Crocin was observed with concentrations ranged between 28 – 32 µg/mL, while safranal was obtained within levels ranged between 4 – 24 µg/mL. Of all saffron samples, Spanish saffron gave the highest concentration of either crocin (32 µg/mL) or safranal (24 µg/mL). 128 03060S1 S2 S3 S4 S5SafranalCrocin Figure 6.9 Concentrations of safranal and crocin in saffron Crocus sativus collected from different regions. Spanish saffron (S4) showed the highest quality of all saffron samples, where the highest level 24 and 32 µg/mL were observed in Spanish saffron for safranal and crocin, respectively. 6.4 Discussion Saffron components have differences in polarity and size of metabolites. Many techniques have been used for saffron analysis such as liquid chromatography tandem mass spectrometry (LC/MS/MS), fourier near infrared spectroscopy, GC/MS (Verma et al., 2010; Tarantilis & Polissiou, 1997; Cullere et al., 2011), UV equipped with HPLC (Haghighi et al., 2007; Gregory, 2005; Lozano, 1999; Tarantilis et al., 1995), HPLC photodiode array detection (Caballero-Ortega et al., 2007), LC/MS (Carmona et al., 2006), MALDI/MS (Zhang et al., 2004), HPLC (Loskutov et al., 2000; Li et al., 1999; Straubinger, 1998; Sujata et al., 1992; Lage & Cantrell, 2009), and FT-IR spectroscopic (Tarantilis et al., 1998; Deng et al., 2013; Mazzotti et al., 2013). To our knowledge, no study on saffron analysis using PSI/MS has been published. The objective in this thesis was to utilize PSI/MS for analysis of mass spectrometry-based metabolomics of natural products using saffron (Crocus sativus L.) as a model. Overall, the data and analyses in the current study demonstrate that metabolomics profile should carefully consider the reproducibility of the technique as well as the potential Percentage of all samples % 129 when interpreting spectra generated from the paper substrate. Utilizing PSI profiling as a fingerprinting technique for identifying compounds is potentially important for a complex samples such as saffron. Previous works used different solvents for saffron extraction such as methanol, water and ethanol (Verma et al., 2010; Lage & Cantrell, 2009; Haghighi et al., 2007; Caballero-Ortega et al., 2007). In the current study, methanol, acetone, acetonitrile, hexane, and toluene were used for extraction process. It was revealed that MeOH extracted the selected metabolites with the highest yield of extraction. These results depend on the polarity and solubility of the metabolites. This factor was discussed in Chapter 2. Polar metabolites were found to be one of the main components of saffron (Chryssanthi et al., 2011; Yilmaz et al., 2010). The polarities of the solvent systems play an important role to extract the selected compounds. Methanol, acetone and acetonitrile have a polarity comparable to other non-polar solvents, therefore, it is compatible for the solubility of polar compounds such as crocin, safranal and others. In this thesis, it is hypothesized that PSI/MS has capacity to identify the metabolomics profile of this complex tissue. To test this hypothesis different triangular papers were used. The results showed a high percent of relative standard deviation for several substrates. It was indicated that the geometric parameters and the content on the substrate affect the ionization efficiency. It was demonstrated that a high value of RSD % results on the accuracy of the technique (Yang et al., 2012; Espy et al., 2012; Jhang et al., 2012; Zhang et al., 2012; Ren et al., 2013).The compounds that might associated on the paper substrate including contaminants from the lab environment and/or manufactory compounds on the paper also affects the RSD % (Espy et al., 2012; Sukumar et al., 2011). For Egyptian saffron, the values of RSD % ranged of 20-40 for 206 metabolites, while only 68 metabolites gave RSD% less than ten percentages. For Indian saffron, most metabolites was obtained at the range of 40-60%, while only 12 metabolites were obtained with RSD % value lower than ten percentages. The results in this thesis demonstrate that PSI/MS has low accuracy to be utilized as a fingerprinting technique. Over 150 compounds have been discovered in saffron spices, including volatile and non-volatile compounds. Several major components investigated are carotene, flavonoid, 130 and/or phenolic acid (Verma & Middha, 2010; Lage & Cantrell, 2009; Moraga et al., 2009). The main constituents of saffron are crocin, safranal, isophorone, crociten and other polar metabolites (Chryssanthi et al., 2011; Yilmaz et al., 2010). In the current study, the results showed the existence of several compounds that were found in previous studies using other techniques such as ESI-ion trap/MS and LC/MS/MS (Lage & Cantrell, 2009; Moraga et al., 2009; Verma and Middha, 2010). The concentrations of several metabolites and the mineral component in saffron are dependent on the geographical classification, quality control and/or soil type (Lage & Cantrell, 2009; Moraga et al., 2009). In the current study, kaempferol, isorhamnetn, picrocrocin, malvidin, lutein, crocetin, glutamic acid, safranal, crocin, lysine, tyrosine, proline, gallic acid, vanillic acid, caffeic acid, syringic acid, quercetin, myricetin, β-ionone, isophorone, petunidin, delphinidin, and other metabolites were found using PSI/MS. Another finding in the current investigations was the diversity of seven major components of Crocus sativus L. Five saffron samples from different countries were used. These compounds were quantified within level in µg/mL with a good regression. The LOD for each tested compound was determined. The previous published work by Verma and Middha was indicated LOD for safranal and crocin at the level microgram per liter similar to the results in this thesis where they used LC/MS/MS (Verma and Middha, 2010). In the previous works, the anther was used for analyzing of saffron quality (Lage and Cantrell, 2009; Goupy et al., 2013; Dar et al., 2012). Saffron has high quality when high levels of major components such as crocin, and/or safranal are obtained (Lage and Cantrell, 2009). Crocin is considered as an important metabolite in saffron. It has different medical applications (Lage and Cantrell, 2009; Goupy et al., 2013). Also, this compound is responsible for the colour in saffron, and is used as a food coloring. In addition, it was discovered that safranal is the main component in saffron responsible for the