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Development of a sensitive, quantitative high-performance liquid chromatographic assay for the measurement… Embree, Leanne 1989

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DEVELOPMENT OF A SENSITIVE, QUANTITATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC ASSAY FOR THE MEASUREMENT OF DIGOXIN IN PATIENT GROUPS WITH HIGH LEVELS OF DIGOXIN-LIKE IMMUNOREACTIVE SUBSTANCES B. Sc. (Pharm), U n i v e r s i t y of B.C., Vancouver, B.C., Canada, 1982 M. Sc., U n i v e r s i t y of B.C., Vancouver, B.C., Canada, 1985 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s D i v i s i o n of P h a r m a c e u t i c a l C h e m i s t r y We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Ja n u a r y 1989 Leanne Embree LEANNE EMBREE DOCTOR OF PHILOSOPHY i n In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pha-r/rta C e is- frctx / S~c ie yt c e J The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date S 9 P ? tf 2^ DE-6G/81) i i A b s t r a c t Digoxin i s the most commonly used d i g i t a l i s glycoside for the treatment of congestive heart f a i l u r e and cert a i n disturbances of cardiac rhythm. The low therapeutic index observed for digoxin and the c l i n i c a l s ignificance of digoxin therapy have necessitated the development of sensitive a n a l y t i c a l methods for the quantitation of digoxin in b i o l o g i c a l samples. Digoxin may be analysed by several methods including immunoassays, chromatographic procedures and various b i o l o g i c a l and chemical methods. Immunoassays, both radioimmunoassay (RIA) and fluorescence p o l a r i z a t i o n immunoassay (FPIA) procedures, are used in the c l i n i c a l laboratory because of the i r speed, prec i s i o n , s e n s i t i v i t y and r e l a t i v e l y low cost. However, reaction of the digoxin antibodies used in the immunoassay methods with digoxin metabolites, endogenous compounds such as digoxin-like immunoreactive substances (DLIS), and other drugs that may be co-administered with digoxin continues to be a major problem. The lack of s p e c i f i c i t y of the immunoassay methods for digoxin has led to d i f f i c u l t i e s in interpretation of assay values. Attempts to compensate for th i s lack of s p e c i f i c i t y have included the use of chromatographic systems as elaborate sample handling methods prior to immunoassay. However, since an immunoassay was used for detection of d i g o x i n i n t h e s e t e c h n i q u e s , t h e s p e c i f i c i t y may s t i l l be ques t i o n a b l e . A s e n s i t i v e and s p e c i f i c a s s a y f o r d i g o x i n u s i n g p h y s i c o - c h e m i c a l methods f o r measurement i s t h e r e f o r e needed. A method was d e v e l o p e d u s i n g pre-column d e r i v a t i z a t i o n of d i g o x i n and i t s m e t a b o l i t e s w i t h 3,5-d i n i t r o b e n z o y l c h l o r i d e f o l l o w e d by HPLC a n a l y s i s w i t h e l e c t r o c h e m i c a l d e t e c t i o n . A maximum s e n s i t i v i t y of 0.883 ng of 3 , 5 - d i n i t r o b e n z o y l d i g o x i n (0.394 ng d i g o x i n ) was ob s e r v e d u s i n g d u a l e l e c t r o d e d e t e c t i o n i n the redox mode. A l t h o u g h r e s o l u t i o n between d e r i v a t i z e d d i g o x i n and i t s m e t a b o l i t e s was o b t a i n e d , the low y i e l d of the d i g o x i n d e r i v a t i v e and the f o r m a t i o n of m e t a b o l i t e s when s m a l l (ng) samples were d e r i v a t i z e d made t h i s method u n s u i t a b l e f o r e v a l u a t i n g p a t i e n t samples. A h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c (HPLC) assay u s i n g post-column d e r i v a t i z a t i o n of d i g o x i n , which s e p a r a t e d d i g o x i n from i t s m e t a b o l i t e s and some commonly c o -a d m i n i s t e r e d d r u g s , was d e v e l o p e d . Post-column (PC) d e r i v a t i z a t i o n of d i g o x i n w i t h c o n c e n t r a t e d h y d r o c h l o r i c a c i d and d e h y d r o a s c o r b i c a c i d , f o l l o w e d by f l u o r e s c e n c e d e t e c t i o n , a l l o w e d f o r q u a n t i t a t i o n w i t h i n the t h e r a p e u t i c range of d i g o x i n . S t e r o i d s which have been r e p o r t e d t o c r o s s - r e a c t w i t h d i g o x i n a n t i s e r a were assay e d u s i n g the HPLC-PC method d e v e l o p e d i n t h i s s t u d y . The s t e r o i d samples e i t h e r d i d not iv elute from the HPLC system or did not produce a fluorescent product under these conditions. Serum samples from d i g i t a l i z e d patients were evaluated using both the HPLC-PC and the FPIA methods. When compared to the HPLC procedure, the FPIA assay results gave, on average, higher digoxin l e v e l s . This may have been due to the inclusion of digoxin metabolites or endogenous compounds with the FPIA assay. Serum samples from u n d i g i t a l i z e d patient groups where high DLIS leve l s have been reported were also evaluated. These included umbilical cord blood samples and samples from hypertensive patients, renal f a i l u r e patients and hepatic f a i l u r e patients. Comparison of the HPLC-PC and FPIA methods demonstrated that the HPLC-PC assay gave fewer false p o s i t i v e results than the FPIA. The HPLC-PC assay developed for analysis of digoxin was unaffected by the presence of digoxin metabolites, numerous steroids, co-administered drugs and endogenous compounds, most of which have been reported to give false p o s i t i v e r e s u l t s with the FPIA. V TABLE OF CONTENTS Chapter Page Abstract i i L i s t of Tables x i i L i s t of Figures xiv Symbols and Abbreviations xvi Acknowledgement xix Introduction 1 1 . Literature Survey 4 1.1 Pharmacodynamic Properties 4 1.1.1 Cardiovascular Actions 4 1.1.1.1 C o n t r a c t i l i t y 4 1.1.1.2 Conduction 5 1.1.2 Mechanism of Action 5 1.1.3 To x i c i t y 6 1.1.4 Pharmacokinetics 8 1.1.4.1 Absorption 9 1.1.4.2 D i s t r i b u t i o n 11 1.1.4.3 Metabolism And Elimination 13 1.1.5 Chemistry 15 1.1.6 Structure-Activity Relationships 16 1.2 Digoxin Therapy 19 1.3 Digoxin-Like Immunoreactive Substance(s) 20 1.4 Digoxin-Drug Interactions 27 1.4.1 Digoxin-Quinidine 27 v i 1.4.2 Other Drug Interactions 29 1.5 Digoxin Analysis 32 1.5.1 Immunoassays 33 1.5.2 B i o l o g i c a l Assays 37 1.5.3 Chemical Methods 38 1.5.4 Chromatographic Techniques 40 1.5.5 HPLC-RIA 43 1.6 HPLC-EC 44 2. EXPERIMENTAL 49 2.1 Supplies 49 2.1.1 Chemicals 49 2.1.2 Solvents 50 2.1.3 Extraction Supplies 50 2.1.4 Post-Column Deri v a t i z a t i o n Supplies 51 2.1.5 Commercial Radioimmunoassay Kits 52 2.1.6 Commercial Fluroescence P o l a r i z a t i o n Immunoassay Kit 53 2.1.7 F i l t r a t i o n of HPLC Mobile Phase 53 2.2 Equipment 53 2.2.1 HPLC Equipment 53 2.2.2 Electrochemical Detector 54 2.2.3 Fluorescence Detector Flow C e l l 56 2.2.4 Post-Column Reactor and Pressure Chamber 56 2.2.5 Extraction Equipment 56 2.2.6 Radioimmunoassay Equipment 59 v i i 2.3 Pre-column 3,5-Dinitrobenzoyl Chloride Deri v a t i z a t i o n Procedures 59 2.3.1 For Gram Quantities of Digoxin 59 2.3.2 For Milligram Quantities of Cardiac Glycosides 59 2.3.3 For Nanogram Quantities of Digoxin 60 2.4 Preparation of Solutions 60 2.4.1 Reagents For 3,5-DNBCl Derivatization 61 2.4.1.1 3,5-DNBCl in Pyridine 61 2.4.1.2 Sodium Bicarbonate (5%) with 4-DMAP 61 2.4.1.3 Dilute Hydrochloric Acid 61 2.4.2 Reagents For Fluorogenic Der i v a t i z a t i o n 61 2.4.2.1 Ascorbic Acid Solution 61 2.4.2.2 Dilute Hydrogen Peroxide Solution 62 2.4.2.3 Dehydroascorbic Acid 62 2.4.2.4 Hydrogen Peroxide with Phosphoric Acid 62 2.4.3 Sodium Acetate Buffer (0.1 M, pH 4.6) 62 2.4.4 Mobile Phase 62 2.4.5 Standard Solutions 63 2.4.5.1 Digoxin in Ethanol 63 2.4.5.2 3,5-Dinitrobenzoyl Digoxin in Methanol 63 2.4.5.2 Digitoxigenin in Ethanol 63 2.4.5.3 Dihydrodigoxin in Ethanol 64 2.4.5.4 Steroid Samples in Methanol 64 2.4.5.5 Preparation of Mexiletine Hydrochloride in 50% Ethanol 65 v i i i 2.4.5.6 P r e p a r a t i o n of Plasma Samples f o r RIA A n a l y s i s 65 2.4.5.7 P r e p a r a t i o n of Serum Samples S p i k e d w i t h D i g o x i n M e t a b o l i t e s f o r FPIA A n a l y s i s 66 2.5 P r e p a r a t i o n of S o l v e n t s f o r Serum E x t r a c t i o n 66 2.5.1 I s o o c t a n e / D i c h l o r o m e t h a n e (20/5) 66 2.5.2 E x t r a c t i o n S o l v e n t 66 2.5.3 P r e p a r a t i o n of P r o t e i n P r e c i p i t a t i o n Reagents 66 2.5.3.1 Z i n c S u l f a t e H e p t a h y d r a t e (5%) 66 2.5.3.2 Barium H y d r o x i d e O c t a h y d r a t e (0.3 N) 67 2.5.3.3 C u p r i c S u l f a t e P e n t a h y d r a t e (10%) 67 2.5.3.4 Sodium H y d r o x i d e (12%) 67 2.6 RIA A n a l y s i s and C r o s s - R e a c t i v i t y C a l c u l a t i o n s 67 2.7 HPLC W i t h E l e c t r o c h e m i c a l D e t e c t i o n 68 2.7.1 O p t i m i z a t i o n of E l e c t r o c h e m i c a l C o n d i t i o n s 68 2.7.2 M o b i l e Phases f o r HPLC w i t h E l e c t r o c h e m i c a l D e t e c t i o n 69 2.8 HPLC Post-Column D e r i v a t i z a t i o n Assay 69 2.8.1 Assembly of S t e e l and PVC P r e s s u r e V e s s e l s 69 2.8.2 O p t i m i z a t i o n of HPLC Post-Column D e r i v a t i z a t i o n 70 2.8.2.1 F l u o r o g e n i c D e r i v a t i z a t i o n w i t h Hydrogen P e r o x i d e , P h o s p h o r i c A c i d and C o n c e n t r a t e d HC1 70 ix 2.8.2.2 Evaluation of the Fluorescence Emission F i l t e r s with Dehydroascorbic Acid and HC1 De r i v a t i z a t i o n 71 2.8.3 F i n a l HPLC-PC Assay Procedure 71 2.9 Serum Extraction Procedure 71 2.9.1 Protein P r e c i p i t a t i o n Methods 71 2.9.2 Centrifree and C e n t r i f l o F i l t r a t i o n 73 2.9.3 Solvent-solvent Extraction 73 2.9.4 F i n a l Procedure for Extraction of Digoxin in Serum 73 2.10 Recovery and Precision of Extraction 74 2.11 Ca l i b r a t i o n Curve 76 2.12 S p e c i f i c i t y 76 2.12.1 Steroids 76 2.12.2 Co-Administered Drugs 76 2.13 Quality Control Procedure 77 2.14 Analysis of D i g i t a l i z e d Patient Serum Samples 77 2.15 Analysis of Serum from Undigitalized Patient Groups Where High DLIS Levels have been Reported 77 2.15.1 Hypertension 77 2.15.2 Renal F a i l u r e 77 2.15.3 Hepatic F a i l u r e 78 2.15.4 Umbilical Cord Blood Samples 78 3. Results and Discussions 79 3.1 Radioimmunoassay of Digoxin and Its Metabolites 79 3.2 Fluorescence P o l a r i z a t i o n Immunoassay of Digoxin Metabolites in Serum 82 3.3 Pre-Column 3,5-Dinitrobenzoyl Derivatization of Cardiac Glycosides with HPLC-EC Analysis 84 3.3.1 U l t r a v i o l e t Detection 84 3.3.2 Detection With a Single Glassy Carbon Electrode 87 3.3.3 Dual Electrode Detection v 90 3.4 Post-Column Fluorogenic HPLC Assay 97 3.4.1 Pressure Vessel 97 3.4.2 Optimization of Post-Column Reactor Conditions 98 3.4.3 Maximum S e n s i t i v i t y 101 3.4.4 Evaluation of Digoxin and i t s Metabolites 103 3.5 Analysis of B i o l o g i c a l Samples Using The HPLC Fluorogenic Assay 105 3.5.1 Optimization of Serum Extraction Method 105 3.5.1.1 Protein P r e c i p i t a t i o n Methods 105 3.5.1.2 Removal of Endogenous Interference 107 3.5.2 Recovery and Precision 109 3.5.3 C a l i b r a t i o n Curve 112 3.5.4 Comparison of HPLC-PC and FPIA Methods in Spiked Serum Samples 114 3.5.5 S p e c i f i c i t y 119 3.5.5.1 Steroids 119 3.5.5.2 Co-administered Drugs 119 3.6 Analysis of Digoxin in D i g i t a l i z e d Patient Serum 120 x i 3.7 Analysis of Serum from Undi g i t a l i z e d Patient Groups Where High DLIS Levels Have Been Reported 122 3.7.1 Hypertensive Patients 122 3.7.2 Renal Failu r e Patients 124 3.7.3 Hepatic Fail u r e Patients 125 3.7.4 Umbilical Cord Blood 128 4. Summary and Conclusions 130 5. References 133 x i i L i s t of Tables Table Page I Summary Of Digoxin Pharmacokinetics 13 II Percent Cross-Reactivity on a Molar Basis for Digoxin Metabolites With Commercial Digoxin RIA Kit s 80 III Evaluation of Digoxin Metabolites in Serum Using the FPIA for Digoxin Analysis 83 IV HPLC Retention Times of 3,5-DNB Derivatives 85 V Resolution of 3,5-DNB Digoxin and 3,5-DNB Dihydro-digoxigenin Using HPLC-EC with Various Mobile Phases 88 VI Eff e c t of Varying the Aqueous Portion of the Mobile Phase on Peak Height Using HPLC-PC Fluorescence Assay 99 VII Eff e c t of Emission F i l t e r s on Peak Height of Digoxin Using HPLC-PC Fluorescence Assay 100 VIII Ef f e c t of HC1 Flow Rate on Peak Height of Digoxin Using HPLC-PC Fluorescence Assay 101 IX Recovery of Digoxin from Spiked Serum Samples 111 X Precision of Digoxin Assay 112 XI Comparison of the HPLC-PC and FPIA Methods for Digoxin Analysis in D i g i t a l i z e d Patients 121 XII Comparison of Undigi t a l i z e d Hypertensive Patient Samples Evaluated by the HPLC-PC and FPIA Methods 123 xi i i XIII Hepatic Failu r e Patient Samples Evaluated by the HPLC-PC and FPIA Methods 126 XIV Mixed Umbilical Cord Blood Samples Evaluated by the HPLC-PC and FPIA Methods 128 x i v L i s t of F i g u r e s F i g u r e Page 1 The Chemical S t r u c t u r e of D i g o x i n 16 2 Reaction Involved i n Immunoassay Procedures 33 3 Schematic of Noise Reducer 55 4 Schematic of HPLC-EC System 57 5 Schematic of HPLC-PC F l u o r o g e n i c System 58 6 Diagram of HPLC-PC Pressure V e s s e l With F i t t i n g s 72 7 Flow Diagram f o r Serum E x t r a c t i o n Procedure 75 8 S t r u c t u r e of 3,5-DNB Digoxin 85 9 Chromatogram of 3,5-DNB Dig o x i n D e r i v a t i z e d i n M i l l i g r a m Q u a n t i t i e s 86 10 Chromatogram of Dig o x i n and i t s M e t a b o l i t e s as T h e i r 3,5-DNB D e r i v a t i v e s using E l e c t r o c h e m i c a l D e t e c t i o n 89 11 Chromatogram of 125 ng of 3,5-DNB Digoxin P u r i f i e d by Double R e c r y s t a l l i z a t i o n 92 12 Chromatogram of 80 ng of Di g o x i n D e r i v a t i z e d i n M i l l i g r a m Q u a n t i t i e s - 93 13 Chromatogram of Dig o x i n and I t s M e t a b o l i t e s by HPLC-PC 104 14 Chromatograms of Digoxin and I t s M e t a b o l i t e s i n E thanol and Blank Serum C o n t a i n i n g I n t e r f e r i n g Peak 108 15 Chromatogram of Blank Serum and Serum Spiked with 1.5 ng Digoxin and I n t e r n a l Standard 110 16 C a l i b r a t i o n Curve f o r HPLC-PC Dig o x i n Assay 113 XV Correlation Between HPLC-PC and FPIA Methods For Digoxin Analysis xvi Symbols and A b b r e v i a t i o n s 3,5-DNBCl 3,5-Dinitrobenzoyl chloride 4-DMAP 4-Dimethylaminopyridine ACN HPLC grade a c e t o n i t r i l e ANP A t r i a l n a t r i u r e t i c peptide ACU U.B.C. Health Sciences Centre Acute Care Unit ATP Adenosine Triphosphate B. C. B r i t i s h Columbia C Centigrade CA C a l i f o r n i a Cl^j. Creatinine clearance cm Centimeter C. V. C o e f f i c i e n t of Variation Da Daltons DLIS Digoxin-like immunoreactive substance(s) EC Electrochemical EMIT Enzyme-multiplied immunoassay EtOH Absolute ethanol FlA Fluorescence immunoassay FPIA Fluorescence p o l a r i z a t i o n immunoassay g gram GFR Glomerular f i l t r a t i o n rate GLC Gas-liquid chromatography HC1 Concentrated hydrochloric acid HPLC High-performance l i q u i d chromatography xv i i HPLC-EC High-performance l i q u i d chromatography-electrochemical detection HPLC-PC High-performance l i q u i d chromatography post-column d e r i v a t i z a t i o n I.D. Internal diameter IL I l l i n o i s i n . Inch I PA HPLC grade isopropanol l b . Pound M Molar MA Massachusetts MD Maryland MeOH HPLC grade methanol MI Michigan min. Minutes MO Missouri Na +,K +-ATPase Sodium-potassium dependent ATPase ng Nanogram NJ New Jersey NV Nevada NY New York Ont. Ontario PC Post-column PTFE Polytetrafluoroethylene PVC Polyvinylchloride Que. Quebec r Correlation c o e f f i c i e n t xvi i i r .c. f . Relative c e n t r i f u g a l force ( g r a v i t i e s ) RIA Radioimmunoassay S.D. Standard deviation TLC Thin-layer chromatography TX Texas U.S.A. United States of America V Volts WI Wisconsin UL M i c r o l i t e r Mm Micrometer xix Acknowledgement I wish to thank my supervisor, Dr. Keith McErlane, for his valuable guidance throughout the course of t h i s study. I am also grateful to the other members of my graduate committee, Drs. Frank Abbott, Helen Burt, Charles Kerr and James Orr for th e i r helpful advice. Furthermore, Dr. James Axelson i s acknowledged for contributing s i g n i f i c a n t ideas for consideration. I am grateful to Drs. C. Kerr, J . Price, U. Steinbrecher, G. Sandor and S. Stordy for their assistance in obtaining c l i n i c a l samples. Fi n a n c i a l support from the Canadian and B r i t i s h Columbia Heart Foundations i s g r a t e f u l l y acknowledged. I would p a r t i c u l a r l y l i k e to thank Roland Burton, Trine Henriksen, Radana Vaughan and Dale Embree for th e i r technical assistance. I am indebted to Dr. A l l a n Goodeve for numerous discussions and proofreading of t h i s document. F i n a l l y , I would l i k e to thank my husband, Dale, for his devotion and encouragement. 