saffron odor (Lage and Cantrell, 2009; Goupy et al., 2013; Dar et al., 2012). Both compounds have been used for several medical applications. Different studies have proposed that these compounds may have a potential effect in mental disorder treatment such as depression, Alzheimer’s disease (AD), agitation and aggression (Yilmaz et al., 2010; Campo et al., 2010; Verma et al., 2010; Pathan et al., 2010). Also, it was discovered that safranal has anxiolytic activities (Yilmaz et al., 2010; Campo et al., 2010; Verma et al., 2010; Lage and Cantrell, 2009; Pathan 131 et al., 2010). The quality of saffron grown under different condition was investigated by Lage and Cantrell (Lage and Cantrell, 2009). Analysis of environmental impact on saffron quality showed that only the altitude affects crocin. Herein, the quality of saffron collected from five regions was accomplished. The analysis in the current study showed the highest levels of crocin and safranal in Spanish saffron. The concentrations of safranal were 8, 15, 8, 24, and 4 µg/mL for Egyptian, Indian, Iranian, Spanish, and Canadian saffron, respectively. For crocin, the concentrations were 29, 28, 29, 32, and 17 µg/mL for the same saffron samples. Based on these results, Spanish saffron has the best quality compared to other four saffron samples. 132 Chapter 7 New Approach for Rapid Analysis of Solid Sample 7.1 Synopsis In PSI there is a trend to try and produce rapid real time analysis from a variety of sample types including those that are composed of heterogeneous complex materials (plants) where analytes can be sequestered into structural compartments within the material. When attempts are made to analyse this complex material either directly or on the surface of a paper, there are always high RSD's associated with the analytical results and these make the approach less desirable. A mechanical Press Extraction (MPE) used to simultaneously disrupt the samples. In this technique, samples are placed on a paper substrate, a solvent is added (few µL), a MPE is used to further crush and disrupt the solid/solvent mixture and this pressure also allows high temperature to be applied without the solvent vaporizing, however, at the end of the process when the pressure is released, the solvent rapidly evaporates leaving a homogenous residue of materials that were extracted from the solid as a film on top of the matrix. However, in practice it was discovered that the best extraction efficiencies were obtained at room temperature and these were the conditions used for the remainder of the experiments. Although, we used this technique to prepare paper spray samples, we suggest this approach has widespread applications in the area of "ambient ionization". 7.2 Experimental 7.2.1 Chemicals and Reagents All compounds and solvents were purchased from Sigma-Aldrich as it was described in the previous chapters. For all compounds (proline, isophorone, safranal, β-ionone, kaempferol, quercetin, β-carotene, astaxanthin, progesterone, cortisone, hydrocortisone, T3, and T4), primary stock solution was prepared using 2 mg, and kept in dark vials. 7.2.2 Apparatus The instrumental procedure of PSI was previously described in Chapter 3. All samples were pressed using mechanical press technique (Gennine Lincoln Part, 112264). 133 7.2.3 Sample Preparation Samples with various hardness were used in this study. Salmon, saffron and foodstuffs were collected from locally store (Kelowna, Canada). In this chapter, six food samples were used, including spinach, dill, lettuce, zucchini, broccoli, and parsley. In order to make a comparison between MPE and another standard extraction method, four saffron samples were prepared according to described extraction method in Chapter 3. In this study saffron from Egypt (S1), India (S2), Iran (S3) and Spain (S4) were ground by mortar and pestle. Then, small weight of the powder was pressed on the paper using mechanical press extraction. For MPE, a piece of paper was cut into circle. Then, a small sample (2-6 mg) was applied to the paper. A solvent was added (6-12 µL), and high pressure was applied (>103 psi), the sample pressed on the paper at room temperature. The MPE technique and the sample that was pressed on the paper are given in Figure 7.1. The paper was cut into a triangle shape prior the analysis. The exact weight of sample that was analyzed calculated as follow: wto= weight of paper before press the sample wt1= weight of paper after press the sample wt2= weight of the paper after cut it into triangle wt1 – wto= weight of pressed sample (wt3) wt2 – wt1= ʜwt4ʜ wt3 – wt4 = weight of analyzed sample 7.2.4 Precision and Recovery Accuracy of the method was assessed as the percentage of recovery by spiking the sample with known amount of analyte solution. A few microliter aliquot of an internal standard solution was added to the middle of the sample using a syringe. After the sample had dried thoroughly, the solid sample was pressed on the paper substrate with few μL of solvent by MPE (Figure 7.1). A comparison between MPE and tradition extraction method (sonication) was accomplished. The standard addition curve was obtained for each compound. Precision was evaluated as the relative standard deviation (RSD) of replicate measurements. Both recovery and precision were calculated according to IUPAC (Miler & Miler, 1994). 134 Figure 7.1 (A) Mechanical press extraction technique. The right images show different food samples, which was pressed on the paper, including spinach, lettuce, saffron, dill, and broccoli. The below images show the proposed apparatus of the technique. (B) The lab-made MPE apparatus, where the sample was placed between the two peices of metal during the process. (C) The paper and the pressed sample (lettuce) after cut the paper into triangle. Metals to pre B C A 135 7.3 Results 7.3.1 Parameters Optimization In order to improve the sensitivity of the new developed method, several parameters needed to be optimized. Amount of extractive solvent, applied pressure, flow rate and total time of analysis were investigated in this study. The range of 6-35 µL was used in order to optimize the amount of solvent extraction. The results showed that the required pressure varied depending on the amount of extractive solvent. At large amounts of extractive solvent (> 12 µL), high pressure could destroy the paper substrate. The optimal pressure was found about 103 psi with volume of solvent less than 12 µL. The variety in the volume of solvent depends on the hardness of the sample. The sample with low hardness property required 6 µL of solvent such as lettuce, while saffron, which has high hardness degree, needed 12 µL of solvent to be extracted. The total applied volume of extractive solvent ranged between 6-12 µL. Two solvents were used for vegetables (acetonitrile) and saffron (methanol) extraction. One of the greatest challenges with the mechanical press method is the selection of the time to give acceptable recoveries. In this work, the stability of carotenoids was accomplished. Several recovery values were investigated within several days, with the aim of achieving one extraction step. The results showed that, the recovery with the highest values was obtained within short time after the sample was pressed on the paper by using MPE. The recovery % values were more than 96% within two days, whereas our results showed low recovery values for astaxanthin and zeaxanthin in lettuce and Zucchini after few days from the extraction process (Figure 7.2). Recovery % was equal to 55% after 10 days. 136 Figure 7.2 The recovery percentage within several days. The recovery decreased with increasing the time after MPE was applied. 7.3.2 Quantitative Analysis of Thyroid Hormones The validation of the PSI technique was assessed by performing the analysis of the tested compounds in various food samples. The solid samples were collected and pressed on the paper substrate with MPE technology. The test samples in the paper were then analyzed by the PSI. The mobile phase extracted the analytes from the paper at flow rate 10 µL/min employing the standard addition procedure. The results of the analysis of the total compounds are summarized in Table 7.1. The standard calibration curves of some tested compounds are shown in Figure 7.3. All standard addition curves were obtained with good linear regressions. 137 Table 7.1 The concentrations (µg/mg) of several organic compounds in eleven solid samples that pressed on the paper substrate using mechanical press extraction technique Compound Spinach Dill Lettuce Zucchini Broccoli Parsley Salmon Proline 1.67 20 106.65 68.4 55 nd nd Isophorone 1.67 20 65 41.65 10 6.65 nd Safranal 3.35 10 25 55 18.35 10 nd Gallic acid 1.67 20 30 65 25 33.5 nd β-ionone 11.65 66.65 30 90 26.65 18.35 nd Kaempferol 3.35 37.35 31.65 45 66.65 nd nd Quercetin 36.65 80 26.65 23.35 33.35 28.35 nd β-carotene 6.65 40 nd 30 70 nd nd Zeaxanthin Nd nd nd 13.35 nd nd nd Astaxanthin Nd nd 33.3 30 nd nd 0.5 Crocin Nd nd 16.7 46.7 nd nd nd T3 Nd nd nd nd nd nd 1.00 T4 Nd nd nd nd nd nd 1.03 nd = not detected. 138 Figure 7.3 Standard addition curve of (a) proline, (b) isophorone, (c) quercetin, (d) gallic acid, (e) β-ionone, (f) kaempferol in zucchini. 7.3.2.1 Method Comparison In order to investigate the accuracy of this method, a comparison between the MPE and standard extraction (SE) method were obtained. Four saffron samples were extracted using both techniques. The obtained concentration was within range of 1-60 µg/mL and 3-40 µg/mL using MPE and standard extraction, respectively. About 23 measurements out of 44 were obtained using MPE with levels higher than the standard extraction method. Five compounds, including gallic acid, proline, β-ionone, and quercetin were observed in saffron samples using MPE, while these compounds show no detectable levels using the standard 0500100015002000-60 -30 0 30 60020004000-55 -25 5 350100020003000-55 -25 5 35Peak response 0400080001200016000-60 -20 20010002000-60 -30 0 30010002000-33 -3 27Concentrations (µg/mL) Concentrations (µg/mL) Concentrations (µg/mL) b c d e f 0500100015002000-60 -30 0 30 60Concentrations (µg/mL) Concentrations (µg/mL) Concentrations (µg/mL) Peak response Peak response Peak response Peak response Peak response a 139 method for the same sample. The concentration was within range of 12-31 µg/mL (Table 7.2). Table 7.2 The concentrations (µg/mL) of several organic compounds using mechanical press extraction and standard extraction method (sonication) Compound MPE Extraction Egyptian Saffron Indian Saffron Iranian Saffron Spanish Saffron Egyptian Saffron Indian Saffron Iranian Saffron Spanish Saffron Proline 4 27 18 nd 23 14 nd nd Isophorone 22 20 1 9 14 9 3 35 Safranal 12 30 33 55 8 15 8 24 Gallic acid 30 20 15 31 10 nd nd 27 β-ionone 22 14 12 16 12 16 nd 10 Kaempferol 16 60 8 13 40 16 20 11 Quercetin 18 31 31 17 17 23 nd 27 β-carotene 22 16 nd nd 40 18 10 nd Zeaxanthin nd nd 24 12 33 17 18 37 Astaxanthin nd nd nd nd nd 14 12 27 Crocin 20 32 42 55 29 28 29 32 7.4 Discussion 7.4.1 Extraction Efficiency, Accuracy and Precision of MPE Previously, published works on paper spray ionization described direct analysis of solid samples using PSI (Wang et al., 2011; Zhang et al., 2012). Wang et al., 2011 placed a piece of adrenal gland and mouse liver tissue on the paper substrate without sample extraction followed by PSI analysis (Wang et al., 2012). In addition, chili and other commercial food samples have been analyzed in a solid phase without extraction (Wang et al., 2011; Zhang et al., 2012; Naccarato, 2013). In both previous studies, analytes were extracted from the outer layer of the solid samples. When attempts are made to analyse such nd= not detected. 140 material directly or on the surface of a paper, there are always high RSDs associated with the analytical results and these make the approach less desirable. The RSD % values approached 20 % for samples with different heterogeneous surface such as powder chili, powder milk, meat, and powder juice (Zhang et al., 2012). These data raise the question of whether these methodologies can be applied for direct quantitative analysis of compounds present in solid samples. In the current study, it was hypothesized that direct and rapid analysis of the compounds from outer and inner surface increased accuracy, and sensitivity and eliminated non-homogeneity issues using MPE. This technique allows solvent molecules to pass through the outer and inner layer of the solid sample under high pressure, which means the target analyte is solubilized in the solvent and extracted from the whole samples layers (Figure 7.4). Based on this data, the motion of the solvent through the sample surfaces affects the extraction efficiency. 