1 INTRODUCTION D i g i t a l i s glycosides form one of the most b e n e f i c i a l group of drugs available to aid the f a i l i n g heart. They are the drugs of choice for the treatment of congestive heart f a i l u r e and certain disturbances in cardiac rhythm. Although digoxin i s the only d i g i t a l i s glycoside available for c l i n i c a l use in Canada and has been employed medically since about 1937, problems with therapy s t i l l exist due to i t s low therapeutic index. At digoxin concentrations greater than 3 ng/mL in serum, there i s a high incidence of t o x i c i t y . However, physiological conditions and i n t e r -patient v a r i a t i o n in response may lead to t o x i c i t y below t h i s value. In an e f f o r t to avoid manifestations of t o x i c i t y while maintaining the desired therapeutic e f f e c t , digoxin serum concentrations are maintained between 0.5 and 2.0 ng/mL. These low concentrations have necessitated the development of extremely sensitive assay techniques. Digoxin has been analysed by several methods including radioimmunoassay, enzyme-multiplied immunoassay, fluorescence p o l a r i z a t i o n immunoassay, high-performance l i q u i d chromatography, and various b i o l o g i c a l and chemical methods. None of the methods developed to date can compete with the immunoassays in terms of speed, pre c i s i o n , s e n s i t i v i t y and cost; therefore immunoassay methods are routinely used in c l i n i c a l laboratories. However, one of the major problems with the immunoassay methods for digoxin 2 analysis i s that of c r o s s - r e a c t i v i t y of the digoxin antibodies with digoxin metabolites, other drugs that may be co-administered with digoxin and endogenous compounds such as digoxin-like immunoactive substance(s). The lack of s p e c i f i c i t y of the immunoassay methods used for digoxin analysis has prompted questions of i t s r e l i a b i l i t y . In order to avoid the p o s s i b i l i t y of over-estimation of digoxin due to interference from endogenous substances and other drugs, investigators have introduced chromatographic methods prior to the immunoassay. However, these methods generally require elaborate sample handling protocols and s t i l l use antibodies with questionable s p e c i f i c i t y . A s e n s i t i v e and s p e c i f i c assay for digoxin using more r e l i a b l e methods for measurement i s therefore needed. High-performance l i q u i d chromatographic (HPLC) methods have been successful in separating digoxin from most of i t s metabolites but lack the s e n s i t i v i t y required for b i o l o g i c a l samples (Bockbrader 1984; Desta 1987; F u j i i 1983; G f e l l e r 1977; Nachtmann 1976a, 1976b). Other HPLC methods do not have s u f f i c i e n t resolution of digoxin from dihydrodigoxin for quantitation (Reh 1985). An HPLC assay for digoxin at therapeutic concentrations in plasma using post-column fluorogenic d e r i v a t i z a t i o n without interference from a number of commonly co-prescribed drugs and the metabolites of digoxin has been reported (Kwong 1986a, 1986b). 3 The s p e c i f i c aims of the p r o j e c t were as f o l l o w s : 1. t o a s c e r t a i n t h e amount of i n t e r f e r e n c e from the m e t a b o l i t e s of d i g o x i n i n radioimmunoassay k i t s c o m m e r c i a l l y a v a i l a b l e . 2. t o i n v e s t i g a t e the p o s s i b i l i t y of i n c r e a s i n g the s e n s i t i v i t y of the HPLC post-column f l u o r o g e n i c a s s a y (Kwong 1986a, 1986b) by d e r i v a t i z a t i o n of d i g o x i n p r i o r t o c h r o m a t o g r a p h i c a n a l y s i s ( F u j i i 1983) c o u p l e d w i t h e l e c t r o c h e m i c a l d e t e c t i o n . 3. t o improve the e x t r a c t i o n p r o c e d u r e and HPLC p o s t -column f l u o r o g e n i c d e r i v a t i z a t i o n method (Kwong 1986a, 1986b) t o a l l o w f a s t e r sample p r o c e s s i n g and e l i m i n a t e p o s s i b l e i n t e r f e r e n c e from endogenous compounds or c o -p r e s c r i b e d m e d i c a t i o n . 4. t o e v a l u a t e serum samples from p a t i e n t s w i t h p a t h o l o g i c a l c o n d i t i o n s where endogenous d i g o x i n - l i k e immunoreactive s u b s t a n c e ( s ) have been r e p o r t e d . 4 1 . L I T E R A T U R E S U R V E Y D i g i t a l i s g l y c o s i d e s a r e the most v a l u a b l e drugs a v a i l a b l e f o r the c l i n i c a l management of c o n g e s t i v e h e a r t f a i l u r e . S i n c e W i t h e r i n g (1937) f i r s t documented the e f f e c t i v e n e s s of d i g i t a l i s p r e p a r a t i o n s i n the t h e r a p y of c e r t a i n forms of d r o p s y , a t t e n t i o n has been d i r e c t e d towards e l u c i d a t i o n of t h e i r mechanism of a c t i o n and c e l l u l a r mechanisms t h a t a f f e c t t h e i r e f f i c a c y . In s p i t e of the s e e f f o r t s , s e r i o u s problems w i t h t h e r a p y s t i l l e x i s t . D i g o x i n , the c a r d i a c g l y c o s i d e a v a i l a b l e i n Canada, i s w i d e l y used i n the t r e a t m e n t of c o n g e s t i v e h e a r t f a i l u r e and c e r t a i n d i s t u r b a n c e s of c a r d i a c rhythm. 1 . 1 Pharmacodynamic P r o p e r t i e s 1.1.1 C a r d i o v a s c u l a r A c t i o n s The main a c t i o n of d i g o x i n and o t h e r d i g i t a l i s g l y c o s i d e s i s the a b i l i t y t o i n c r e a s e the c o n t r a c t i l e f o r c e of the b e a t i n g h e a r t ( H a u s t e i n 1983; Hoffman 1980). The c o n d u c t i o n , r e f r a c t o r i n e s s and a u t o m a t i c i t y of the h e a r t a r e a l s o a f f e c t e d . T h e r a p e u t i c use of d i g o x i n i s based on changes i n c o n t r a c t i l i t y and c o n d u c t i o n . 1.1.1.1 C o n t r a c t i l i t y The c l i n i c a l and hemodynamic changes obse r v e d i n the t r e a t m e n t of c o n g e s t i v e h e a r t f a i l u r e a r e the r e s u l t of a 5 d i r e c t p o s i t i v e i n o t r o p i c e f f e c t of d i g o x i n on t h e myocardium ( B l a u s t e i n 1985; Do h e r t y 1975; H a u s t e i n 1983). T h i s i n c r e a s e i n c o n t r a c t i l i t y l e a d s t o an i n c r e a s e d c a r d i a c o u t p u t , d e c r e a s e d h e a r t s i z e , d e c r e a s e d venous p r e s s u r e and b l o o d volume, d i u r e s i s and r e l i e f of edema i n p a t i e n t s w i t h h e a r t f a i l u r e ( H a u s t e i n 1983; Hoffman 1980). 1.1.1.2 C o n d u c t i o n The d i r e c t e f f e c t s of d i g o x i n on e l e c t r i c a l a c t i v i t y a r e s t r o n g l y dependent on the p h y s i o l o g i c a l c o n d i t i o n of the h e a r t . A l s o , d i f f e r e n c e s i n e l e c t r i c a l r esponse t o d i g o x i n were found between muscle f i b e r t y p e s ( H a u s t e i n 1983; Hoffman 1980). G e n e r a l l y , h i g h d i g o x i n l e v e l s a r e thought t o d e p r e s s the c o n d u c t i o n v e l o c i t y and t o d e l a y or b l o c k t h e a t r i o v e n t r i c u l a r n o d a l c o n d u c t i o n (Endou 1982). Low doses of d i g o x i n i n d i r e c t l y d e c r e a s e the r a t e a t which a t r i a l i m p u l s e s can be t r a n s m i t t e d t o the v e n t r i c l e s and p r o l o n g the r e f r a c t o r y p e r i o d of the a t r i o v e n t r i c u l a r node (Hoffman 1980) . 1.1.2 Mechanism of A c t i o n T h e r a p e u t i c c o n c e n t r a t i o n s of d i g i t a l i s g l y c o s i d e s s e l e c t i v e l y i n h i b i t the plasma membrane sodium pump ( t h e Na +,K +-ATPase) i n a v a r i e t y of c e l l t y p e s i n c l u d i n g c a r d i a c and v a s c u l a r smooth muscle c e l l s , neurons and r e n a l t u b u l e c e l l s . T h i s i n h i b i t i o n of outward sodium t r a n s p o r t l e a d s t o an i n c r e a s e i n the sodium c o n c e n t r a t i o n i n s i d e the c e l l . 6 These c e l l s a l s o have s o d i u m - c a l c i u m exchanger mechanisms which d e c r e a s e t h e i n t r a c e l l u l a r sodium by en h a n c i n g the c a l c i u m i n f l u x or r e d u c i n g the c a l c i u m e f f l u x o r b o t h . The r e s u l t a n t r i s e i n i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n r e s u l t s i n more c a l c i u m b e i n g a v a i l a b l e t o the c o n t r a c t i l e e l ements and t h e r e f o r e t o a p o s i t i v e i n o t r o p i c e f f e c t (Akera 1985; B l a u s t e i n 1985; Hoffman 1980; Repke 1984; Smith 1984a). These e f f e c t s can account f o r the c a r d i o t o n i c a c t i v i t y of d i g o x i n as w e l l as e f f e c t s on the k i d n e y s , nervous t i s s u e and v a s c u l a r smooth muscle. 1.1.3 T o x i c i t y I t has been noted t h a t 15 t o 20% of h o s p i t a l i z e d p a t i e n t s r e c e i v i n g d i g o x i n e x h i b i t symptoms of t o x i c i t y , and t h a t t h e m o r t a l i t y r a t e of such p a t i e n t s i s from 7 t o 50% (Doherty 1975). At d i g o x i n c o n c e n t r a t i o n s g r e a t e r than 3 ng/mL plasma (Aronson 1983), t h e r e i s a h i g h i n c i d e n c e of t o x i c i t y . However, p h y s i o l o g i c a l c o n d i t i o n s and i n t e r -p a t i e n t v a r i a t i o n i n response may l e a d t o t o x i c i t y below t h i s v a l u e . I n an e f f o r t t o a v o i d m a n i f e s t a t i o n s of t o x i c i t y , t he t h e r a p e u t i c c o n c e n t r a t i o n i n plasma i s m a i n t a i n e d between 0.5 and 2.0 ng/mL. Depending on p a t i e n t p a r a m e t e r s , such as r e n a l f u n c t i o n and age, maintenance doses of d i g o x i n a r e u s u a l l y 0.125 mg t o 0.50 mg d a i l y . The c a r d i a c and n o n - c a r d i a c m a n i f e s t a t i o n s of d i g o x i n t o x i c i t y have been r e c e n t l y r e v i e w e d (Antman 1985; Aronson 1983; H a u s t e i n 1983). D i s o r d e r s of the g a s t r o i n t e s t i n a l 7 t r a c t due t o c e n t r a l nervous system e f f e c t s (nausea, v o m i t i n g , a n o r e x i a and d i a r r h e a ) and c a r d i a c rhythm d i s o r d e r s a r e u s u a l l y the e a r l i e s t s i d e e f f e c t s o b s e r v e d (Antman 1985; Aronson 1983; H a u s t e i n 1983; Mason 1981). The c e n t r a l nervous system e f f e c t s of d i g i t a l i s a r e numerous and i n c l u d e headache, weakness, d r o w s i n e s s , d e p r e s s i o n , d i z z i n e s s , v e r t i g o , a t a x i a , c o n f u s i o n , h a l l u c i n a t i o n s , p s y c h o s i s , n e u r a l g i a , s e i z u r e , s t u p o r and coma (Antman 1985; H a u s t e i n 1983). V i s u a l symptoms such as b l u r r i n g , dimness, f l i c k e r i n g or f l a s h i n g l i g h t s , and c o l o r d i s t u r b a n c e s w i t h y e l l o w v i s i o n a r e most common (Aronson 1983) and may be the e a r l i e s t symptoms of t o x i c i t y ( C l o s s o n 1983). At t h e r a p e u t i c plasma l e v e l s , d i g o x i n may produce a p r o g r e s s i v e d e t e r i o r a t i o n i n a u d i t o r y v e r b a l l e a r n i n g and s h o r t - t e r m memory (Tucker 1983). V a r i o u s a r r h y t h m i a s , b l o c k a g e i n c o n d u c t i o n , and w o r s e n i n g h e a r t f a i l u r e a r e c a r d i a c e v i d e n c e of t o x i c i t y (Antman 1985; Aronson 1983; H a u s t e i n 1983; Mason 1981; Takayanagi 1986). R e c e n t l y , the mechanisms and m a n i f e s t a t i o n s of d i g i t a l i s t o x i c i t y have been r e v i e w e d (Smith 1984a, 1984b, 1984c). The s i g n s and symptoms of d i g i t a l i s t o x i c i t y a r e u s u a l l y n o n - s p e c i f i c , making d i a g n o s i s d i f f i c u l t . D i g o x i n - s p e c i f i c a n t i b o d y fragments have been s u c c e s s f u l l y used f o r the t r e a t m e n t of d i g i t a l i s i n t o x i c a t i o n (Cohen 1982; Friedman 1983; Murphy 1982; Rozkovec 1982; Zucker 1982). Agents t h a t d e c r e a s e a b s o r p t i o n of d i g o x i n from t h e g a s t r o i n t e s t i n a l t r a c t such 8 as c h o l e s t y r a m i n e and a c t i v a t e d c h a r c o a l , have a l s o been used i n t r e a t i n g d i g o x i n t o x i c i t y (Hoffman 1980). T i s s u e d i g o x i n c o n c e n t r a t i o n s may not be u s e f u l i n t h e d e t e r m i n a t i o n of d i g o x i n t o x i c i t y . Postmortem d i g o x i n l e v e l s i n c a s e s of d i g i t a l i s t o x i c i t y ranged from 43 t o 283 ng/mL w h i l e t h o s e from c a s e s where t o x i c i t y was not the cause of d e a t h were from 0 t o 463 ng/mL ( A d e r j a n 1979; Andersson 1975; Margot 1983). U s i n g immunoassay methods f o r d i g o x i n a n a l y s i s , postmortem b l o o d samples appear t o have e l e v a t e d d i g o x i n l e v e l s when compared t o antemortem d a t a ( A d e r j a n 1979; H a s t r e i t e r 1983; Kim 1975; Margot 1983; McKercher 1986; V o r p a h l 1978). S i m i l a r r e s u l t s have been found u s i n g a r u b i d i u m uptake assay (Andersson 1975). The i n t e r p r e t a t i o n of postmortem d i g o x i n b l o o d c o n c e n t r a t i o n s must t a k e i n t o c o n s i d e r a t i o n the time of b l o o d c o l l e c t i o n a f t e r d e a t h and the s a m p l i n g s i t e (Margot 1983) s i n c e t h e s e f a c t o r s a f f e c t the c o n c e n t r a t i o n s o b s e r v e d . 