141 Figure 7.4 (A) Few drops of solvent is added on the solid sample, the sample is pressed on the paper under high pressure (about 103 psi). The paper is cut into triangle shape for the next analysis. (B) The solvent pass through the layers (outer and inner) of the solid sample, which affects the extraction efficiency. The specific research questions that guided this section were: (1) can homogenizing the sample with paper substrate increase the extraction efficiency and (2) can decreasing the sample size from gram to milligram, with concurrent decrease in solvent volume from mL to µL, retain adequate accuracy and precision of extraction? The goal of MPE was to homogenize solid sample on the paper substrate, increasing the area of the sample that is available to be analyzed (Figure 7.4). In a previous study, Wang et al., 2011 analyzed a solid sample with PSI but they did not report the precision or accuracy for their study (Wang et al., A B 142 2011). The accuracy and the suitability of the developed MPE method compared to the values obtained by the standard extraction method (sonication) gave an average recovery close to 100%. Recovery was obtained in several samples using MPE and it was higher than standard extraction method. In addition, the results showed that the analyzed compounds were obtained at level ranged between 1-60 µg/mL and 3-40 µg/mL using MPE and standard extraction for the same sample, respectively. About 23 measurements out of 44 were obtained using MPE with levels higher than the standard extraction method. Furthermore, gallic acid, proline, β-ionone, and quercetin were observed in saffron samples using MPE, while these compounds show no detectable levels using the standard method for the same sample. It is suggested that these results was obtained by losing a portion of samples during the extraction process (sonication), which affects the observed level of the analytes. Some analytes were not detected because these analytes might exist at low level (lower than LOD) in the extraction solution based on how much of the sample are lost during the extraction process with sonication method. Furthermore, recovery was obtained for different samples in order to determine the accuracy of MPE. A previous work showed recovery varied from 98.56 to 100.31 % using traditional extraction method (Dar et al., 2012), while it was close to 100 % in the current study. The % recovery between the two studies were close to each other, indicating good accuracy of the developed method (MPE). Further addition, RSD% was accomplished within three replicates in order to study the precision of MPE. The values approached 7 % using MPE, while it was more than 13 % using traditional extraction method (sonication). Based on these results, MPE showed a good extraction efficiency, accuracy and precision. 7.4.2 Stability of Carotenoids in MPE Paper The stability of samples under the relevant storage and processing conditions must be known prior to analysis. To determine whether delay the analysis affects the stability of carotenoids on the pressed sample, the recovery values of zeaxanthin and astaxanthin were investigated over ten days. A small volume of standard solutions (3-5 µL) were injected to vegetable samples. Lettuce and zucchini were pressed on the paper with few volume of acetonitrile (6 µL) and standard solution of carotenoids. The papers were kept openly in light for further analysis. The results showed that the recoveries varied during 10 days. The 143 highest values (> 96 %) were obtained within two days from the extraction process, while after 10 days the recovery downwards trend to 55 %. The results showed that the recovery values decreased with increasing the time after the samples were pressed on the paper using MPE. It is hypothesized that the stability of these carotenoids changed with time under the light. Previous works demonstrated that quantitative and qualitative determination of carotenoids is limited due to the oxidation of these compounds after extraction process especially under light (Havaux et al., 2004). The main cause of extensive losses of carotenoids is oxidative degradation, which depends on the availability of oxygen and is stimulated by light. Thus, carotenoids must be kept under faint light. (Klein et al., 2004). Previously, it was investigated that astaxanthin significantly decreased in a 24-weeks conducted by Li et al. (Li et al., 2004). Other investigators studied the stability of zeaxanthin in blood sample. The results showed downwards in the concentration of zeaxanthin after 24 hours from extraction process (Su et al., 1999). In addition, Craft et al. studied the stability of individual carotenoids in extracted plasma left for several hours (0, 4, 12, and 24 h). They indicated the changes in the concentration between 0 and 4 hours (Craft et al., 1988). Both previous studies showed that the stability of carotenoids changed during the time, which was revealed in the current study as well. Based on these results, it was suggested that the stability of the carotenoids affects the recovery and accuracy of MPE. However, further research is required to determine whether these results are unique to carotenoids and whether the delay of analysis affects the accuracy of MPE using other compounds. The advantage of MPE is few milligram of sample could be analyzed using few microliter of solvent. A previous study extracted saffron using 200 mL of extractive solvent with 10 g of saffron (Dar et al., 2012). Another researchers used 5 mL of solvent to extract 0.05 g of saffron (Verma et al., 2010). Two to six milligrams of the samples were extracted with approximately 6-12 µL of extractive solvent using MPE. This novel approach showed a significant decrease in the volume of solvent and the amount of sample. Another advantage of this new approach is the extraction could be finished within few seconds. Previously Verma et al. found that the total extraction process needed 25 h to be finished (Verma et al., 2010). In contrast, the total process took maximum 120 second with MPE. Thus, MPE saved time and is a more environmentally-friendly technique. 144 Chapter 8 Silica Nanoparticles-Paper Spray Ionization Mass Spectrometry 8.1 Synopsis The rapid development of nanotechnological products raises concern on their possible adverse effects on the ionization efficiency (Gulin et al., 2014; Choi et al., 2014). Several nanoparticles have been used with different surface/ionization mass spectrometry techniques (Tsao et al, 2012, Ullah et al., 2013; Wen et al., 2007; Gulin et al., 2014). Recently, SiO2 nanoparticles have also been utilized with surface ionization technique such as surface assisted laser desorption ionization (SALDI), and MALDI (Wen et al., 2007; Kinumi et al., 2000). Modification of a commercially available silicon nanoparticles powder on the surface was accomplished. The best sensitivity for some drugs was obtained by the smaller size silicon particles approximately 50 nm (Wen et al., 2007). Here we further explore the application of commercially available silicon nanoparticles solution for paper spray ionization mass spectrometry. The impact of the physical and chemical properties of the paper substrate on the release of chemical compounds from the paper, the spread of the silica nanoparticles through the paper substrate, and ionization efficiency for mass spectrometry analysis were determined. It is known that the porosity, polarity, and analyte/surface interaction on the paper substrate affected the ionization efficiency (Chapter 2; Zhang et al., 2001; Ren et al., 2013; Jhang et al., 2012). We hypothesized that using a silicon dioxide nanoparticles film may lead to an increase ionization efficiency on paper. These particles might fill the pores in the paper and decrease the analyte/surface interaction. Thus, the analyte will not held on the paper, which leads to boost the ionization efficiency. In addition, it was investigated that improved ionization was observed from the surface with better proton donating capabilities (Liu and He, 2008; Tsao et al., 2012). Silicon dioxide nanoparticles could serve as a proton source on the paper surface. The formation of acidic Si-OH groups was investigated in different applications (Liu and He, 2009; Tsao et al., 2012; Wen et al., 2007; Ullah et al., 2013; Gulin et al., 2014), which benefits analyte ionization. Based on this data, it is hypothesized that the ionization efficiency increases on the paper substrate that coated with silica nanoparticles film. 145 8.2 Experimental 8.2.1 Chemicals and Reagents Silica dioxide nanoparticles solution was received from Sigma-Aldrich Canada (Oakville, ON, Canada). All compounds were purchased from Sigma-Aldrich as it was described in the previous chapters. 8.2.2 Apparatus The instrumental procedure of PSI was previously described in Chapter 3. 8.2.3 Silica Nanoparticles Film Numerous paper substrates, which were cut into triangle shape, were immersed in silica dioxide nanoparticles solution. The substrates was left in the solution for several hours (0.25, 24, 72, 96 h) in order to study the distribution efficiency of silica nanoparticles on the paper. 8.2.4 Imaging of Paper Substrate Images of surfaces of paper substrate with silica nanoparticles film were obtained using scanning electron microscopy. The substrates were coated with silica nanoparticles by immersing the paper in the silica nanoparticles solution as was described in the last section. Then, a small amount of analyte was loaded on the paper substrate for further analysis. 8.3 Results 8.3.1 Paper Substrate Characterization SEM images of the paper substrate surfaces with silica nanoparticles film are shown in Figure 8.1. In order to create the nanoparticles film each paper substrate was immersed in the silicon dioxide nanoparticles solution within several days as described in the experimental section. The results showed that the distribution efficiency of silicon varied over the paper substrate. SEM spectra were obtained for the paper substrate within 0.25-96 hours. Silicon was found within range of 2.2-36 % wt at different positions on the paper substrate within 15 minutes, while it was obtained with level of 30-36 % wt after 72 hours. The SEM spectra are given in Figure 8.2 and Figure 8.3. 146 Figure 8.1 Scanning electron microscopy images of paper substrate (whatman filter paper grade 1) after it was coated with silica nanoparticles film. The lower image shows the silica nanoparticles coated the surface of cellulose fiber. 147 Figure 8.2 Scanning electron microscopy image of paper substrate (whatman filter paper grade 1) after immersion in silica nanoparticles solution (15 minutes). The spectra showed the existence of silica nanoparticles within range of 2.2 – 36 %wt. Many pores on the paper were not filled by silica nanoparticles. 148 Figure 8.3 Scanning electron microscopy image of paper substrate (whatman filter paper grade 1) after immersion in silica nanoparticles solution (72 h). The spectra showed the existence of silica nanoparticles within range of 30 – 36 %wt. Most pores on the paper were filled by silica nanoparticles. 149 8.3.2 Method Comparison The proposed method was compared with the standard method (absence of silica nanoparticles film) in order to investigate the effect of the film. Applied potential was optimized for each method. A wide range of 1.5-4.5 KV was used in order to optimize the applied potential. A range of 3.8-4.1 KV was found to be optimal with the presence of silica nanoparticles film while it was within range of 3.3-3.5 KV without the film. The difference in the applied potential with and without the film was only 0.5 KV (Table 8.1). PSI mass spectra were also compared with and without the silica nanoparticles. The results showed that the protonated peaks were the major peak for all tested compounds using the standard paper and the paper coated with the film except for kaempferol, β-carotene, and quercetin. These compounds gave a radical cation as a predominant ion. In addition, the peak responses varied using the standard and coated paper. The results showed peak responses with coated paper higher than the standard paper (Figure 8.4). 150 Table 8.1 Applied voltages and major ions using standard and coated paper Compound With SiO2 nanoparticles Without SiO2 nanoparticles Ion formation Capillary Ion formation Capillary Isophorone [M+H]+ 3.8 [M+H]+ 3.3 Safranal [M+H]+ 3.8 [M+H]+ 3.3 Kaempferol [M].+ 3.8 [M+H]+ 3.3 Quercetin [M].+ 4.1 [M+H]+ 3.5 β-carotene [M].+ 4.1 [M+H]+ 3.5 Astaxanthin [M+H]+ 4.1 [M+H]+ 3.5 Progesterone [M+H]+ 3.8 [M+H]+ 3.3 Cortisone [M+H]+ 4.1 [M+H]+ 3.5 Hydrocortisone [M+H]+ 4.1 [M+H]+ 3.5 T4 [M+H]+ 4.1 [M+H]+ 3.5 151 Figure 8.4 Comparison of peak responses with standard and coated papers. The plot show peak responses with coated paper higher than standard paper. 