1.1.4 P h a r m a c o k i n e t i c s Large i n t e r s u b j e c t v a r i a t i o n i n the p h a r m a c o k i n e t i c b e h a v i o r of d i g o x i n (Aronson 1983; C l a r k 1974; G a u l t 1979; Kramer 1974; L u c h i 1968; M o v s e l l i 1977; R i e t b r o c k 1981, 1985) and i n t e r f e r e n c e w i t h the most common d i g o x i n assay method by o t h e r compounds ( B u t l e r 1978, 1979; D i P i r o 1980; Holtzman 1974; Kramer 1978; Ku b a s i k 1974a, 1974b; L i c h e y 1979; Lindenbaum 1975; M a l i n i 1982; M u l l e r 1978; O s t e r l o h 1982; Pudek 1983a, 1983b; R a v e l 1975; Scherrmann 1980; 9 S c h r e i b e r 1981a, 1981b, 1981c; S i l b e r 1979; Smith 1973) make i t d i f f i c u l t t o g e n e r a l i z e about d i g o x i n p h a r m a c o k i n e t i c s . Due t o t h e i n t r a p a t i e n t and i n t e r p a t i e n t v a r i a t i o n i n t h e p h a r m a c o k i n e t i c s as w e l l as problems w i t h the immunoassays of d i g o x i n , nomograms f o r d e t e r m i n a t i o n of a dosage regimen have not been p o p u l a r . R e c e n t l y , however, i t was s u g g ested t h a t a nomogram ( B j o r n s s o n 1986) may be u s e f u l f o r a d j u s t i n g the dosage of d i g o x i n and o t h e r c a r d i o v a s c u l a r drugs t h a t are p a r t i a l l y e l i m i n a t e d by the k i d n e y s i n p a t i e n t s w i t h reduced r e n a l f u n c t i o n . 1.1.4.1 A b s o r p t i o n O r a l l y a d m i n i s t e r e d d i g o x i n i s a d e q u a t e l y a b s o r b e d from the g a s t r o i n t e s t i n a l t r a c t , even i n v a s c u l a r c o n g e s t i o n of the g a s t r i c mucosa, h y p o x i a and d i a r r h e a t h a t may e x i s t i n the c a r d i a c p a t i e n t ( S m i t h 1984a). W h i l e i t has been su g g e s t e d ( G a u l t 1977; Lindenbaum 1981; Loo 1977; Magnusson 1982a, 1982b; Sonobe 1980) t h a t d i g o x i n can be degraded by a c i d and/or enzymes t o d i g o x i g e n i n and i t s mono- and b i s -d i g i t o x o s i d e s and t h a t t h i s may o c c u r i n the g a s t r o i n t e s t i n a l t r a c t , the l e v e l s of t h e d e g r a d a t i o n p r o d u c t s i n plasma cannot be d i f f e r e n t i a t e d from t h o s e m e t a b o l i c a l l y formed. Thus, f i r m c o n c l u s i o n s r e g a r d i n g the in vivo d e g r a d a t i o n have not been e s t a b l i s h e d . I t has been found t h a t a lowered g a s t r i c pH l e a d s t o - lower plasma d i g o x i n l e v e l s and t h i s has been assumed t o be due t o the s e n s i t i v i t y of t h e s e compounds t o a c i d ( H o s s i e 1977; 10 S t e r n s o n 1978). Some i n v e s t i g a t o r s (Brown 1978; G r e e n b l a t t 1976) r e g a r d the l o w e r i n g of plasma d i g o x i n l e v e l s w i t h h y p e r a c i d i t y t o be s u f f i c i e n t r e a son t o m o n i t o r t h e s e p a t i e n t s more c l o s e l y . I t has a l s o been s u g g e s t e d t h a t the i n t e s t i n a l f l o r a may be r e s p o n s i b l e f o r t h e c o n v e r s i o n of d i g o x i n t o d i h y d r o d i g o x i n (Dobkin 1983; Lindenbaum 1981). The e f f e c t of food i n g e s t i o n p r i o r t o drug t h e r a p y has a l s o been r e p o r t e d ( G r e e n b l a t t 1976; Wagner 1974) t o r e s u l t i n l a r g e d i s c r e p a n c i e s i n d i g o x i n plasma l e v e l s . The b i o a v a i l a b i l i t y of d i g o x i n i n a c a p s u l e form ( M a l i n i 1983) was about 20% h i g h e r than t h a t of t a b l e t s , i n d i c a t i n g t h a t the dosage f o r m u l a t i o n of d i g o x i n may a l s o a f f e c t a b s o r p t i o n . S o f t g e l a t i n c a p s u l e s of d i g o x i n as a s o l u t i o n gave 90 t o 100% a b s o r p t i o n (Johnson 1986). Johnson et at. (1986) a l s o n o t i c e d no s i g n i f i c a n t v a r i a b i l i t y i n d i g o x i n t r o u g h c o n c e n t r a t i o n s nor i n u r i n a r y e x c r e t i o n between t a b l e t and c a p s u l e f o r m u l a t i o n s . They f u r t h e r n o t e d an i n v e r s e r a t i o of me t a b o l i s m w i t h b i o a v a i l a b i l i t y . T h i s was c o n s i d e r e d t o be due t o c o l o n i c b a c t e r i a , a l t h o u g h t h i s a s p e c t was not s t u d i e d i n d e t a i l . Plasma l e v e l s of d i g o x i n have a l s o been found t o v a r y when o t h e r drugs and s u b s t a n c e s a r e a d m i n i s t e r e d ( H a u s t e i n 1983; Manninen 1981). A r t e r i a l b l o o d pH i n the range 7.25 t o 7.50 was found t o be d i r e c t l y c o r r e l a t e d w i t h d i g o x i n plasma l e v e l s a t stea d y s t a t e (Catenazzo 1985). These d a t a ( C a t e n a z z o 1985) may e x p l a i n the s t r o n g l y reduced a c t i v i t y of d i g o x i n i n p a t i e n t s w i t h r e s p i r a t o r y a c i d o s i s . 11 P e r m e a t i o n of d i g o x i n t h r o u g h mouse and human s k i n ( C a i r n c r o s s 1985) s t u d i e d u s i n g an in vitro system i n d i c a t e s t h a t i t may be p o s s i b l e t o d e v e l o p a c l i n i c a l l y e f f i c i e n t t r a n s d e r m a l t h e r a p e u t i c system. 1.1.4.2 D i s t r i b u t i o n A f t e r o r a l a d m i n i s t r a t i o n , the serum d i g o x i n c o n c e n t r a t i o n reaches a peak between 45 minutes and 3 hours (Doherty 1975; Hoffman 1980). The d i s t r i b u t i o n of d i g o x i n t o c e n t r a l nervous t i s s u e i s thought t o produce the n e u r o l o g i c a l s i g n s of t o x i c i t y such as d r o w s i n e s s , d i s o r i e n t a t i o n , h a l l u c i n a t i o n s and v i s u a l d i s t u r b a n c e s (Aronson 1980; H a u s t e i n 1983; R i e t b r o c k 1981). I t has a l s o been s u g g e s t e d t h a t the r e s i s t a n c e t o the a r r h y t h m o g e n i c and v a s o c o n s t r i c t o r e f f e c t s of d i g o x i n may i n v o l v e the hypothalamus (Otsuka 1982). D i g o x i n i s a l s o d i s t r i b u t e d t o o t h e r t i s s u e s : about 65% i s found i n the l i v e r , 4% i n the h e a r t , 3% i n the b r a i n and 1.5% i n the k i d n e y s (Aronson 1980). Serum p r o t e i n b i n d i n g of d i g o x i n , p r i n c i p a l l y t o a l b u m i n , v a r i e s from 10 t o 40% (Kramer 1974; M o v s e l l i 1977; Smith 1984a) and appears t o be u n a f f e c t e d by c o n c e n t r a t i o n (Aronson 1980). D i g o x i n i s a l s o bound t o ^ - l i p o p r o t e i n s (Brock 1976) but w i t h a lower a f f i n i t y than d i g i t o x i n . In comparison t o a l b u m i n , the / 3 - l i p o p r o t e i n c o n c e n t r a t i o n i n b l o o d i s so s m a l l t h a t the c o n c e n t r a t i o n of l i p o p r o t e i n - b o u n d d i g o x i n t o the t o t a l bound i s m i n i m a l . Thus, i t has been s u g g e s t e d , the f r a c t i o n 12 of unbound d i g o x i n i n serum can be p r e d i c t e d s o l e l y from the a l b u m i n c o n c e n t r a t i o n and pH (Brock 1976). A model w i t h a t l e a s t two k i n e t i c a l l y d i s t i n c t compartments can be used t o d e s c r i b e d i g o x i n ' s d i s p o s i t i o n (Keys 1980; Nyberg 1974; R i e t b r o c k 1981) where the c e n t r a l compartment r e p r e s e n t s the b l o o d and w e l l - p e r f u s e d body f l u i d s and t i s s u e and the p e r i p h e r a l compartment r e p r e s e n t s the s l o w l y - p e r f u s e d body space. A r e c e n t s t u d y s u g g e s t s t h a t the d a t a best f i t a n o n - l i n e a r , two-compartment model w i t h a deep t i s s u e compartment (Kramer 1979). D i g o x i n may a l s o undergo s i g n i f i c a n t b i l i a r y e x c r e t i o n ( R e i s s e l l 1982) and e n t e r o h e p a t i c c i r c u l a t i o n or i n t e s t i n a l s e c r e t i o n ( R e i s s e l l 1982; S c h a f e r 1985). T h i s a r e a i s s t i l l d i s p u t e d i n the l i t e r a t u r e . D u r i n g the d i s t r i b u t i o n phase of d i g o x i n , r a p i d removal from the c e n t r a l compartment i s o b s e r v e d . E x t e n s i v e d i s t r i b u t i o n of d i g o x i n i n t i s s u e i s i n d i c a t e d by a l a r g e s t e a d y s t a t e volume of d i s t r i b u t i o n . U s i n g t r i t i a t e d d i g o x i n , t h e d i s t r i b u t i o n h a l f - l i f e was 60 minutes (Doherty 1975). The r e p o r t e d s t e a d y - s t a t e volume of d i s t r i b u t i o n of d i g o x i n was e x t r e m e l y v a r i a b l e : between 5.1 and 8.1 L/kg i n h e a l t h y s u b j e c t s , 5.0 L/kg i n p a t i e n t s w i t h c a r d i a c f a i l u r e and between 3.3 and 4.4 L/kg i n p a t i e n t s w i t h r e n a l f a i l u r e ( R i e t b r o c k 1981). M o r p h o l o g i c a l changes i n t h e h e a r t due t o c a r d i a c d i s e a s e may a l t e r d i g o x i n b i n d i n g and produce a change i n the volume of d i s t r i b u t i o n (Keys 1980). The volume of d i s t r i b u t i o n appears t o v a r y w i t h r e n a l f u n c t i o n 13 but l a r g e v a r i a t i o n e x i s t s i n i n d i v i d u a l s w i t h comparable r e n a l f u n c t i o n (Keys 1980). A d e c r e a s e i n the number of d i g i t a l i s r e c e p t o r s has been o b s e r v e d i n r e n a l f a i l u r e p a t i e n t s ( M a l i n i 1985), which may e x p l a i n the d e c r e a s e i n a c t i v i t y of d i g i t a l i s g l y c o s i d e s i n c h r o n i c r e n a l f a i l u r e . A summary of the p h a r m a c o k i n e t i c d a t a f o r d i g o x i n i s shown i n T a b l e I . Table I. Summary Of Digoxin Pharmacokinetics P h a r m a c o k i n e t i c Parameter A v a i l a b i l i t y ( O r a l ) 70 + 13% U r i n a r y E x c r e t i o n 60 + 11% Bound i n P l a s m a 2 25 + 5% Volume of D i s t r i b u t i o n ( L / K g ) 3 ' 4 7 + 30% H a l f - l i f e ( H o u r s ) 4 3 9 + 1 3 Time t o Maximum C o n c e n t r a t i o n (Hours) 2 t o 3 Time t o Maximum E f f e c t (Hours) 4 t o 6 1 From Gilman et al. ( 1988). 2 D e c r e a s e s w i t h u remia. 3 D e c r e a s e s w i t h h y p o t h y r o i d i s m and i n c r e a s e s w i t h hyper-t h y r o i d i s m . 4 D e c r e a s e s w i t h h y p e r t h y r o i d i s m and i n c r e a s e s w i t h u r e m i a , c o n g e s t i v e h e a r t f a i l u r e and h y p o t h y r o d i s m . 1.1.4.3 M e t a b o l i s m And E l i m i n a t i o n M e t a b o l i s m of d i g o x i n o c c u r s m a i n l y i n the l i v e r ( A b e l 1965) and the drug i s e x c r e t e d l a r g e l y by the k i d n e y s v i a g l o m e r u l a r f i l t r a t i o n and t u b u l a r s e c r e t i o n ( S t e i n e s s 1982). A l t h o u g h many r e p o r t s have e v a l u a t e d t h e m e t a b o l i c t u r n - o v e r of d i g o x i n , t h i s a r e a i s s t i l l one of c o n s i d e r a b l e d i s p u t e . Some i n v e s t i g a t o r s have s u g g e s t e d t h a t o n l y a s m a l l 14 p r o p o r t i o n of d i g o x i n was m e t a b o l i z e d ( A s h l e y 1958; Beerman 1972; Doherty 1970; G i b s o n 1980; Marcus 1966), w h i l e o t h e r s have shown t h a t 57 t o 60% ( C l a r k 1974; L u c h i 1968) was e x c r e t e d as a m e t a b o l i c p r o d u c t , l a r g e l y d i h y d r o d i g o x i n . A r e c e n t r e p o r t (Aronson 1980) n o t e d a l a r g e i n t e r p a t i e n t v a r i a t i o n i n m e t a b o l i t e e x c r e t i o n such t h a t from 20 t o 55% of t h e drug was m e t a b o l i z e d i n a few p a t i e n t s , but the m a j o r i t y e x c r e t e d the drug 80% unchanged. U s i n g o r a l l y a d m i n i s t e r e d t r i t i a t e d d i g o x i n , G a u l t et al. ( 1979) r e p o r t e d t h a t , w i t h normal r e n a l f u n c t i o n , d i g o x i n was e x c r e t e d 54.5% unchanged, 2.0% as d i g o x i g e n i n b i s d i g i t o x o s i d e , 0.8% as d i g o x i g e n i n m o n o d i g i t o x o s i d e , 0.25% as d i g o x i g e n i n and 0.3% as d i h y d r o d i g o x i n . S u b j e c t s w i t h minimum r e n a l f u n c t i o n ( G a u l t 1979; G i b s o n 1980) e x c r e t e d o n l y t r a c e l e v e l s of m e t a b o l i t e s . C l a r k and Kalman (1974) s p e c u l a t e d t h a t the v a r i a b i l i t y i n d i g o x i n m e t a b o l i s m and the l a c k of s p e c i f i c i t y of the RIA f o r d i g o x i n would i n c r e a s e the c o m p l e x i t y of e v a l u a t i n g RIA r e s u l t s f o r p a t i e n t samples. The m e t a b o l i s m of d i g o x i n by c l e a v a g e of the d i g i t o x o s e r e s i d u e s produces an i n c r e a s e i n l i p i d s o l u b i l i t y w h i l e c o n j u g a t i o n r e a c t i o n s would cause the l i p i d s o l u b i l i t y t o d e c r e a s e . Lage and S p r a t t (1966) r e p o r t e d t h a t s t e p w i s e c l e a v a g e of the d i g i t o x o s e r e s i d u e s p r o g r e s s i v e l y d e c r e a s e d the c a r d i o a c t i v i t y . R e d u c t i o n of the l a c t o n e r i n g a l s o s u b s t a n t i a l l y d e c r e a s e d the a c t i v i t y (Keys 1980) w i t h the c a r d i o a c t i v i t y of d i h y d r o d i g o x i n b e i n g o n l y 1/20 t h a t of d i g o x i n ( R i e t b r o c k 1981). E p i m e r i z a t i o n of d i g o x i g e n i n and 15 c o n j u g a t i o n r e a c t i o n s w i t h s u l f a t e and g l u c u r o n i d e r e s u l t e d i n a l o s s of almost a l l a c t i v i t y (Keys 1980). U s i n g a d u l t male mice, the potency r a t i o s of d i g o x i g e n i n b i s d i g i t o x o s i d e , d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n and 3 - e p i d i g o x i g e n i n compared t o d i g o x i n were found t o be 1/1.33, 1/1.47, 1/4.84 and 1/6.45 r e s p e c t i v e l y . With r e s p e c t t o d i g o x i n , the potency r a t i o f o r d i h y d r o d i g o x i g e n i n ( l e s s than 1/46) (Brown 1962) has a l s o been r e p o r t e d . 1.1.5 Chemi s t r y D i g o x i n , a n a t u r a l l y o c c u r r i n g c a r d i o a c t i v e s t e r o i d , has t h r e e s t r u c t u r a l components; a s e r i e s of sugar r e s i d u e s , a s t e r o i d n u c l e u s and a five-membered u n s a t u r a t e d l a c t o n e r i n g . Removal of the sugar r e s i d u e s l e a v e s t h e g e n i n or a g l y c o n e . T h i s g l y c o s i d e i s found i n the l e a v e s of the f o x g l o v e s p e c i e s Digitalis Ianata ( H a u s t e i n 1983) which c o n t a i n a number of o t h e r d i g i t a l i s g l y c o s i d e s ( d i g i t o x i n , g i t o x i n , d i g i n a t i n and g i t a l o x i n ) . The a g l y c o n e of d i g i t a l i s g l y c o s i d e s c o n s i s t s of a c y c l o p e n t a n o -p e r hydrophenanthrene n u c l e u s w i t h a five-membered u n s a t u r a t e d l a c t o n e r i n g a t t a c h e d i n the 17-/3 p o s i t i o n . M e t h y l groups a r e a t t a c h e d a t carbons 10 and 13 (0 p o s i t i o n ) , a h y d r o x y l group a t carb o n 14 (/3 p o s i t i o n ) and the five-membered l a c t o n e r i n g (0 p o s i t i o n ) i s a t t a c h e d a t carbo n 17 i n a l l d i g i t a l i s a g l y c o n e s ( H a u s t e i n 1983). In a d d i t i o n , the a g l y c o n e f o r d i g o x i n , d i g o x i g e n i n , has a h y d r o x y l group a t c a r b o n 12. 