8.3.3 Accuracy The recovery percent of the proposed method was obtained in order to study the accuracy of the new approach. This experiment was accomplished with pure solution of the analytes at small level (≤ 10 µg/mL). The results showed that the recovery reached 100 % (for isophorone and safranal) and 97 % (for proline) in the presence of silica nanoparticles film, while it reached 94 % with the standard paper. 8.4 Discussion 8.4.1 Ionization Efficiency It was indicated that the physical and chemical properties of the paper substrate affect the ionization efficiency of PSI (Chapter 2; Ren et al., 2013; Jhang et al., 2012). Analytes might be adsorbed surfaces the paper, which affects the ionization efficiency (Chapter 2). Previously published works indicated that the type of residual solvent left and the type of surface functional groups serves as the major proton source in surface ionization techniques (Liu and He, 2008). Improved ionization was observed from the surface with better proton donating capabilities (Liu and He, 2008; Tsao et al., 2012). Recently, several chemical modifications, such as silicon nanoparticles, were used with different surface ionization 020Si nanoparticleStandard paperPeak response (103) % 152 techniques such as LDI (Tsao et al., 2012; Liu et al., 2008), MALDI (Wen et al., 2007), and secondary ion mass spectrometry (Gulin et al., 2014). These nanoparticles could serve as a proton source on the surface. The SiO2 nanoparticles can undergo hydrolysis process in the presence of water or organic solvent, leading to form silicic acid Si (OH)4 (Liu and He, 2008; Gulin et al, 2014; Ullah et al., 2013). Therefore, it was hypothesized that the present of these nanoparticles on the paper surface could increase the concentration of proton, which improves the ionization efficiency. Furthermore, it is important to note that silicon nanoparticles not only might increase the proton level, but also might change the physical properties of the porous surface because the nano size allows these particles to fill the pores, which has approximate size 11 µm, in the paper instead of the target analyte. Silica nanoparticles have been used in various applications with mass spectrometry. Previously, silica nanoparticles was utilized in order to determine protein in any biological sample. The adsorption of protein on the surface of silica nanoparticles was identified. These particles increased the concentration of proton, resulting on the peak responses of the protein (Omanovic-Miklicanin et al., 2014). Tsao et al. (2012) used silica nanoparticles with LDI in order to identify the effects of these particles on the sensitivity of desorption ionization techniques. Good sensitivities were obtained (Tsao et al., 2012; Liu and He, 2008). Porous silica substrate was used with surface/ionization mass spectrometry. This approach has been utilized to profile metabolites, identify active drug components, and determine small molecules in biological sample (Deng and Sanyal, 2006; Go et al., 2007; Kraj et al., 2006; Okuno and Wada, 2005; Shen et al., 2001; Xu et al., 2004). In the current study, a paper substrate was modified with silicon dioxide nanoparticles to understand the interaction between the analyte, silica nanoparticles and the cellulosic fiber. It is hypothesized that the pores in the Whatman filter paper grade 1could be filled with the silica nanoparticles. Thus, the amount of the analyte that adsorbed on the paper might decrease, which positively affects the ionization efficiency. In addition, the concentration of proton could increase with the present of -Si-OH group. In order to study the effect of silica nanoparticles film on the ionization efficiency, peak responses were compared for various analytes. The results showed peak responses with the coated paper higher than the standard paper for various compounds, including isophorone, safranal, β-ionone, kaempferol, astaxanthin, progesterone, cortisone, 153 hydrocortisone, and T4. Based on this result, either the concentration of proton increased or the analytes were less strongly bound to the paper because the nanoparticles filled the pores in the paper. Silica nanoparticles boost the ionization efficiency on the paper. This result is in agreement with a previous work where the researchers observed increasing in the peak response of protein with silica nanoparticles using secondary ion mass spectrometry (Omanovic-Miklicanin et al., 2014). Further addition, the recovery values were calculated for several analytes in order to determine the accuracy of the developed method. The values were obtained with pure solution of the analyte using coated paper. The results revealed recovery values in the presence of silica nanoparticles film reached to 100 %. Based on this result, the coated paper has a good accuracy. It was indicated that Si/cellulose interaction (hybrid) is an important factor that affects the silica nanoparticles film formation on the substrate. Poor interaction leads to variation in the ionization efficiency (Tsao et al., 2012). In order to produce an efficient Si/cellulose hybrid on the paper, the nanoparticles must be strongly adsorbed on the paper whilst avoiding damage of the paper. To study the distribution of nanoparticles on the paper four papers were immersed in the silica nanoparticles solution for several days. The SEM images showed that SiO2 nanoparticles spread over the papers with high efficiency with increasing the time of immersion of the paper substrate in the silica nanoparticles solution. At short time (15 minutes), the silicon ranged between 2.2-36 % wt., where not all pores were filled by silica nanoparticles. For longer time, the silicon ranged between 30-36 % wt. Most pores were filled with the nanoparticles. Based on these results, the paper substrates were immersed in the solution for 72 h, and used for the rest experiments. However, the formation of an over-oxidized silicon nanoparticles layer (after 72 h) slows down overall peak intensity for some compounds in the current study. The aggregation state of nanoparticles might affect the ionization efficiency. Therefore, the degree of the silica nanoparticles distribution on the paper needs to be carefully monitored to achieve optimal MS results. 