16 N a t u r a l l y o c c u r r i n g d i g i t a l i s g l y c o s i d e s have a sugar component a t t a c h e d a t p o s i t i o n 3 v i a a g l y c o s i d i c l i n k a g e (Myerson 1967). These s u g a r s a r e u s u a l l y six-membered r i n g s i n t he p r e f e r r e d c h a i r c o n f o r m a t i o n and a r e l i n k e d t o g e t h e r by 0 - g l y c o s i d i c 1 , 4 - l i n k a g e s . F o r d i g o x i n , a t h r e e sugar c h a i n of d i g i t o x o s e r e s i d u e s i s j o i n e d t o the a g l y c o n e a t p o s i t i o n 3. The c h e m i c a l s t r u c t u r e of d i g o x i n i s shown i n F i g u r e 1. F i g u r e 1. The C h e m i c a l S t r u c t u r e of D i g o x i n 1.1.6 S t r u c t u r e A c t i v i t y R e l a t i o n s h i p s D e s p i t e t h e l e n g t h of ti m e the c a r d i a c g l y c o s i d e s have been i n use, problems due t o t h e low t h e r a p e u t i c index and i n t e r - and i n t r a - p a t i e n t v a r i a b i l i t y a r e s t i l l p r e s e n t . Numerous s t u d i e s have examined the c h e m i s t r y and s t r u c t u r e -a c t i v i t y r e l a t i o n s h i p s of t h e s e g l y c o s i d e s . For OH 17 c a r d i o a c t i v i t y t o be p r e s e n t , the g l y c o s i d e s must have the f o l l o w i n g ( G u n t e r t 1981; H a u s t e i n 1983): i ) a c i s - c i s j u n c t i o n of the C and D r i n g s of the g e n i n m o i e t y , i i ) an u n s a t u r a t e d l a c t o n e moiety a t t a c h e d a t c a r b o n 17 i n the j3 c o n f i g u r a t i o n , i i i ) a sugar component a t t a c h e d t o the 3-/3-hydroxyl on the g e n i n v i a a g l y c o s i d i c l i n k a g e . The C and D r i n g s of c a r d i a c g l y c o s i d e s a r e u s u a l l y f u s e d c i s w i t h the h y d r o x y l a t p o s i t i o n 14 i n the 0 p o s i t i o n . Comparison of 14-a a g l y c o n e s ( G u n t e r t 1981) w i t h the c o r r e s p o n d i n g 14-0 compounds i n d i c a t e t h a t a c t i v i t y i s l o s t when r i n g s C and D are f u s e d i n the t r a n s p o s i t i o n . R i n g s C and D a r e thought t o undergo Van der Waals and/or h y d r o p h o b i c i n t e r a c t i o n s w i t h the proposed r e c e p t o r (Thomas 1980) and c h a n g i n g the c o n f i g u r a t i o n l e a d s t o a d e c r e a s e i n b i n d i n g and t h e r e f o r e a d e c r e a s e i n a c t i v i t y . An u n s a t u r a t e d l a c t o n e r i n g composed of f o u r or f i v e c a r b o n members a t t a c h e d t o the g e n i n a t carbon 17 i n the 0 c o n f i g u r a t i o n i s almost e s s e n t i a l f o r c a r d i a c a c t i v i t y . A change i n c o n f i g u r a t i o n of t h i s s i d e - c h a i n y i e l d s i n a c t i v e compounds ( G u n t e r t 1981; S a i t o 1970; Tamm 1963). R e p l a c i n g the l a c t o n e r i n g w i t h open-chain analogues ( F u l l e r t o n 1976; G e l b a r t 1978; Smith 1982; Thomas 1974a, 1974b) i n d i c a t e d t h a t t h e r e a r e both s t e r i c and e l e c t r o n i c r e q u i r e m e n t s f o r t h e carbon 17 s i d e - c h a i n . R e c e n t l y , G r i f f i n et al. (1986) s u g g e s t e d t h a t t h e r e was an a d d i t i o n a l r e c e p t o r b i n d i n g s i t e f o r d i g i t a l i s g l y c o s i d e s s i n c e 16-/3 formate and a c e t a t e 18 e s t e r s showed i n c r e a s e d a c t i v i t y w i t h r e s p e c t t o the p a r e n t compound. P h a r m a c o l o g i c a l a c t i v i t y r e s i d e s i n the a g l y c o n e but the s u g a r s a t t a c h e d a t p o s i t i o n 3 mod i f y the water and l i p i d s o l u b i l i t y and potency of the r e s u l t i n g g l y c o s i d e (Tamm 1963). P r o g r e s s i v e removal of the sugar m o i e t i e s produced a de c r e a s e i n a c t i v i t y (Keys 1980). Removal of the l a s t sugar g r e a t l y reduced a c t i v i t y (Keys 1980; L u c h i 1965; Repke 1963; Thomas 1980). M e t a b o l i s m b e f o r e and a f t e r a b s o r p t i o n from the g a s t r o i n t e s t i n a l t r a c t can l e a d t o the a g l y c o n e w i t h a f r e e h y d r o x y l group a t carbon 3 ( G a u l t 1977; Repke 1963). F o r m a t i o n of the 3-dehydro-genin f o l l o w e d by ' r e -h y d r o g e n a t i o n ' may y i e l d e i t h e r the 3 0 - h y d r o x y l - g e n i n or the 3 a - h y d r o x y l - g e n i n . T h i s e p i m e r i z a t i o n a t carbon 3 i s p r e v e n t e d when a minimum of one sugar r e s i d u e i s p r e s e n t ( L u c h i 1965; Repke 1963). P a r t of the d e c r e a s e i n a c t i v i t y o b s e r v e d when the l a s t sugar r e s i d u e i s removed from the ge n i n may be due t o e p i m e r i z a t i o n t o the 3 a - h y d r o x y l form which i s almost i n a c t i v e ( G u n t e r t 1981). B i n d i n g s t u d i e s w i t h Na +,K +-ATPase (Akera 1981; T a k i u r a 1974; W a l l i c k 1974; Yoda 1974) suggest t h a t b o t h the g e n i n and the sugar component c o n t r i b u t e t o b i n d i n g . C o n f l i c t i n g r e s u l t s have been r e p o r t e d c o n c e r n i n g the r e q u i r e m e n t s f o r the oxygen f u n c t i o n a t carbon 3. U s i n g an i s o l a t e d f r o g ' s h e a r t p r e p a r a t i o n , S a i t o et al. ( 1 970) found t h a t t h e oxygen was not r e q u i r e d f o r a c t i v i t y . I n h i b i t i o n s t u d i e s w i t h Na +,K +-19 ATPase ( W i t t y 1975), however, show t h a t the oxygen a t p o s i t i o n 3 i s r e q u i r e d f o r f u l l a c t i v i t y of the a g l y c o n e . Replacement of the d i g i t o x o s e r e s i d u e s w i t h v a r i o u s branched groups ( S i e b e n e i c k 1978) gave compounds w i t h lower Na +,K +-ATPase i n h i b i t o r y a c t i v i t y . Aminosugar d e r i v a t i v e s were a p p r o x i m a t e l y t e n ti m e s more p o t e n t i n h i b i t o r s than the p a r e n t g l y c o s i d e ( C a l d w e l l 1978). N a t u r a l l y o c c u r r i n g a m i n o - d e o x y g l y c o - c a r d e n o l i d e s (Choay 1978) have a l s o been i d e n t i f i e d . 1.2 D i g o x i n Therapy D i g i t a l i s g l y c o s i d e s a r e i n d i c a t e d f o r the t r e a t m e n t of a r r h y t h m i a s and c o n g e s t i v e h e a r t f a i l u r e . These compounds were c o n s i d e r e d t h e best a g e n t s f o r c o n t r o l of the v e n t r i c u l a r r a t e i n a t r i a l f i b r i l l a t i o n or f l u t t e r (Doherty 1985). The c a l c i u m a n t a g o n i s t v e r a p a m i l was a l s o e f f e c t i v e i n s l o w i n g the v e n t r i c u l a r r a t e i n a t r i a l f i b r i l l a t i o n ( K l e i n 1986). P r e s e n t l y , b o t h d i g i t a l i s g l y c o s i d e s and v e r a p a m i l a r e drugs of c h o i c e i n the t r e a t m e n t of a t r i a l f i b r i l l a t i o n . F o r c o n g e s t i v e h e a r t f a i l u r e , the d i g i t a l i s g l y c o s i d e s a r e u s e f u l i n e i t h e r r i g h t , l e f t or combined v e n t r i c u l a r f a i l u r e (Doherty 1985). R e p o r t s comparing the c l i n i c a l response of d i g o x i n t h e r a p y w i t h i t s a s s o c i a t e d plasma l e v e l s , have i n d i c a t e d t h a t 30% of the p a t i e n t s s t u d i e d d i d not improve t h e i r c a r d i a c e f f i c i e n c y i n a p r e d i c t a b l e manner (Kramer 1979). Whether t h e l a t t e r group was unimproved due t o ina d e q u a t e 20 t h e r a p y o r due t o an i n a b i l i t y t o t i t r a t e the drug a d m i n i s t r a t i o n was not c l e a r . A l s o , problems w i t h the d i g o x i n plasma a s s a y have been suggested i n t h i s s t u d y . There i s g r e a t c o n t r o v e r s y over the p r o l o n g e d t r e a t m e n t of c o n g e s t i v e h e a r t f a i l u r e i n s p e c i f - i c p a t i e n t groups w i t h d i g i t a l i s g l y c o s i d e s ( A p p l e f e l d 1986; Doherty 1985; Yusuf 1986). Long-term t h e r a p y w i t h d i g o x i n has been r e p o r t e d ( S p e c t o r 1979) t o b e n e f i t o n l y a s m a l l number of p a t i e n t s , but a t p r e s e n t i t i s not c o n s i d e r e d good t h e r a p y t o withdraw the drug once adequate c o n t r o l has been a c h i e v e d . The e f f e c t s of d i g o x i n t h e r a p e u t i c w i t h d r a w a l a r e u n d e r s c o r e d by a s t u d y (Bowman 1983) i n which p a t i e n t s who were a t reduced r i s k had t h e i r d i g o x i n t h e r a p y d i s c o n t i n u e d . W h i l e o n l y 14% of the p a t i e n t s s t u d i e d d e t e r i o r a t e d , the a u t h o r s s u g g e s t e d t h a t o n l y those p a t i e n t s who had l i t t l e i n d i c a t i o n f o r d i g o x i n t h e r a p y s h o u l d be c o n s i d e r e d f o r d i s c o n t i n u a t i o n of t h e r a p y . A more r e c e n t r e v i e w of the l i t e r a t u r e i n t h i s a r e a ( A p p l e f e l d 1986) i n d i c a t e d t h a t c a u t i o n s h o u l d be e x e r c i s e d w i t h s t o p p i n g d i g o x i n t h e r a p y i n tho s e p a t i e n t s w i t h moderate h e a r t f a i l u r e and t h a t c l o s e m o n i t o r i n g of c a r d i a c f u n c t i o n i s e s s e n t i a l . 1.3 D i g o x i n - L i k e Immunoreactive S u b s t a n c e ( s ) Endogenous f a c t o r s , d i s t i n c t from a l d o s t e r o n e , t h a t p l a y a r o l e i n sodium e x c r e t i o n and e x t r a c e l l u l a r f l u i d r e g u l a t i o n , have been r e p o r t e d ( W i l k i n s 1985). These f a c t o r s a r e s e c r e t e d i n response t o h y p e r v o l e m i a , c i r c u l a t e 21 i n plasma and s t i m u l a t e n a t r i u r e s i s i n o r d e r t o r e s t o r e sodium and water b a l a n c e . R e c e n t l y , an a t r i a l n a t r i u r e t i c p e p t i d e (ANP), which has p o t e n t n a t r i u r e t i c a c t i v i t y , has been found i n the mammalian a t r i a (Lang 1985; M i l l s 1984; W i l k i n s 1985). A second endogenous f a c t o r t h a t i n h i b i t e d t h e Na +,K +-ATPase enzyme t o b l o c k sodium t r a n s p o r t i n the r e n a l t u b u l e s (De Wardener 1977, 1982a; W i l k i n s 1985) a l s o a f f e c t e d the sodium and water b a l a n c e . S i n c e b o t h d i g o x i n and t h i s second f a c t o r i n h i b i t the a c t i v i t y of Na +,K +-ATPase and r e a c t w i t h d i g o x i n a n t i b o d i e s , the endogenous d i g i t a l i s -l i k e f a c t o r was termed e n d o x i n (Diamandis 1985; S c h r e i b e r 1982). Some r e s e a r c h e r s (De Wardener 1982a; Diamandis 1985; Hnatowich 1984; W i l k i n s 1985) have c a l l e d t h i s f a c t o r d i g o x i n - l i k e immunoreactive s u b s t a n c e ( s ) or DLIS. The p r o p e r t i e s of ANP and DLIS have r e c e n t l y been compared ( W i l k i n s 1985). The s t r u c t u r e of DLIS i s unknown but thought t o be l e s s than 500 Da i n m o l e c u l a r mass. DLIS i n h i b i t s Na +,K +-ATPase (ANP does n o t ) , causes v a s o c o n s t r i c t i o n (ANP causes v a s o d i l a t i o n ) , i s e x c r e t e d i n the u r i n e and may be produced i n the hypothalamus (ANP i s produced i n c a r d i a c a t r i a ) . DLIS has been found i n t i s s u e s and b i o l o g i c a l f l u i d s of normal a d u l t s u b j e c t s who never r e c e i v e d d i g o x i n ( B a l z a n 1984; C l e r i c o 1985; Diamandis 1985; Hamlyn 1982; K l i n g m u l l e r 1982; V a l d e s 1983a; Vinge 1988). T h i s s u b s t a n c e has been found i n u m b i l i c a l c o r d b l o o d and p l a c e n t a l homogenates (Besch 1976; Diamandis 1985; K e l l y 1981; Ng 1985; Pudek 22 1983a, 1983b; Scherrmann 1986a, 1986b; Y a t s c o f f 1984), n e o n a t a l serum ( C l e r i c o 1985; Heazlewood 1984; Koren 1984; Pudek 1983a, 1983b; V a l d e s 1983b; Y a t s c o f f 1984), s e r a from pregnant women ( B a r b a r a s h 1984; Boink 1977; Graves 1984; Gusdon 1984; L o n g e r i c h 1988; P h e l p s 1988) and a m n i o t i c f l u i d ( D r o s t 1977; V a l d e s 1983b). Go n z a l e z et al. (1987) and Koren et al. (1988) r e p o r t t h a t DLIS l e v e l s i n u m b i l i c a l c o r d b l o o d (both venous and a r t e r i a l b l o o d ) were s i g n i f i c a n t l y g r e a t e r than i n m a t e r n a l venous b l o o d . A l s o , DLIS l e v e l s i n h i g h - r i s k p r e g n a n c i e s were s i g n i f i c a n t l y h i g h e r than i n normal p r e g n a n c i e s (Koren 1988). P h e l p s and co-workers (1988) found DLIS t o appear i n m a t e r n a l serum and l e v e l s i n c r e a s e d w i t h i n c r e a s i n g g e s t a t i o n a l age but t h a t t h e r e was no s i g n i f i c a n t d i f f e r e n c e between DLIS l e v e l s i n p a t i e n t s w i t h and w i t h o u t p r e e c l a m p s i a . A l t h o u g h no c a u s a l r e l a t i o n s h i p between DLIS and p r e e c l a m p s i a has been found, i t may s t i l l have an e t i o l o g i c r o l e i n development of t h i s c o n d i t i o n ( P h e l p s 1988). DLIS has been r e p o r t e d i n b i l e and meconium ( K i e v a l 1988; Pudek 1984) and i n p a t i e n t s w i t h r e n a l impairment ( B o u r g o i g n i e 1972; C r a v e r 1983; D'Arcy 1984; Graves 1983a, 1983b; Kramer 1985b; O l d f i e l d 1985; Y a t s c o f f 1984) and i n h e a l t h y s u b j e c t s who have been s a l t l o a d e d (Kramer 1985a). The serum from p a t i e n t s w i t h h e p a t i c f a i l u r e has a l s o been r e p o r t e d t o c o n t a i n DLIS ( D i P i r o 1980; Greenway 1985; N a n j i 1985, 1986; Yang 1988). I n i t i a l l y , a 2 t o 3 f o l d i n c r e a s e i n DLIS l e v e l s was found i n p a t i e n t s w i t h a l c o h o l i c 23 c i r r h o s i s ( D i P i r o 1980). DLIS l e v e l s up t o 0.8 ng/mL were r e p o r t e d i n p a t i e n t s w i t h i n f e c t i o u s h e p a t i t i s , a c u t e f a t t y l i v e r of pregnancy and m e t a s t a t i c l i v e r d i s e a s e (Greenway 1985; N a n j i 1985). O t h e r s , however, f a i l e d t o f i n d e l e v a t e d DLIS l e v e l s i n p a t i e n t s w i t h a l c o h o l i c and m e t a s t a t i c l i v e r d i s e a s e s ( F r e w i n 1986). Recent r e p o r t s suggest t h a t DLIS, or a f r a c t i o n t h e r e o f , may be the n a t r i u r e t i c hormone and i n v o l v e d i n h y p e r t e n s i o n (Buckalew 1984; C l o i x 1987; De Wardener 1982a, 1982b; Grantham 1984; W i l k i n s 1985) and may a l s o be p r e s e n t i n n o r m o t e n s i v e s u b j e c t s ( C l o i x 1987; Hamlyn 1982). Peak serum DLIS l e v e l s i n p r e - t e r m and term i n f a n t s c o r r e l a t e d w i t h the e x c r e t i o n of sodium (Ebara 1986a), i n d i c a t i n g t h a t DLIS may be among the p a t h o l o g i c a l f a c t o r s i n v o l v e d i n hyponatremia i n p r e - t e r m i n f a n t s . The s o u r c e , s t r u c t u r e and c o m p o s i t i o n of DLIS a r e the s u b j e c t s of a number of