8.4.2 Ionization Mechanism Further, the MS spectrum was obtained using the coated and standard papers. The results showed that the protonated ion was the major ion for the selected compounds using both standard and coated paper. The exception is kaempferol, β-carotene, and quercetin gave radical cation with the coated paper, which means the silica nanoparticles affects the ion 154 formation of the kaempferol and quercetine. However, more experiments are required to determine whether these nanoparticles affect the ionization mechanism of compounds with various polarities. The interaction between the silica nanoparticles and the cellulose is suggested as follow. The macromolecular structure of cellulose has a lot of hydroxyl groups. The cellulose/silica hybrid form new molecular structures containing silica nanoparticles. In this process inorganic nanoparticles are dispersed in the cellulosic fiber, which binds to the cellulose through the O-Si- bonds, therefore, forming network organic-inorganic bonds. The interaction between inorganic nanoparticles and cellulose molecules was accomplished through oxygen atom and H-bond affect (Ullah et al., 2013). The proposed schematic diagram of Si/cellulose hybrid is given in Figure 8.5. The results in this thesis showed that mass spectra generated from paper coated with silica nanoparticles were higher than standard paper. Thus, silica nanoparticles improve the ionization efficiency. O O O O OO OSi Si Si Si Si Si SiO O O O OOOH OH OH OH OH OH OH Figure 8.5 The schematic of the proposed interaction between silicon dioxide nanoparticles and cellulose molecules. 155 Chapter 9 Conclusion With the ongoing interest of the paper spray ionization mass spectrometry (PSI/MS) technique for direct analyses without sample preparation, it is crucial that characterization and applications are determined to better understand several insights of this technique. In this thesis, I discussed seven bodies of work to characterize and utilize PSI/MS new developed ionization method for direct analyses. 9.1 The Role of Paper Surface on Paper Spray Ionization Mass Spectrometry The critical first objective of this thesis was to develop new understanding of the ionization mechanism. Mass spectrometry and geometric parameters were optimized for several compounds. Interestingly, it was shown that water molecules are inherently associated with the paper, and are the main reason for induction ionization with pure non-polar solvents. Many water cluster peaks were found with pure either polar or non-polar solvents. When an oven-dried paper was used, no peak response was found with non-polar solvents. When a non-polar solvent was used on paper acclimatized to normal lab humidity, ionization was observed indicating that residual water on the paper surface played a role in the ionization mechanism. This approach added a very important insight to the PSI/MS mechanism. To date no one has investigated the phenomenon. Furthermore, the results of this study demonstrated that the geometric parameters, including tip position, and the elements that present on the paper substrate affected the ionization efficiency. Corona discharge produced at short distance between the paper tip and MS inlet, which affects the ionization efficiency. It is demonstrated a cone shaped distribution of the sprayed jets. Further addition, the effect of the porosity and analyte/surface interaction on the ionization efficiency was demonstrated. The reproducibility of the paper tip changed with a high number of replicates. These data added new insight on the fundamental concept of surface ionization techniques. 156 9.2 Separation of Compounds from Complex Sample Matrices by Paper Substrate The second objective of the thesis was adaptation of PSI as chromatography for sample components separation. Paper substrates with different lengths were prepared for this purpose. A mixture of crocin, astaxanthin and zeaxanthin was prepared for this study. The PSI technique was compared with two standard chromatography methods, including paper chromatography and thin layer chromatography. The results showed that Rf values for each tested analyte were close to each other using the three separation methods. This novel approach provides an interesting possibility for the future by reducing the time of analysis (maximum 3 min.) and no need to use high-pressure pump for sample separation, allowing for rapid high throughput analysis, and identify unknowns on TLC plate. In addition, the PSI efficiency for quantitative analysis was demonstrated by calculating the recovery percentage with both standard and washed papers. Approximately all recovery values were more than 95 % using washed paper. Based on these results, it is indicated that the washed paper makes PSI more adequate for quantitative analysis. 9.3 Comparison of Three Ionization Techniques/Corona Discharge The objective of this section was designed in two parts. First, continuous flow of the solvent, using either pump syringe or infused injection, inhibits the possibility to produce corona discharge. Another factor that might boost the production of corona discharge is the distance between paper tip and MS inlet. Mass spectra showed that PSI is consistent with electrospray ionization (no corona discharge) more than electrospray chemical ionization (in the presence of corona discharge) at distance 5 mm. At distance lower than 5 mm, the results showed that PSI is more consistences with ESCI in the ion formation. It was demonstrated that corona discharge produced at the paper tip with a shorter distance. There were two ways to inhibit the production of corona discharge at the paper tip. First, the voltage can be made low enough on the paper substrate to stop the spreading of the discharge somewhere before the MS inlet is reached. Second, lower the field strength between the paper tip and MS inlet cone by increasing the distance between the paper tip and MS inlet cone. This novel achievement added more insight on the fundamental concept of the ionization mechanism. 