i n v e s t i g a t i o n s . The a d r e n a l g l a n d s have been suggested as a s o u r c e of DLIS ( P e r n o l l e t 1986; S c h r e i b e r 1981a, 1981b, 1981c; S h i l p 1987) as has the f e t a l a d r e n a l c o r t e x (Pudek 1983b). One m a n u s c r i p t (Diamandis 1985) noted t h a t p r o g e s t e r o n e and c o r t i s o n e a r e p r e s e n t i n HPLC f r a c t i o n s of c o r d b l o o d , p l a c e n t a and m a t e r n a l b l o o d and t h a t the c h r o m a t o g r a p h i c e l u t i o n volumes of th e s e f r a c t i o n s were p a r t i c u l a r l y r e a c t i v e w i t h RIA methods of d i g o x i n a n a l y s i s . L o n g e r i c h et al. (1988) r e p o r t e d the i d e n t i f i c a t i o n of C o r t i s o l and p r o g e s t e r o n e i n a p o r t i o n of the plasma DLIS i n pregnant women. Wh i l e t h e s e s u b s t a n c e s 24 i n t h e m s e l v e s a r e u n l i k e l y t o be the DLIS m a t e r i a l , t h e i r h i g h r e a c t i v i t y c auses f u r t h e r d i f f i c u l t i e s w i t h the v a l i d i t y of the RIA p r o c e d u r e . D e h y d r o e p i a n d r o s t e r o n e -s u l f a t e , c o r t i s o n e , C o r t i s o l , d e o x y c o r t i s o n e , A ^ a n d r o s t e n e -d i o n e , p r o g e s t e r o n e and g l y c o c h e n o d e o x y c h o l i c a c i d c r o s s -r e a c t e d w i t h d i g o x i n a n t i s e r a and had HPLC r e t e n t i o n t i m e s s i m i l a r t o D L I S - c o n t a i n i n g f r a c t i o n s (Matthewson 1988). Dasgupta and co-workers (1987, 1988) e s t a b l i s h e d t h a t p a r t of t h e DLIS found i n h a e m o d i a l y s i s p a t i e n t s was a p h o s p h o l i p i d . F a s t atom bombardment mass s p e c t r a of t h i s compound (DLIS-2) (Dasgupta 1988) s u p p o r t e d the p r e s e n c e of a p h o s p h o s e r i n e group i n the m o l e c u l e . Another group, u s i n g mass s p e c t r o m e t r y of HPLC f r a c t i o n s of plasma from a p a t i e n t w i t h r e n a l and l i v e r impairment, r e p o r t e d the p r e s e n c e of b i l e a c i d s i n the f r a c t i o n s c o n t a i n i n g DLIS ( T o s e l a n d 1988). U s i n g r e v e r s e d - p h a s e HPLC, DLIS has been s e p a r a t e d i n t o s e v e r a l immunoreactive f r a c t i o n s w hich i n d i c a t e d t h a t DLIS i s not a s i n g l e s u b s t a n c e (Diamandis 1985). DLIS has been s u g g e s t e d t o be d e h y d r o e p i a n d r o s t e r o n e (Vasdev 1985), however, t h i s has been d i s p u t e d (Pudek 1983a). Recent m a n u s c r i p t s (Braquet 1986a, 1986b; Fagoo 1986) noted t h a t e n t e r o l a c t o n e which c o n t a i n s a l a c t o n e r i n g s i m i l a r t o t h a t of d i g o x i n , may c o n t r i b u t e t o the d i g i t a l i s - l i k e a c t i v i t y of t h e s e samples and mono and d i g l y c e r i d e s have a l s o been i m p l i c a t e d ( S o l d i n 1986a). V a l d e s and co-workers (1985a, 1985b) r e p o r t e d the p r o t e i n b i n d i n g of endogenous d i g o x i n - i m m u n o r e a c t i v e f a c t o r s 25 i n human serum and u r i n e . These f a c t o r s a r e water s o l u b l e , heat s t a b l e and n e u t r a l i n m o l e c u l a r c h a r g e . Serum DLIS has an a p p a r e n t m o l e c u l a r mass of 200 Da w h i l e t h a t from u r i n e appears t o be 400 Da ( V a l d e s 1985a, 1985b). U r i n a r y DLIS has a h i g h e r a f f i n i t y f o r d i g o x i n a n t i s e r a and l e s s r e s i s t a n c e t o a c i d h y d r o l y s i s than t h a t i s o l a t e d from serum. I t has been suggested ( V a l d e s 1985a, 1985b) t h a t the u r i n a r y DLIS may r e p r e s e n t a c o n j u g a t e d m e t a b o l i t e of the f a c t o r found i n serum. In a d d i t i o n , serum DLIS appears t o be bound n o n c o v a l e n t l y t o serum p r o t e i n ( V a l d e s 1985a, 1985b). N o r m a l l y over 90% of the t o t a l endogenous i m m u n o r e a c t i v i t y i n serum i s t i g h t l y but r e v e r s i b l y bound t o p r o t e i n and i s t h e r e f o r e not d e t e c t e d by d i r e c t measurement w i t h c o n v e n t i o n a l RIA methods ( V a l d e s 1985a, 1985b). The r e m a i n i n g serum DLIS i s weakly p r o t e i n bound or unbound ( V a l d e s 1985a, 1985b). V a l d e s (1985a, 1985b) suggested t h a t the i n c r e a s e i n DLIS l e v e l s , seen i n p a t i e n t s w i t h r e n a l f a i l u r e , i n neonates and pregnant women, was due t o an i n c r e a s e i n the amount of weakly p r o t e i n bound DLIS r a t h e r than an i n c r e a s e i n t o t a l DLIS. Other r e p o r t s have i n d i c a t e d t h a t an i n c r e a s e i n serum DLIS l e v e l s may be a t l e a s t p a r t l y due t o a d e c r e a s e i n i t s removal v i a the k i d n e y ( C l e r i c o 1988a) and t h a t the u r i n a r y e x c r e t i o n can be p o s i t i v e l y c o r r e l a t e d w i t h p h y s i c a l a c t i v i t y ( C l e r i c o 1988a, 1988b). The p r e s e n c e of s u f f i c i e n t DLIS i n p a t i e n t samples, p a r t i c u l a r l y n eonates, s e r i o u s l y compromises the a c c u r a c y 26 and i n t e r p r e t a t i o n of t h e r e s u l t s from immunoassay methods. S e v e r a l i n v e s t i g a t o r s ( G o r t n e r 1985; McCarthy 1985; Ng 1985; Pudek 1985; Scherrmann 1986a; W i t h e r s p o o n 1987) have t e s t e d c o m m e r c i a l k i t s and have recommended p a r t i c u l a r brands as h a v i n g lower r e a c t i v i t y t o DLIS. A l t e r a t i o n s i n the i n c u b a t i o n c o n d i t i o n s w i t h commercial RIA methods a l s o e f f e c t e d the apparent d i g o x i n l e v e l s i n the p r e s e n c e of DLIS (Sm i t h 1987; Yannakou 1987). U l t r a f i l t r a t i o n of serum, which has been shown t o remove a p p r o x i m a t e l y 90% of the DLIS p r e s e n t ( C h r i s t e n s o n 1987; Graves 1986), may be used t o m i n i m i z e the i n t e r f e r e n c e w i t h d i g o x i n immunoassays. The use of f l u o r e s c e n c e p o l a r i z a t i o n immunoassays f o r d i g o x i n may reduce i n t e r f e r e n c e from DLIS ( Y a t s c o f f 1984) but w i l l not e l i m i n a t e i t e n t i r e l y ( B i a n c h i 1986; S o l d i n 1986b). W h i l e the above p r o c e d u r e s may appear t o be a s o l u t i o n t o the problem, b a t c h - t o - b a t c h v a r i a n c e s i n immunoassay k i t s make t h i s an u n r e l i a b l e s o l u t i o n over t i m e . In a d d i t i o n t o the presence of DLIS, a d i g i t o x i n - l i k e i mmunoreactive s u b s t a n c e was d e t e c t e d i n a m n i o t i c f l u i d and c o r d b l o o d (Ebara 1986b). A s i g n i f i c a n t c o r r e l a t i o n between the l e v e l s of DLIS and d i g i t o x i n - l i k e immunoreactive s u b s t a n c e was a l s o found. The p h y s i o l o g i c a l r o l e of DLIS has y e t t o be e n t i r e l y i n v e s t i g a t e d ( C l e r i c o 1987). H i g h l e v e l s of DLIS, whether due t o e x c e s s p r o d u c t i o n or reduced e x c r e t i o n , may produce e f f e c t s on the h e a r t and the autonomic nervous system s i m i l a r t o those seen w i t h d i g i t a l i s t o x i c i t y ( K i e v a l 1988). 27 U s i n g DLIS i s o l a t e d from human b i l e and c a n i n e P u r k i n j e f i b e r s , K i e v a l et al. (1988) demonstrated c a r d i o t o x i c i t y s i m i l a r t o o u a b a i n . 1.4 D i g o x i n - D r u g I n t e r a c t i o n s The c l i n i c a l management of p a t i e n t s on d i g o x i n i s c o m p l i c a t e d by the v a r i a b i l i t y of d i g o x i n p h a r m a c o k i n e t i c s and problems w i t h the assay methods. F u r t h e r m o r e , a low t h e r a p e u t i c index has been r e p o r t e d f o r d i g o x i n , w i t h the t h e r a p e u t i c range b e i n g 0.5 t o 2.0 ng/mL serum. The i n t e r a c t i o n of d i g o x i n w i t h o t h e r a gents which may produce s m a l l changes i n d i g o x i n d i s p o s i t i o n may l e a d t o t o x i c or s u b - t h e r a p e u t i c plasma d i g o x i n c o n c e n t r a t i o n s . S i n c e d i g o x i n t o x i c i t y and u n t r e a t e d c o n g e s t i v e h e a r t f a i l u r e or a r r h y t h m i a s may be f a t a l , i t i s e s s e n t i a l t h a t t h e s e p a t i e n t s be c a r e f u l l y o b s e r v e d and a p p r o p r i a t e changes i n t h e i r d i g o x i n regimen be made when o t h e r drugs a r e c o -a d m i n i s t e r e d . 1.4.1 D i g o x i n - Q u i n i d i n e M u l t i p l e drug t h e r a p y i s o f t e n used t o c o n t r o l edema or a r r h y t h m i a s t h a t may accompany c o n g e s t i v e h e a r t f a i l u r e . U n f o r t u n a t e l y , many drug i n t e r a c t i o n s have been found between d i g o x i n and o t h e r c o - t h e r a p e u t i c a g e n t s . The most noted drug i n t e r a c t i o n of d i g o x i n i s w i t h q u i n i d i n e , a w i d e l y used a n t i a r r h y t h m i c a g e n t . One of the f i r s t r e p o r t s of a problem w i t h t h i s c o m b i n a t i o n was i n 1932 (Gol d 1932) 28 when an i n c r e a s e d r i s k of d r u g - i n d u c e d a r r h y t h m i a s i n dogs was n o t i c e d . In g u i n e a - p i g s , g u i n i d i n e was found t o i n h i b i t the d i s t r i b u t i o n of d i g o x i n ( O k u d a i r a 1986) by d e c r e a s i n g b o t h the ATP-dependent b i n d i n g i n h e a r t , muscle and l i v e r and the ATP-independent b i n d i n g i n h e a r t . A l s o , q u i n i d i n e i n h i b i t e d the i n t e s t i n a l s e c r e t i o n of d i g o x i n i n g u i n e a p i g s ( S c h a f e r 1985). F u r t h e r s t u d i e s of t h i s i n t e r a c t i o n i n h e a l t h y v o l u n t e e r s (one i n t r a v e n o u s d i g o x i n dose) and c a r d i a c p a t i e n t s ( a t s t e a d y s t a t e d i g o x i n l e v e l s ) have suggested t h a t more than one mechanism was i n v o l v e d . Q u i n i d i n e d e c r e a s e d the t o t a l body c l e a r a n c e and n o n - r e n a l c l e a r a n c e of d i g o x i n i n h e a l t h y v o l u n t e e r s (Ochs 1981) and c a r d i a c p a t i e n t s ( S c h e n c k - G u s t a f s s o n 1981a, 1982). I t a l s o i n h i b i t e d r e n a l t u b u l a r s e c r e t i o n i n p a t i e n t s t h u s p r o d u c i n g a d e c r e a s e i n the r e n a l c l e a r a n c e of d i g o x i n (Schenck-G u s t a f s s o n 1982). R e c e n t l y , F i c h t l et al. (1983) r e p o r t e d t h a t serum d i g o x i n l e v e l s i n c r e a s e d t o about the same amount i r r e s p e c t i v e of the degree of r e n a l impairment. The r e l a t i v e c o n t r i b u t i o n s of i m p a i r e d r e n a l and n o n - r e n a l c l e a r a n c e s , and r e - d i s t r i b u t i o n of d i g o x i n t o the i n t e r a c t i o n w i t h q u i n i d i n e a r e unknown a t the p r e s e n t t i m e . S c h e n c k - G u s t a f s s o n et al. (1981b) r e p o r t e d a r e d u c t i o n of t h e r a t i o of s k e l e t a l muscle t o serum d i g o x i n l e v e l s i n c a r d i a c p a t i e n t s which c o n t r i b u t e d t o the q u i n i d i n e - i n d u c e d d e c r e a s e i n the a p p arent volume of d i s t r i b u t i o n f o r d i g o x i n . S t e a d y - s t a t e serum d i g o x i n c o n c e n t r a t i o n s a f t e r q u i n i d i n e 29 a d m i n i s t r a t i o n c o r r e l a t e d w e l l w i t h serum d i g o x i n c o n c e n t r a t i o n s b e f o r e q u i n i d i n e t h e r a p y i n c a r d i a c p a t i e n t s (Friedman 1982), a l l o w i n g f o r the e s t i m a t i o n of d i g o x i n l e v e l s t h a t w i l l be reached a f t e r q u i n i d i n e a d m i n i s t r a t i o n . 1.4.2 Other Drug I n t e r a c t i o n s Many o t h e r drugs i n t e r a c t w i t h d i g o x i n t o produce a change i n the plasma l e v e l s of d i g o x i n or a l t e r a t i o n s of i t s i n o t r o p i c e f f e c t i n humans. Burgess and Crane (1986) o b s e r v e d a q u a n t i t a t i v e l y s i m i l a r p o s i t i v e i n o t r o p i c response f o r food and i n t r a v e n o u s d i g o x i n i n v o l u n t e e r s . T h i s i n o t r o p i c response e x h i b i t e d p o t e n t i a t i o n when d i g o x i n and food were a d m i n i s t e r e d t o g e t h e r . Q u i n i n e has been r e p o r t e d t o i m p a i r the e x t r a r e n a l c l e a r a n c e of d i g o x i n which caused an i n c r e a s e i n plasma d i g o x i n l e v e l s (Pedersen 1985a). Diazepam produced an i n c r e a s e i n plasma d i g o x i n c o n c e n t r a t i o n s ( C a s t i l l o - F e r r a n d o 1980) w h i l e a n o t h e r b e n z o d i a z e p i n e , a l p r a z o l a m , d i d not s i g n i f i c a n t l y a l t e r d i g o x i n c l e a r a n c e (Ochs 1985). C h o l e s t y r a m i n e , a b a s i c a n i o n exchange r e s i n t h a t i s not a b s orbed by the g a s t r o i n t e s t i n a l t r a c t , has been noted t o b i n d d i g o x i n and p r e v e n t a b s o r p t i o n (Hoffman 1980), t h e r e b y r e d u c i n g plasma d i g o x i n l e v e l s by 69% from t h o s e of c o n t r o l s u b j e c t s (Brown 1976). A c t i v a t e d c h a r c o a l has a l s o been o b s e r v e d t o d e c r e a s e the a b s o r p t i o n of d i g o x i n from the g a s t r o i n t e s t i n a l t r a c t ( R e i s s e l l 1982). M e t o c l o p r a m i d e , a g a s t r i c m o t i l i t y m o d i f i e r , has been found by one group of 30 i n v e s t i g a t o r s t o lower plasma d i g o x i n l e v e l s (Lindenbaum 1971), w h i l e a more r e c e n t s t u d y (Johnson 1984) noted t h a t m e t o c l o p r a m i d e r e d u c e d both the time t o r e a c h peak plasma l e v e l s and the a b s o r p t i o n when t a b l e t f o r m u l a t i o n s were used. However, t h e o v e r a l l e f f e c t on d i g o x i n a b s o r p t i o n by me t o c l o p r a m i d e was m i n i m i z e d when c a p s u l e s ( c o n t a i n i n g d i g o x i n i n s o l u t i o n ) r a t h e r than t a b l e t s were a d m i n i s t e r e d (Johnson 1984). The serum c o n c e n t r a t i o n of c e r t a i n e l e c t r o l y t e s i n f l u e n c e m y o c a r d i a l s e n s i t i v i t y t o d i g o x i n . Hypokalemia has been shown t o r e s u l t i n c a r c i a c t o x i c i t y when serum d i g o x i n c o n c e n t r a t i o n s were i n the t h e r a p e u t i c range (Keys 1980). Some of t h e d i u r e t i c s t h a t cause h y p o k a l e m i a ( t h i a z i d e s , f u r o s e m i d e and e t h a c r y n i c a c i d ) may a f f e c t d i g o x i n t o x i c i t y due t o t h i s e l e c t r o l y t i c e f f e c t (Hoffman 1980). The r e n a l e x c r e t i o n of d i g o x i n , however, was not a f f e c t e d by f u r o s e m i d e (Brown 1976; Malcolm 1977; T i l s t o n e 1 977). S p i r o n o l a c t o n e reduces t h e volume of d i s t r i b u t i o n , r e n a l t u b u l a r s e c r e t i o n and n o n - r e n a l c l e a r a n c e of d i g o x i n (Bussey 1982; W a l d o r f f 1978). C a u t i o n was a l s o s u g g e s t e d when c o m b i n i n g t r i m e t h o p r i m w i t h d i g o x i n (D'Arcy 1985). P h e n y t o i n was found t o s i g n i f i c a n t l y i n c r e a s e the t o t a l d i g o x i n c l e a r a n c e s u g g e s t i n g t h a t , d u r i n g c o - a d m i n i s t r a t i o n , t h e d i g o x i n serum c o n c e n t r a t i o n s s h o u l d be m o n i t o r e d and i f n e c e s s a r y , t h e d i g o x i n dose i n c r e a s e d (Rameis 1985). 31 The a n t i a n g i n a l and a n t i a r r h y t h m i c a g e n t s v e r a p a m i l , n i f e d i p i n e and amiodarone cause an i n c r e a s e i n the serum d i g o x i n c o n c e n t r a t i o n i n c a r d i a c p a t i e n t s and h e a l t h y v o l u n t e e r s ( B e l z 1981a, 1983; George 1982; K l e i n 1982; Pedersen 1981, 1982, 1985b; Venkatesh 1985, 1986). The c a l c i u m a n t a g o n i s t , b e p r i d i l , a l s o produced an i n c r e a s e i n the serum d i g o x i n c o n c e n t r a t i o n s i n h e a l t h y s u b j e c t s ( B e l z 1986). D i g o x i n i n t o x i c a t i o n i n p a t i e n t s t r e a t e d w i t h amiodarone has been r e p o r t e d ( B e n - C h e t r i t 1985). In r a t s , Koren et al. (1983) found t h a t v e r a p a m i l produced no change i n t i s s u e uptake of d i g o x i n , which s u g g e s t e d t h a t an i n h i b i t i o n of d i g o x i n e l i m i n a t i o n o c c u r r e d . The t i s s u e / s e r u m r a t i o s of d i g o x i n c o n c e n t r a t i o n s i n r a t myocardium, s k e l e t a l muscle, and b r a i n were d e c r e a s e d when amiodarone or i t s major m e t a b o l i t e , d e s e t h y l a m i o d a r o n e , were added t o the regimen (Venkatesh 1985, 1986). However, amiodarone had no e f f e c t on the k i n e t i c s of a s i n g l e dose of d i g o x i n i n the r a b b i t (Buss 1985). The e f f e c t of d i s o p y r a m i d e , a n o t h e r a n t i a r r h y t h m i c a g e n t , on serum d i g o x i n l e v e l s has been s t u d i e d w i t h c o n f l i c t i n g r e s u l t s . Some i n v e s t i g a t o r s (Leahey 1980; W e l l e n s 1980) have found no change i n the serum d i g o x i n c o n c e n t r a t i o n i n c a r d i a c p a t i e n t s when t h e r a p e u t i c l e v e l s of d i s o p y r a m i d e were c o - a d m i n i s t e r e d w i t h d i g o x i n . R e l a t i v e l y h i g h l e v e l s of d i s o p y r a m i d e (mean of 5.05 j ig/mL compared t o t h e r a p e u t i c c o n c e n t r a t i o n s of 2.8 t o 3.2 Mg/mL f o r a t r i a l a r r h y t h m i a s ) (Gilman 1980) produced a 15% i n c r e a s e i n serum 32 d i g o x i n c o n c e n t r a t i o n but was thought t o be of l i m i t e d c l i n i c a l s i g n i f i c a n c e (Manolas 1980). The a n t i a r r h y t h m i c ethmozine produced no s t a t i s t i c a l l y s i g n i f i c a n t change i n serum d i g o x i n c o n c e n t r a t i o n s of c a r d i a c p a t i e n t s w i t h normal r e n a l f u n c t i o n (Kennedy 1986). 1.5 D i g o x i n A n a l y s i s The low plasma l e v e l s of d i g o x i n and i t s m e t a b o l i t e s have prompted the development of e x t r e m e l y s e n s i t i v e a ssay p r o c e d u r e s . S i n c e a l a r g e v a r i a b i l i t y i n the m e t a b o l i s m and e x c r e t i o n of d i g o x i n has been r e p o r t e d , i t i s i m p e r a t i v e t h a t any as s a y p r o c e d u r e s f o r t h e measurement of d i g o x i n be s p e c i f i c f o r the i n t a c t drug s u b s t a n c e . D i g o x i n may be a n a l y s e d by s e v e r a l methods i n c l u d i n g radioimmunoassay (RIA) ( B u t l e r 1978, 1979), e n z y m e - m u l t i p l i e d immunoassay (EMIT) (Brunk 1977; B u t l e r 1978; E r i k s e n 1978; L i n d a y 1983; R o s e n t h a l 1976; Sun 1976), f l u o r e s c e n c e p o l a r i z a t i o n immunoassay (FPIA) ( B u t l e r 1978; E r i c k s o n 1984; Rawal 1983), h i g h - p e r f o r m a n c e l i q u i d chromatography (HPLC) ( B e a s l e y 1983; Davydov 1982; Desta 1982a, 1982b, 1987; E r i k s s o n 1981a; Kwong 1986a, 1986b), and v a r i o u s b i o l o g i c a l and c h e m i c a l methods ( H a u s t e i n 1983; L o w e n s t e i n 1965; Simson 1962; St e w a r t 1981). None of the methods d e v e l o p e d t o d a t e can compete w i t h the immunoassay methods i n terms of speed, p r e c i s i o n , s e n s i t i v i t y and c o s t . As a r e s u l t t h e s e methods a r e most f r e q u e n t l y used i n c l i n i c a l l a b o r a t o r i e s . FPIA methods have r e p l a c e d RIA p r o c e d u r e s i n many c l i n i c a l 33 l a b o r a t o r i e s s i n c e t h e FPIA system p r o v i d e s more r a p i d a n a l y s i s (20 m i n . ) , g r e a t e r p r e c i s i o n and r e c o v e r y of d i g o x i n , t h e r e a g e n t s have a l o n g e r s h e l f - l i f e and r a d i o a c t i v e i s o t o p e s a r e not r e q u i r e d ( E r i c k s o n 1984). 1.5.1 Immunoassays The a n a l y t i c a l p r o c e d u r e based on the r e a c t i o n between an a n t i g e n and an a n t i b o d y t o t h e a n t i g e n i s termed an immunoassay. T h i s r e a c t i o n obeys t h e Law of Mass A c t i o n and i s shown i n F i g u r e 2. A n t i g e n + A n t i b o d y ^ A n t i g e n - A n t i b o d y — ( F r e e F r a c t i o n ) (Bound Complex) F i g u r e 2. Reaction Involved i n Immunoassay Procedures Immunoassays use a l a b e l l e d a n t i g e n t o i n c r e a s e t h e s e n s i t i v i t y of the a s s a y . C o m p e t i t i v e b i n d i n g of the l a b e l l e d and n o n - l a b e l l e d a n t i g e n w i t h the a n t i b o d y form l a b e l l e d and n o n - l a b e l l e d bound complexes. The l a b e l l e d a n t i g e n i n e i t h e r t h e f r e e f r a c t i o n or the bound f r a c t i o n i s then d e t e r m i n e d . By comparison t o a s e r i e s of samples of known c o n c e n t r a t i o n of a n a l y t e ( a s s a y e d a t t h e same t i m e ) , t h e c o n c e n t r a t i o n of a n a l y t e i n an unknown sample can be de t e r m i n e d . R a d i o i s o t o p e s , enzymes, coenzymes, r e d b l o o d c e l l s , l a t e x p a r t i c l e s and m e t a l s as w e l l as f l u o r e s c e n t , 34 b i o l u m i n e s c e n t and c h e m i l u m i n e s c e n t m o l e c u l e s have been used f o r l a b e l l i n g a n t i g e n s . For d i g o x i n a n a l y s i s , r a d i o a c t i v i t y , enzymes and f l u o r e s c e n t m o l e c u l e s a r e the most common l a b e l l i n g methods ( B u t l e r 1978). One of the major problems w i t h immunoassay p r o c e d u r e s f o r d i g o x i n a n a l y s i s has been t h e c r o s s - r e a c t i v i t y of the a n t i b o d i e s w i t h t h e m e t a b o l i t e s of d i g o x i n and o t h e r endogenous compounds. T h i s may l e a d t o e r r o n e o u s r e s u l t s , p a r t i c u l a r l y i n view of the f a c t t h a t DLIS i s p r e s e n t i n many p a t i e n t groups and t h a t d i g o x i g e n i n and i t s mono- and b i s - d i g i t o x o s i d e s a r e known t o have c a r d i a c a c t i v i t y , a l t h o u g h a t s u b s t a n t i a l l y lower p o t e n c i e s than d i g o x i n (Aronson 1980). N a t u r a l l y o c c u r r i n g and s y n t h e t i c s t e r o i d s a l s o e x h i b i t some c r o s s - r e a c t i v i t y w i t h d i g o x i n a n t i b o d i e s i s o l a t e d from the s e r a of a n i m a l s when o n l y r e l a t i v e l y s h o r t p e r i o d s of i m m u n i z a t i o n w i t h the d i g o x i n c o n j u g a t e a r e used ( B u t l e r 1979; Pudek 1983a; S c h r e i b e r 1981a, 1981b, 1981c; Smith 1970, 1973). Radioimmunoassay t e c h n i q u e s , u s i n g a r a d i o i s o t o p e l a b e l , have been d e v e l o p e d f o r the a n a l y s i s of d i g o x i n i n serum or plasma. Other c a r d i a c g l y c o s i d e s (Besch 1975; B u t l e r 1978; Lenz 1975; R e i s s e l l 1982; W e i l e r 1980) and some of the m e t a b o l i t e s of d i g o x i n ( B u t l e r 1978, 1982; E i c h h o r s t 1981) have a l s o been a s s a y e d by s i m i l a r radioimmunoassay t e c h n i q u e s . The d e t e r m i n a t i o n of d i g o x i n i n u r i n e samples by RIA has a l s o been r e p o r t e d ( C h r i s t e n s o n 1982). 35 A n t i b o d y s p e c i f i c i t y i s a major problem w i t h t h e RIA f o r d i g o x i n . S p i r o n o l a c t o n e , a s y n t h e t i c s t e r o i d a n a l o g u e , has produced i n t e r f e r e n c e w i t h some RIA k i t s ( D i P i r o 1980; L i c h e y 1 979; S c h r e i b e r 1981c; S i l b e r 1 979). S i l b e r et al. (1979) s u g g e s t e d t h a t m e t a b o l i t e s of s p i r o n o l a c t o n e ( o t h e r than canrenone) may be r e s p o n s i b l e f o r t h i s i n t e r f e r e n c e . S t i l l o t h e r r e p o r t s have i n d i c a t e d t h a t s p i r o n o l a c t o n e d i d not i n t e r f e r e w i t h the RIA method ( H a n s e l l 1979; M u l l e r 1978; R a v e l 1975). Furosemide has not been found t o i n t e r f e r e w i t h the d e t e r m i n a t i o n of d i g o x i n u s i n g the RIA method ( H a n s e l l 1979). In a d d i t i o n t o exogenous s u b s t a n c e s , endogenous s u b s t a n c e s such as a l b u m i n (Holtzman 1974; Lindenbaum 1975) and DLIS ( V a l d e s 1983a, 1983b), as w e l l as b a c t e r i a l c o n t a m i n a t i o n of p a t i e n t samples (Boone 1977) have caused a l t e r a t i o n s i n apparent d i g o x i n v a l u e s u s i n g t h e RIA p r o c e d u r e . V a r i a t i o n s i n c r o s s - r e a c t i v i t y w i t h d i g o x i n m e t a b o l i t e s and endogenous s u b s t a n c e s has been t r a c e d t o d i f f e r e n c e s i n the c o m m e r c i a l s o u r c e s of the RIA k i t s ( H a n s e l l 1979; K u b a s i k 1974a, 1974b; K u c z a l a 1976; L a r s o n 1977; Loo 1977; MacKinney 1975; Smith 1976). Comparing RIA k i t s u s i n g t r i t i u m or r a d i o i o d i n e l a b e l l e d d i g o x i n show t h a t the t r i t i u m method was l e s s v a r i a b l e ( K u b a s i k 1975; P i p p i n 1976; Vemuri 1980). However, r a d i o i o d i n e l a b e l l i n g has some adv a n t a g e s . I o d i n e - 1 2 5 i s a gamma e m i t t e r and c o u n t i n g i s f a s t e r , more c o n v e n i e n t and l e s s e x p e n s i v e than l i q u i d s c i n t i l l a t i o n c o u n t i n g which i s n e c e s s a r y w i t h t r i t i u m 36 ( B u t l e r 1978). In a d d i t i o n , quenching i s not o b s e r v e d w i t h i o d i n e - 1 2 5 ( K r o e n i n g 1976). FPIA i s a fluoroimmunoassay (FIA) where a f l u o r e s c e n t t r a c e r i s e x c i t e d w i t h p o l a r i z e d l i g h t . The p o l a r i z a t i o n of the e m i t t e d l i g h t w i l l depend on the e x t e n t of random Brownian r o t a t i o n of the m o l e c u l e s d u r i n g t h e i r e x c i t e d s t a t e (Landon 1981). S i n c e the p o l a r i z a t i o n of the e m i t t e d f l u o r e s c e n c e i s d i f f e r e n t f o r a n t i b o d y - b o u n d and f r e e a n t i g e n , FPIA methods do not r e q u i r e a s e p a r a t i o n s t e p ( D a n d l i k e r 1964; J o l l e y 1981; Landon 1981). The c o m m e r c i a l l y a v a i l a b l e FPIA f o r d i g o x i n from Abbott L a b o r a t o r i e s i s the TDx a s s a y . T h i s a s s a y r e q u i r e s p r e c i p i t a t i o n of serum p r o t e i n s w i t h t r i c h l o r o a c e t i c a c i d ( E r i c k s o n 1984; P o r t e r 1984) or 5 - s u l f o s a l i c y l i c a c i d (Skogen 1987). The r e s u l t s a r e s i g n i f i c a n t l y i n f l u e n c e d by the t o t a l p r o t e i n c o n c e n t r a t i o n ( P o r t e r 1984) such t h a t i n c r e a s e d p r o t e i n d e c r e a s e s the measured d i g o x i n l e v e l s . The use of a c i d f o r p r o t e i n p r e c i p i t a t i o n , however, has been s u g g e s t e d t o r e s u l t i n the t r a n s f o r m a t i o n of d i g o x i n and i t s m e t a b o l i t e s t o d i g o x i g e n i n and/or d i h y d r o d i g o x i g e n i n ( G a u l t 1977; Sonobe 1980; S t e r n s o n 1978). T h e r e f o r e w i t h t h i s method, d i g o x i n can not be d i f f e r e n t i a t e d from i t s m e t a b o l i t e s p r e s e n t i n the serum sample. The TDx FPIA has been r e p o r t e d t o be s u b j e c t t o m i n i m a l i n t e r f e r e n c e from DLIS as compared t o the RIA p r o c e d u r e ( Y a t s c o f f 1984). However, numerous r e p o r t s i n the l i t e r a t u r e ( B i a n c h i 1986; F r y e 1987; Kanan 1987; Skogen 1987; S o l d i n 1986b; Weiner 37 1987) have shown t h a t the TDx a s s a y i n d i c a t e d i n c r e a s e d a p p a r e n t d i g o x i n l e v e l s i n the p r e s e n c e of DLIS. In a d d i t i o n , i t has been s u g g e s t e d t h a t t h e p r o t e i n p r e c i p i t a t i o n s t e p used w i t h t h e FPIA may enhance DLIS i n t e r f e r e n c e by d i s r u p t i n g the D L I S - p r o t e i n i n t e r a c t i o n and a l l o w i n g f r e e DLIS t o be d e t e c t e d (Skogen 1987; S o l d i n 1986b). 1.5.2 B i o l o g i c a l A s s a y s D i g o x i n i n h i b i t s the a c t i v i t y of Na +,K +-ATPase by b i n d i n g t o the enzyme and ATPase i s o l a t e d from h e a r t and b r a i n t i s s u e s has been used t o d e v e l o p enzyme b i n d i n g a s s a y s f o r the d e t e r m i n a t i o n of c a r d i a c g l y c o s i d e c o n c e n t r a t i o n s (Gundert-Remy 1981). However, the m e t a b o l i t e s of d i g o x i n a l s o i n h i b i t Na +,K +-ATPase a c t i v i t y , w i t h d i h y d r o d i g o x i n h a v i n g t e n t i m e s the b i n d i n g a f f i n i t y of d i g o x i n (Gundert-Remy 1981; Marcus 1975). As a r e s u l t , the ATPase assay p r o c e d u r e s , l i k e the immunoassay methods, l a c k s p e c i f i c i t y f o r d i g o x i n . The rubidium-86 uptake a s s a y i s based on the i n h i b i t i o n of r u b i d i u m uptake i n t o r e d b l o o d c e l l s in vitro by d i g i t a l i s g l y c o s i d e s ( B e l z 1981b; L o w e n s t e i n 1965). D e t e r m i n a t i o n of d i g i t a l i s g l y c o s i d e s i n plasma u s i n g t h i s t e c h n i q u e r e q u i r e s a p p r o x i m a t e l y 8 h o u r s , hence t h i s assay method i s more time consuming than c o n v e n t i o n a l immunoassay methods ( B e l z 1981b). 38 A number of t e s t a n i m a l s , i n c l u d i n g g u i n e a p i g s , f r o g s , c a t s and p i g e o n s , have been used i n the b i o a s s a y of d i g i t a l i s ( T y l e r 1976; USP 1985). In the USP as s a y f o r d i g o x i n , p i g e o n s a r e i n j e c t e d w i t h f i x e d volumes of a d i l u t e d d i g i t a l i s p r e p a r a t i o n a t f i v e minute i n t e r v a l s u n t i l t h e p i g e o n d i e s from c a r d i a c a r r e s t (USP 1985). Problems w i t h t h i s b i o a s s a y a r e found s i n c e i t assumes t h a t t o x i c i t y i s a s s o c i a t e d w i t h , and p r o p o r t i o n a l t o , the t h e r a p e u t i c e f f e c t . I n g e n e r a l , b i o a s s a y s a r e unable t o p r e d i c t the o r a l p o t e n cy of the g l y c o s i d e b e i n g t e s t e d and l a c k s p e c i f i c i t y (Simson 1962; T y l e r 1976). A l o n g w i t h a l a c k of s e n s i t i v i t y , b i o a s s a y methods a r e i m p r a c t i c a l f o r e v a l u a t i n g d i g i t a l i z e d p a t i e n t samples (Simson 1962; T y l e r 1976). 