157 9.4 Comparison of Two Mass Analyzers and Two Surface Ionization Sources The fourth objective of the research was to utilize the paper spray ionization mass spectrometry technique to investigate the sensitivity and accuracy across two mass analyzers for the same PSI lab-made apparatus. It was shown that both mass analyzers gave the same mass spectra with paper spray ionization. The ions of the tested compounds were obtained using TQ with responses higher than TOF by two to three orders of magnitude. So far, lower detection limits of µg/L and µg/mL for six compounds have been determined using triple quadrupole and time of flight mass analyzers, respectively. These results might be depending on the accuracy of each instrument. TQ/MS can detect only two decimal points of ion mass, while TOF could detect mass with four decimal points. Thus, the ions that have the same integer mass with different decimal points gave the same peak with TQ/MS, while a unique peak can be observed for each ion using TOF. This makes the peak intensity with TQ/MS higher than TOF. However, this approach still leaves room for investigation of several instruments to determine whether these results are applicable to the class of instruments or whether the results are unique to these individual instruments. The second part was designed to provide insight into solubility, and micro-flow of the sample on the paper surface. Furthermore, to determine the relative qualitative efficacy of the new developed technique as compared to another solid surface ionization technique liquid extraction surface analysis (LESA). It was found that the peak response with PSI higher than LESA by one to two orders of magnitude. Mass spectra showed that ions were produced with the same mechanism using the two surface ionization techniques. The spray was produced through the paper substrate with PI while it was produced from the ESI probe with LESA. The limit of detection was accomplished at level µg/L(PSI), while it was at level µg/mL using LESA. Thus, trace concentrations can be detected using PSI while it is not possible using LESA. Based on these results, the analyte was extracted and mobilized from the paper surface in PSI with extraction efficiency higher than LESA. However, more investigations with different apparatus, either PSI or LESA, are required to determine whether these results are unique to these individual apparatus. 158 9.5 PSI Profile of a Model Plant Extract: Saffron Crocus sativus The next objective was designed to determine the accuracy of PSI to be utilized as a metabolites fingerprinting technique of saffron samples. It was demonstrated that most metabolites were obtained within less than five minutes. The repeatability was determined by using different substrates. It was demonstrated that most metabolites were observed with high values of RSD %, which were more than 20 %. In addition, more than 10,000 compounds were observed in the blank using different paper substrates. Based on these results, PSI has low accuracy to determine a fingerprint profile of complex sample such as saffron. Further addition, a comprehensive discussion was given on the diversity of several major bioactive metabolites in saffron over saffron collected from five regions and over three parts of Crocus sativus L. tissues. The quality of saffron samples was determined as well. The results showed that Spanish saffron has the highest quality based upon the highest levels of crocin and safranal. Based on these results PSI is suitable technique for quantitative analysis. 9.6 New Approach for Rapid Analysis of Solid Sample The objective of this section was designed to develop new achievement for solid sample analysis with surface/ionization mass spectrometry techniques. Mechanical press extraction (MPE) was introduced for solid sample analysis without the requirement of sample preparation. Twelve compounds were estimated over eleven solid samples. Accordingly, a completely new approach for PSI/MS was developed within this work. The recovery values of MPE were successfully compared with different previous works as well as with standard extraction method (sonication). The key and novelty of this approach is that it overcomes the limitations of existing methods for analyzing solid samples with less time consuming, less amount of either extractive solvent or sample, and provides an excellent recoveries. This approach has widespread applications in the rapidly developing area of "ambient ionization" techniques. 9.7 Silica Nanoparticles-Paper Spray Ionization Mass Spectrometry The last objective was developed to enhance the ionization efficiency by creating silica nanoparticles film on the paper substrate. These nanoparticles filled the pores on the paper, decreased the analyte/surface interaction, and increased the concentration of proton on the paper, which enhance the ionization efficiency with the PSI technique. The results 159 showed that ion response was enhanced for the selected compounds. The recoveries were indicated with values approaching 100 %. These results show that silica nanoparticles improved the ionization efficiency with good accuracy. This approach will be vital for the developing area of ambient ionization research. 9.8 Strengths and Limitations of the Thesis Research Paper spray ionization mass spectrometry is a promising technique for direct analyses or ambient ionization. Water molecules inherently associated with the paper play an important role to induce ionization efficiency and thus enable even non-polar solvents to be used as eluent. This thesis is the first to clearly demonstrate how this process works and this adds new insight on the PSI/MS mechanism. Furthermore, this thesis develop new achievement to inhibit the production of corona discharge, which adds more insight on the fundamental concept of this technique. The effects of pore size and analyte/surface interaction were demonstrated. This thesis is the first to demonstrate how this process works and this adds new insight on the PSI fundamental concept. Another important development for the rapid analysis of solid sample is MPE. This novel approach has widespread application in the direct analysis developing area of ambient ionization techniques. We also demonstrated that paper spray could be used in a manner that matched Thin Layer Chromatography (TLC) data, so that this approach could aid in the identification of unknown species detected by TLC. Finally, we also demonstrated that silicon nanoparticles film could increase ionization efficiency. When attempting to apply PSI to metabolomics of plants, it was discovered that the current repeatability was insufficient to get a reproducible fingerprint of complex plant tissue such as saffron. 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Thesis/Dissertation
eng
Chemistry
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivs 2.5 Canada
Graduate
Characterization and applications of paper spray ionization mass spectrometry
Text