1.5.3 C h e m i c a l Methods D i g o x i n has an u l t r a v i o l e t a b s o r p t i o n maximum a t 217 nm w i t h a molar e x t i n c t i o n c o e f f i c i e n t of 16595 ( F l a s c h 1981). D e r i v a t i z a t i o n p r o c e d u r e s , t o i n c r e a s e b oth the i n t e n s i t y of a b s o r p t i o n and a l t e r t he wave l e n g t h of maximum absorbance, have been r e p o r t e d f o r q u a n t i t a t i v e a n a l y s i s of d i g i t a l i s g l y c o s i d e s ( F l a s c h 1981; Rowson 1952a, 1952b). These methods have not been s u f f i c i e n t l y s e n s i t i v e f o r the e v a l u a t i o n of b i o l o g i c a l samples ( F l a s c h 1981) and have not been s p e c i f i c f o r a p a r t i c u l a r d i g i t a l i s g l y c o s i d e ( E a s t l a n d 1952; F l a s c h 1981; Rowson 1952a, 1952b). The u n s a t u r a t e d c a r b o n y l and t h e d i g i t o x o s e sugar r e s i d u e s of d i g i t a l i s g l y c o s i d e s can be d e r i v a t i z e d . In 39 a l k a l i n e s o l u t i o n , p i c r i c a c i d ( B a l j e t r e a c t i o n ) , d i n i t r o b e n z o i c a c i d (Kedde r e a c t i o n ) , t e t r a n i t r o b i p h e n y l ( R a b i t z s c h r e a c t i o n ) and s e v e r a l o t h e r d i n i t r o a r o m a t i c compounds r e a c t w i t h the c a r b o n y l f u n c t i o n of the l a c t o n e r i n g . Reviews i n the l i t e r a t u r e ( F l a s c h 1981; Rowson 1952a, 1952b) have i n d i c a t e d t h a t , a l t h o u g h t h e s e r e a g e n t s were r e a s o n a b l y s p e c i f i c f o r the l a c t o n e r i n g , t hey d i d not d i f f e r e n t i a t e between the v a r i o u s g l y c o s i d e s , a g l y c o n e s , and m e t a b o l i t e s w i t h u n s a t u r a t e d l a c t o n e m o i e t i e s . C o l o r e d d e r i v a t i v e s of the d i g i t o x o s e s u g a r s have been formed by the K e l l e r - K i l a n i , t h i o b a r b i t u r i c a c i d and x a n t h y d r o l r e a c t i o n s ( F l a s c h 1981; Rowson 1952a). U s i n g f e r r i c c h l o r i d e , a c e t i c a c i d and s u l f u r i c a c i d , the K e l l e r -K i l a n i p r o c e s s removes the d i g i t o x o s e r e s i d u e s and produces d e r i v a t i v e s w i t h a b s o r p t i o n maxima a t 470 nm and 590 nm. S i m i l a r l y , the x a n t h y d r o l r e a c t i o n h y d r o l y s e s the d i g i t o x o s e r e s i d u e s w i t h a c i d , f o l l o w e d by d e r i v a t i z a t i o n t o a r e d p r o d u c t ( a b s o r p t i o n maximum a t 520 nm). The t h i o b a r b i t u r i c r e a c t i o n i n v o l v e s o x i d a t i o n of d i g i t o x o s e s u g a r s t o d i a l d e h y d e s , opening of the sugar and f o r m a t i o n of a c o l o r e d complex w i t h 2 - t h i o b a r b i t u r i c a c i d (maximum absorbance a t 532 nm). These r e a c t i o n s a r e s p e c i f i c f o r t h e d i g i t o x o s e s u g a r s and t h e r e f o r e the a g l y c o n e p o r t i o n of t h e m o l e c u l e would not be d e t e c t e d ( F l a s c h 1981; Rowson 1952a). The r e p o r t e d lower l i m i t of d e t e c t i o n w i t h t h e s e c o l o r i m e t r i c p r o c e d u r e s was i n t h e ng range ( F l a s c h 1981). 40 F l u o r e s c e n t methods f o r the a n a l y s i s of c a r d i a c g l y c o s i d e s lower the d e t e c t i o n l i m i t t o the ng range. F or example, t h e r e a c t i o n of d i g o x i n w i t h s t r o n g a c i d s such as p h o s p h o r i c , h y d r o c h l o r i c or t r i c h l o r o a c e t i c , causes d e h y d r a t i o n and h y d r o l y s i s t o the 14,1 6 - d i a n h y d r o g e n i n ( F l a s c h 1981; Jensen 1952). S i n c e t h e s e f l u o r e s c e n t assay methods a r e s p e c i f i c f o r the s t e r o i d p a r t of d i g o x i n , m e t a b o l i t e s of the a g l y c o n e would not be d i f f e r e n t i a t e d . C h e m i c a l methods of a n a l y s i s a r e not s p e c i f i c , but have f r e q u e n t l y been used i n c o m b i n a t i o n w i t h c h r o m a t o g r a p h i c methods t o a l l o w i s o l a t i o n and p u r i f i c a t i o n of i n d i v i d u a l g l y c o s i d e s . For example, the sugar and c a r b o n y l c o l o r d e r i v a t i v e s , as w e l l as the f l u o r e s c e n t d e r i v a t i v e s , have been used i n c o m b i n a t i o n w i t h paper, t h i n - l a y e r and l i q u i d c h r o m a t o g r a p h i c t e c h n i q u e s ( F l a s c h 1981). 1.5.4 Chromatographic Techniques G a s - l i q u i d c h r o m a t o g r a p h i c (GLC) methods of a n a l y s i s of d i g o x i n have been r e p o r t e d i n the l i t e r a t u r e ( F l a s c h 1981). F o r m a t i o n of t r i m e t h y l s i l y l ( J e l l i f f e 1963) and h e p t a f l u o r o b u t y r y l (Watson 1972) d e r i v a t i v e s p r i o r t o GLC a n a l y s i s have been r e p o r t e d . Flame i o n i z a t i o n ( K i b b e 1973), mass s p e c t r o m e t r y ( F l a s c h 1981) and e l e c t r o n c a p t u r e (Watson 1972) d e t e c t o r systems have been used i n the GLC a n a l y s i s of d i g o x i n . The GLC a s s a y methods l a c k e d s p e c i f i c i t y , s i n c e d i g o x i n and i t s m e t a b o l i t e s were c o n v e r t e d t o the a g l y c o n e 41 p o r t i o n of the m o l e c u l e d u r i n g d e r i v a t i z a t i o n or e x t r a c t i o n ( F l a s c h 1981; J e l l i f f e 1963; K i b b e 1973; Watson 1972). T h i n - l a y e r c h r o m a t o g r a p h i c (TLC) methods of a n a l y s i s of d i g o x i n , u s i n g s i l i c a g e l , c e l l u l o s e and r e v e r s e d - p h a s e p l a t e s have been r e p o r t e d ( F l a s c h 1981; J e l l i f f e 1969; Sabatka 1976). B i o l o g i c a l samples have been e v a l u a t e d u s i n g TLC methods but l a r g e sample volumes were r e q u i r e d and the s e n s i t i v i t y was inadequate f o r t h e r a p e u t i c m o n i t o r i n g ( F l a s c h 1981). Numerous r e p o r t s on the a n a l y s i s of d i g i t a l i s g l y c o s i d e s by hi g h - p e r f o r m a n c e l i q u i d chromatography have appeared i n the l i t e r a t u r e . These methods o f f e r an a t t r a c t i v e a l t e r n a t i v e s i n c e , u n l i k e GLC methods, d i g o x i n and i t s m e t a b o l i t e s a r e not degraded by t h e c o n d i t i o n s r e q u i r e d . V a r i o u s t y p e s of s t a t i o n a r y phases have been used, i n c l u d i n g s i l i c a g e l (Bockbrader 1984; E r i k s s o n 1981b; Loo 1977; Nachtmann 1976a, 1976b), ion-exchange phases ( F l a s c h 1981) and p a r t i t i o n mode phases ( B e a s l e y 1983; Davydov 1982; Desta 1982a, 1982b, 1987; Diamandis 1985; E r i k s s o n 1981b; G a u l t 1982; G f e l l e r 1977; G i b s o n 1980; Kwong 1986a, 1986b; Loo 1981; P e k i c 1983; Plum 1986). Both p r e -column and post-column d e r i v a t i z a t i o n t e c h n i q u e s have been used f o r the a n a l y s i s of d i g i t a l i s g l y c o s i d e s i n an attempt t o i n c r e a s e the s e n s i t i v i t y of HPLC as s a y t e c h n i q u e s . S e i b e r et al. (1981) r e v i e w e d the t e c h n i q u e s and a p p l i c a t i o n s of HPLC f o r t h e a n a l y s i s of d i g i t a l i s g l y c o s i d e s and r e l a t e d s t e r o i d s . 42 Pre-column d e r i v a t i z a t i o n t e c h n i q u e s u s i n g 3,5-d i n i t r o b e n z o y l c h l o r i d e (Bockbrader 1984; F u j i i 1983) has been r e p o r t e d . Bockbrader and Reuning (1984) e x t r a c t e d the g l y c o s i d e s from u r i n e , formed the 3 , 5 - d i n i t r o b e n z o y l d e r i v a t i v e and a n a l y s e d t h e s e u s i n g a s i l i c a g e l s t a t i o n a r y phase. The s e n s i t i v i t y was 100 ng/mL i n plasma w i t h d e t e c t i o n a t 254 nm. F u j i i et al . (1983) d e r i v a t i z e d d i g o x i n and i t s m e t a b o l i t e s w i t h 3 , 5 - d i n i t r o b e n z o y l c h l o r i d e and d e v e l o p e d a m i c r o HPLC assay system w i t h u l t r a v i o l e t d e t e c t i o n a t 230 nm. Wi t h t h i s l a t t e r p r o c e d u r e , the l i m i t of d e t e c t i o n of d i g o x i n was 2 ng. U s i n g pre-column d e r i v a t i z a t i o n w i t h 4 - n i t r o b e n z o y l c h l o r i d e f o l l o w e d by s e p a r a t i o n on a s i l i c a g e l s t a t i o n a r y phase, Nachtmann el al. (1976a, 1976b) found a d e t e c t i o n l i m i t f o r d i g o x i n of 11 ng/mL i n plasma w i t h d e t e c t i o n a t 254 nm. H i g h l y f l u o r e s c e n t d e r i v a t i v e s have been formed by post-column d e r i v a t i z a t i o n of d i g o x i n and i t s m e t a b o l i t e s . G f e l l e r et al. (1977) d e v e l o p e d a p r o c e d u r e f o r post-column d e r i v a t i z a t i o n of d i g o x i n , d i g o x i g e n i n , l a n a t o s i d e C and d e s a c e t y l l a n a t o s i d e C w i t h h y d r o c h l o r i c a c i d and d e h y d r o a s c o r b i c a c i d u s i n g an a i r - s e g m e n t a t i o n p r o c e d u r e f o r d e l i v e r i n g the r e a g e n t s . A d e t e c t i o n l i m i t of 0.5 ng f o r d e s a c e t y l l a n a t o s i d e C was found w i t h t h i s system ( G f e l l e r 1977). F u r t h e r m o d i f i c a t i o n of t h i s HPLC-PC a i r seg m e n t a t i o n method (Desta 1987) produced complete r e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n but the 43 s e n s i t i v i t y (10 ng of d i g o x i n ) was i n a d e q u a t e f o r development of a r o u t i n e c l i n i c a l a s s a y . Recent m a n u s c r i p t s (Kwong 1986a, 1986b; Reh 1985) have d e s c r i b e d the development of HPLC a s s a y s f o r d i g o x i n a t t h e r a p e u t i c c o n c e n t r a t i o n s i n plasma u s i n g post-column f l u o r o g e n i c d e r i v a t i z a t i o n . The p r o c e d u r e r e p o r t e d by Kwong and M c E r l a n e (1986a) s e p a r a t e d d i g o x i n from s e v e r a l drugs f r e q u e n t l y c o - p r e s c r i b e d w i t h d i g o x i n . P a r t i a l r e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n was a c h i e v e d which a l l o w e d f o r a peak h e i g h t q u a n t i t a t i v e a s s a y . The minimum d e t e c t a b l e q u a n t i t y was found t o be 0.5 ng digoxin/mL i n plasma (Kwong 1986b). A post-column f l u o r o g e n i c d e r i v a t i z a t i o n method was a l s o d e v e l o p e d by Reh and J o r k (1985) but s e p a r a t i o n of d i g o x i n from d i h y d r o d i g o x i n and i n t e r f e r e n c e from p o s s i b l e c o - p r e s c r i b e d drugs were not e v a l u a t e d . 1.5.5 HPLC-RIA The s e p a r a t i o n of d i g o x i n from i t s m e t a b o l i t e s and endogenous s t e r o i d s u s i n g HPLC p r i o r t o the a n a l y s i s of the d i g o x i n f r a c t i o n u s i n g RIA (Gibson 1980; Loo 1977, 1981; Margot 1983; M o r a i s 1981; Plum 1986; Vasdev 1985; Wagner 1983) i n c r e a s e d s p e c i f i c i t y . The HPLC p r o c e d u r e s r e p o r t e d s e p a r a t e d d i g o x i n from t h r e e m e t a b o l i t e s ; d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e ( G i b s o n 1980; Plum 1986; Wagner 1983). These m e t a b o l i t e s have been shown t o e x h i b i t h i g h c r o s s - r e a c t i v i t y w i t h 44 d i g o x i n RIA p r o c e d u r e s . W h i l e the s e n s i t i v i t y of t h e s e HPLC-RIA methods was s u f f i c i e n t t o m o n i t o r t h e r a p e u t i c c o n c e n t r a t i o n s of d i g o x i n i n plasma, the c o l l e c t i o n of t i m e d e l u a t e f r a c t i o n s w i t h subsequent e v a p o r a t i o n and RIA measurement may i n t r o d u c e m e t h o d o l o g i c a l e r r o r s . 1.6 HPLC-EC E l e c t r o c h e m i c a l (EC) d e t e c t i o n methods r e q u i r e an e l e c t r o a c t i v e a n a l y t e t h a t can be o x i d i z e d or reduced. G e n e r a l l y , a c o n s t a n t p o t e n t i a l i s a p p l i e d and the c u r r e n t f l o w can be measured as a f u n c t i o n of time ( F l e e t 1974). F u n c t i o n a l groups t h a t have o x i d a t i v e e l e c t r o a c t i v i t y i n c l u d e amines, p h e n o t h i a z i n e s , p h e n o l s , a r o m a t i c h y d r o x y l s and c a t e c h o l a m i n e s ( F l e e t 1974; V o l k e 1983). F u n c t i o n a l groups t h a t e x h i b i t r e d u c t i v e e l e c t r o a c t i v i t y i n c l u d e e s t e r s , k e t o n e s , a l d e h y d e s , e t h e r s , d i a z o compounds and n i t r o groups ( F l e e t 1974; V o l k e 1983). The c o m b i n a t i o n of HPLC w i t h EC d e t e c t i o n (HPLC-EC) f o r drug measurement has l e d t o v e r y s e n s i t i v e a s s a y methods (Brooks 1983; Hu 1986; K r e u z i g 1981; L e e l a v a t h i 1986; Lund 1979; S t e w a r t 1981, 1986; V o l k e 1983; Wang 1986, 1987). Reviews of EC d e t e c t i o n methods and f l o w c e l l geometry f o r HPLC have been p r e p a r e d by White (1984) and F l e e t and L i t t l e ( 1 9 7 4 ) . Numerous drugs and b i o l o g i c a l l y a c t i v e compounds have been e v a l u a t e d u s i n g HPLC-EC i n the o x i d a t i v e mode. B i o g e n i c amines and c a t e c h o l a m i n e s have f r e q u e n t l y been as s a y e d by HPLC-EC ( B a u e r s f e l d 1986; D a v i s 1981; H o l l y 1983; 45 K r s t u l o v i c 1982; Zaczek 1982). Drugs e v a l u a t e d u s i n g o x i d a t i v e HPLC-EC i n c l u d e t h e o p h y l l i n e and i t s d e r i v a t i v e s (Greenberg 1979), d o p a z i n o l (Mazzo 1986), p h e n o t h i a z i n e s [ t r i m e p r a z i n e (Hu 1986), p e r p h e n a z i n e and f l u p h e n a z i n e (Tjaden 1976), c h l o r p r o m a z i n e and t h i o r i d a z i n e (Svendsen 1986) ] , i n d o r a m i n ( L e e l a v a t h i 1986), t r i c y c l i c a n t i d e p r e s s a n t s [ d e s i p r a m i n e , i m i p r a m i n e and t r i m i p r a m i n e (Wang 19 8 6 ) ] , g u a n e t h i d i n e ( S t e w a r t 1986), a t r o p i n e (Leroy 1987) , p h y s o s t i g m i n e ( I s a k s s o n 1987), c o d e i n e (Shah 1987), p e n t a z o c i n e ( S h i b a n o k i 1987), a n t h r a c y c l i n e a n t i b i o t i c s ( R i l e y 1987), e r y t h r o m y c i n ( C r o t e a u 1987), h y d r o c h l o r o t h i a z i d e ( S t e w a r t 1986), and 0 - r e c e p t o r a n t a g o n i s t s [ b o p i n d o l o l (Humbert 1987) and m e p i n d o l o l (Krause 1 9 8 0 ) ] . R e d u c t i v e HPLC-EC poses numerous pro b l e m s . The r e d u c t i o n of oxygen and t r a c e m e t a l s i n t e r f e r e w i t h EC a n a l y s i s ( C a u d i l l 1985; Lund 1979). T h i s n e c e s s i t a t e s the use of c o n t i n u o u s s p a r g i n g of t h e m o b i l e phase w i t h argon or h e l i u m gas i n o r d e r t o m a i n t a i n a de-oxygenated s t a t e ( C a u d i l l 1985). At p o t e n t i a l s more n e g a t i v e than -0.4 V, r e d u c t i o n of d i s s o l v e d oxygen can s i g n i f i c a n t l y i n c r e a s e b a s e l i n e n o i s e . P a s s i v a t i o n of t h e HPLC pump, i n j e c t o r and a s s o c i a t e d t u b i n g w i t h 20% n i t r i c a c i d removes t r a c e m e t a l s t h a t can be washed o f f by the m o b i l e phase c r e a t i n g b a s e l i n e n o i s e . R e d u c t i v e mode HPLC-EC has been used f o r the a n a l y s i s of b e n z o d i a z e p i n e s such as n i t r a z e p a m , diazepam and 46 c h l o r d i a z e p o x i d e (Lund 1979). The d e t e c t i o n l i m i t s were found t o depend s t r o n g l y on the p o t e n t i a l used; such t h a t as the p o t e n t i a l became more n e g a t i v e the d e t e c t i o n l i m i t i n c r e a s e d (Lund 1979). Brooks (1983) r e v i e w e d the HPLC-EC d e t e r m i n a t i o n of a l a r g e number of b e n z o d i a z e p i n e s . U s i n g o x i d a t i v e and r e d u c t i v e e l e c t r o d e s i n s e r i e s , b o t h modes of d e t e c t i o n were used t o observe c h l o r p r o m a z i n e , t h i o r i d a z i n e and m e t a b o l i t e s from b r a i n t i s s u e (Svendsen 1986). With HPLC and r e d u c t i v e EC, Me e r i n g et al. (1984) found d e t e c t i o n l i m i t s of 2 t o 4 pg f o r m i s o n i d a z o l e and d e s m e t h y l -m i s o n i d a z o l e . The e l e c t r o c h e m i c a l r e d u c t i o n of n i t r o groups has been e s t a b l i s h e d by a number of i n v e s t i g a t o r s f o r many y e a r s ( F l e e t 1974; M o r a l e s 1987; White 1984). N i t r o g r o u p s , i n p a r t i c u l a r a r o m a t i c n i t r o groups, a r e e a s i l y reduced i n a c i d i c s o l u t i o n s ( M o r a l e s 1987; Plambeck 1982; Ryan 1984). Mousty et al. (1986) r e p o r t e d t h a t p - n i t r o p h e n o x y a l k y l ammonium s a l t s a r e reduced i n two s u c c e s s i v e s t e p s i n a c i d i c medium (pH 2.7) u s i n g p o t e n t i a l s from -0.25 t o -1.0 V. Many r e p o r t s i n the l i t e r a t u r e on the a n a l y s i s of n i t r o groups u s i n g r e d u c t i v e EC have not used f l o w - t h r o u g h d e t e c t o r s . U s i n g column chromatography and EC d e t e c t i o n (0 t o -1.15 V ) , B r i l m y e r et al. (1975) s e p a r a t e d o r t h o and p a r a n i t r o b e n z o i c a c i d s i n 75% aqueous b u f f e r a t pH 6.0. C a u d i l l et al. (1985) a n a l y s e d the t r i n i t r o b e n z e n e s u l f o n i c a c i d d e r i v a t i v e s of 7 - a m i n o b u t y r i c a c i d w i t h HPLC-EC a t -0.55 V w i t h a m o b i l e phase of 50% aqueous b u f f e r a t pH 3.0. 47 HPLC w i t h e l e c t r o c h e m i c a l d e t e c t i o n i s becoming w i d e l y used f o r the a n a l y s i s of t r a c e q u a n t i t i e s of e l e c t r o a c t i v e compounds. A l t h o u g h t h e u t i l i t y of a r o m a t i c n i t r o d e r i v a t i v e s f o r use w i t h r e d u c t i v e HPLC-EC has been suggested ( K i s s i n g e r 1979), 3 , 5 - d i n i t r o b e n z o y l d i g o x i n ( Bockbrader 1984; F u j i i 1983) or o t h e r n i t r o c o n t a i n i n g d e r i v a t i v e s of d i g o x i n (Nachtmann 1976a) have not been e v a l u a t e d by r e d u c t i v e EC or HPLC-EC. E x c e p t i o n a l l y low l e v e l s of d i g o x i n a r e used t h e r a p e u t i c a l l y . T h i s n e c e s s i t a t e s u s i n g the most s e n s i t i v e a ssay methods f o r e v a l u a t i n g p a t i e n t samples f o r d i g o x i n . The e x p e c t e d s e n s i t i v i t y of e l e c t r o c h e m i c a l d e t e c t o r s t o a r o m a t i c n i t r o groups make i n v e s t i g a t i o n of u s i n g HPLC-EC f o r a n a l y s i s of the 3 , 5 - d i n i t r o b e n z o y l d e r i v a t i v e of d i g o x i n imperat i v e . G f e l l e r et al. (1977) d e v e l o p e d an HPLC pr o c e d u r e f o r post-column d e r i v a t i z a t i o n of c a r d i a c g l y c o s i d e s u s i n g h y d r o c h l o r i c a c i d . M o d i f i c a t i o n s of t h i s HPLC-PC assay l a c k e i t h e r the s e n s i t i v i t y (Desta 1987) or the r e s o l u t i o n of d i g o x i n from i t s m e t a b o l i t e s (Reh 1985) r e q u i r e d f o r a s p e c i f i c and s e n s i t i v e d i g o x i n a s s a y . Problems w i t h r e l i a b l e d e l i v e r y of the post-column r e a g e n t s w i t h one method (Kwong 1986a) have a l s o been o b s e r v e d . F u r t h e r i n v e s t i g a t i o n of post-column d e r i v a t i z a t i o n of d i g o x i n t o combine s e n s i t i v i t y , r e s o l u t i o n from d i g o x i n m e t a b o l i t e s , s p e c i f i c i t y f o r d i g o x i n and r e l i a b l e d e l i v e r y of post-column r e a g e n t s i n one HPLC as s a y i s r e q u i r e d . Samples from 48 p a t i e n t groups where h i g h l e v e l s of DLIS have been r e p o r t e d s h o u l d a l s o be e v a l u a t e d by the HPLC assay t o ensure t h a t s p e c i f i c i t y f o r d i g o x i n i s m a i n t a i n e d i n the pr e s e n c e of endogenous compounds. These were the p r i m a r y aims of t h i s t h e s i s . 49 2. E X P E R I M E N T A L 2.1 S u p p l i e s 2.1.1 C h e m i c a l s D i g o x i n , d i g o x i g e n i n b i s d i g i t o x o s i d e , d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n , d i h y d r o d i g o x i n , d i h y d r o -d i g o x i g e n i n , d i g i t o x i g e n i n and g i t o x i n were o b t a i n e d from B o e h r i n g e r (Mannheim, GFR). R , S , - M e x i l e t i n e h y d r o c h l o r i d e was p u r c h a s e d from B o e h r i n g e r I n g e l h e i m L t d . ( B u r l i n g t o n , Ont., Canada). Sodium b i c a r b o n a t e , L - a s c o r b i c a c i d , sodium h y d r o x i d e ( A r i s t a r g r a d e ) , h y d r o c h l o r i c a c i d ( H C l ) , hydrogen p e r o x i d e ( 3 0 % ) , sodium s u l f a t e anhydrous and z i n c s u l f a t e h e p t a h y d r a t e were p u r c h a s e d from BDH C h e m i c a l s ( T o r o n t o , Ont., Canada). Sodium a c e t a t e t r i h y d r a t e ( G o l d l a b e l ) , g l a c i a l a c e t i c a c i d ( G o l d l a b e l ) and 3 , 5 - d i n i t r o b e n z o y l c h l o r i d e (3,5-DNBCl) were p u r c h a s e d from A l d r i c h C h e m i c a l Company, I n c . (Milwaukee, WI, U.S.A.). Barium h y d r o x i d e o c t a h y d r a t e was p u r c h a s e d from J . T. Baker C h e m i c a l ( P h i l l i p s b u r g , NJ, U.S.A.). C u p r i c s u l f a t e p e n t a h y d r a t e was o b t a i n e d from A l l i e d C h e m i c a l ( M o r r i s t o w n , NJ, U.S.A.). 4-D i m e t h y l a m i n o p y r i d i n e (4-DMAP), n o r e t h i n d r o n e , I 7 a - e t h y n y l e s t r a d i o l , e s t r o n e , 6 a - m e t h y l - 1 7 o - h y d r o x y l - p r o g e s t e r o n e a c e t a t e , e s t r a d i o l , e s t r o n e - 3 - s u l f a t e , t e s t o s t e r o n e , 19-nor-t e s t o s t e r o n e , 1 7 a - m e t h y l - t e s t o s t e r o n e , a d r e n o s t e r o n e , 5a-a n d r o s t a n e - 3 , 1 7 - d i o n e , A 4 - a n d r o s t e n e - 3 , 1 7 - d i o n e , A 4-androsten-11/3-ol-3,1 7-dione, d e o x y c o r t i c o s t e r o n e , 21-50 d e o x y c o r t i s o n e , e s t r i o l , h y d r o c o r t i s o n e , 17a-hy d r o x y p r e g n e n o l o n e , 1 7 a - h y d r o x y p r o g e s t e r o n e , A^-pregnene-3/3,20a-diol, 5-pregnen-3j3-ol-20-one, c o r t i s o n e , d e h y d r o i s o a n d r o s t e r o n e , d e h y d r o i s o a n d r o s t e r o n e - 3 - s u l f a t e , 5/3-pregnane-3a, 2 0 a - d i o l , p r o g e s t e r o n e and R e i c h s t e i n ' s Substance S were o b t a i n e d from Sigma C h e m i c a l Co. ( S t . L o u i s , MO, U.S.A.). 1 7 a - E s t r a d i o l and 1 7 / 3 - e s t r a d i o l were samples from A y e r s t L a b o r a t o r i e s ( M o n t r e a l , Que., Canada). 2.1.2 S o l v e n t s Reagent grade a b s o l u t e e t h a n o l was pu r c h a s e d from Commercial A l c o h o l s L t d . ( T o r o n t o , Ont., Canada). A c e t o n e , methanol and p r o p a n - 1 - o l were reagent grade and o b t a i n e d from BDH C h e m i c a l s ( T o r o n t o , Ont., Canada). I s o o c t a n e ( 2 , 2 , 4 - t r i m e t h y l p e n t a n e ) was g l a s s d i s t i l l e d q u a l i t y and pur c h a s e d from BDH C h e m i c a l s ( T o r o n t o , Ont., Canada). HPLC grade water was produced u s i n g the M i l l i - Q Water System ( M i l l i p o r e Corp., M i l f o r d , MA, U.S.A.). P y r i d i n e and the r e m a i n i n g HPLC grade s o l v e n t s were Omnisolv grade from BDH C h e m i c a l s ( T o r o n t o , Ont., Canada). P r i o r t o use, p y r i d i n e was d i s t i l l e d and s t o r e d over sodium h y d r o x i d e . 2.1.3 E x t r a c t i o n S u p p l i e s A V o l a c p i p e t t e c o n t r o l l e r f o r volumes of 1 t o 20 mL ( S c i e n c e w a r e , Pequannock, NJ, U.S.A.) and R e p i p e t D i s p e n s e r ( L a b i n d u s t r i e s , B e r k e l e y , CA, U.S.A.) were used f o r d i s p e n s i n g o r g a n i c s o l v e n t s d u r i n g t h e e x t r a c t i o n p r o c e d u r e . 51 The f i l t r a t i o n u n i t c o n s i s t e d of a N y l o n 66 membrane (0.45 umr 13 mm d i a m e t e r ) f i l t e r d i s c ( R a i n i n Instrument Co., I n c . , Woburn, MA, U.S.A.) w i t h a Swinnex 13 M i l l i p o r e f i l t e r h o l d e r ( M i l l i p o r e Corp., M i l f o r d , MA, U.S.A.) a t t a c h e d t o a 5 mL Luer-Lock M u l t i f i t B-D g l a s s s y r i n g e (Becton D i c k i n s o n Canada, M i s s i s s a u g a , Ont., Canada). F i l t r a t i o n of serum samples p r i o r t o e x t r a c t i o n was a c h i e v e d u s i n g the Amicon C e n t r i f r e e m i c r o p a r t i t i o n system and the C e n t r i f l o u l t r a f i l t r a t i o n membrane cones (CF25) (Amicon D i v . , W.R. Grace and Co., Danvers, MA, U.S.A.). 2.1.4 Post-Column D e r i v a t i z a t i o n S u p p l i e s Two 15.2 cm (6 i n . ) d i a m e t e r weld neck f l a n g e s ( S c h e d u l e 80 A-53 Grade A s t e e l w i t h 150 l b . f l a n g e d r i l l i n g ) and two 15.2 cm (6 i n . ) d i a m e t e r b l i n d f l a n g e s ( S c h e d u l e 80 A-53 Grade A s t e e l w i t h 150 l b . f l a n g e d r i l l i n g ) ( G r i n n e l l S a l e s L t d . , Vancouver, B.C., Canada) were used f o r the s t e e l p r e s s u r e v e s s e l . The g a s k e t m a t e r i a l was T e f l o n G o r e - T e x T M j o i n t s e a l a n t (W.L. Gore and A s s o c i a t e s , I n c . , E l k t o n , MD, U.S.A.). Two 5.1 cm (2 i n . ) d i a m e t e r PVC s o c k e t weld f l a n g e s ( S c h e d u l e 80 PVC w i t h 150 l b . f l a n g e d r i l l i n g ) , two 5.1 cm (2 i n . ) d i a m e t e r b l i n d f l a n g e s (Schedule 80 PVC w i t h 150 l b . f l a n g e d r i l l i n g ) and 36.8 cm of 5.1 cm (2 i n . ) d i a m e t e r PVC p i p e ( S c h e d u l e 80) ( S c e p t e r Mfg. Co. L t d . , Vancouver, B.C., Canada) were used i n c o n s t r u c t i o n of the PVC p r e s s u r e v e s s e l . Two 5.1 cm (2 i n . ) d i a m e t e r f u l l f a c e 0.32 cm (1/8 52 i n . ) Hypalon g a s k e t s (Custom G a s k e t s , Vancouver, B.C., Canada) were used between the p i p e s e c t i o n f l a n g e s and the b l i n d f l a n g e s . For bo t h the s t e e l and PVC p r e s s u r e v e s s e l s the n o n - m e t r i c e q u i v a l e n t component p a r t s were used. See F i g u r e 6 f o r diagram of PVC p r e s s u r e v e s s e l w i t h f i t t i n g s . P o l y t e t r a f l u o r o e t h y l e n e (PTFE) t u b i n g (0.16 cm d i a m e t e r , 0.3 mm I.D.) was used f o r the r e a c t o r and c o n n e c t i o n of the column and p r e s s u r e v e s s e l t o the r e a c t o r . A three-way PTFE v a l v e ( p a r t number 1102 O m n i f i t L t d . , Cambridge, England) was between the p r e s s u r e v e s s e l , column and r e a c t o r . The PTFE t u b i n g and c o n n e c t o r s were o b t a i n e d from O m n i f i t L t d . (Cambridge, E n g l a n d ) . 2.1.5 Commercial Radioimmunoassay K i t s The s i x commercial RIA k i t s used f o r d i g o x i n a n a l y s i s were as f o l l o w s : D i g i - T a b RIA ( N u c l e a r - M e d i c a l L a b o r a t o r i e s D i v . , Warner-Lambert T e c h n o l o g i e s I n c . , I r v i n g , TX, U.S.A.), Bio-RIA 1-125 D i g o x i n ( I n s t i t u t e of B i o - E n d o c r i n o l o g y I n c . , M o n t r e a l , Que., Canada), Amerlex D i g o x i n RIA K i t (Amersham Corp., A r l i n g t o n H e i g h t s , I L , U.S.A.), D i g o x i n S o l i d Phase RIA (Becton D i c k i n s o n Immunodiagnostics, Orangeburg, NY, U.S.A.), GammaCoat D i g o x i n RIA ( C l i n i c a l A s says D i v . , T r a v e n o l L a b o r a t o r i e s I n c . , Cambridge, MA, U.S.A.) and D i g o x i n 1-125 Imusay (Abbott L a b o r a t o r i e s D i a g n o s t i c s D i v . , N o r t h C h i c a g o , I L , U.S.A.). 53 2.1.6 Commercial F l u o r e s c e n c e P o l a r i z a t i o n Immunoassay K i t The A b b o t t L a b o r a t o r i e s TDx f l u o r e s c e n c e p o l a r i z a t i o n immunoassay f o r d i g o x i n was used (A b b o t t L a b o r a t o r i e s D i a g n o s t i c s D i v . , N o r t h C h i c a g o , I L , U.S.A.). 2.1.7 F i l t r a t i o n of HPLC M o b i l e Phase FP V e r i c e l 47 mm 0.45 Mm membrane f i l t e r s (Gelman S c i e n c e s I n c . , Ann A r b o r , MI, U.S.A.) were used w i t h the M i l l i p o r e a l l - g l a s s f i l t e r a p p a r a t u s ( M i l l i p o r e Waters A s s o c i a t e s , M i l f o r d , MA, U.S.A.) f o r f i l t r a t i o n of the m o b i l e phase. 2.2 Equipment 2.2.1 HPLC Equipment A Beckman Model 100 A d u a l p i s t o n s o l v e n t m e t e r i n g system (Beckman I n s t r u m e n t , I n c . , F u l l e r t o n , CA, U.S.A.) was used as the HPLC pump. The r e m a i n i n g HPLC system c o n s i s t e d of e i t h e r a model U6K i n j e c t o r (Waters A s s o c i a t e s , M i l f o r d , MA, U.S.A.) or a model 210 A l t e x i n j e c t o r (Beckman I n s t r u m e n t , I n c . , F u l l e r t o n , CA, U.S.A.), a Waters f l u o r e s c e n c e d e t e c t o r model 420 AC w i t h a q u a r t z f l o w c e l l (1 mm I.D. by 40 mm q u a r t z t u b i n g ) ( s e c t i o n 2.2.3) and an A l t e x CR1A Chromatopac Data P r o c e s s o r (Beckman I n s t r u m e n t , I n c . , F u l l e r t o n , CA, U.S.A.). The n o i s e r e d u c e r shown i n 54 F i g u r e 3 was c o n s t r u c t e d and used w i t h the Waters 420 f l u o r e s c e n t d e t e c t o r . A f i x e d w a v e l e n g t h (254 nm) u l t r a v i o l e t d e t e c t o r (Beckman model 153 d e t e c t o r , Beckman Instru m e n t I n c . , " F u l l e r t o n , CA, U.S.A.) was used t o a s s a y 3 , 5 - d i n i t r o b e n z o y l d e r i v a t i v e s of d i g o x i n and i t s m e t a b o l i t e s . A NewGuard h o l d e r equipped w i t h a 1.5 cm x 3.2 mm I.D. ODS c a r t r i d g e (Brownlee Labs I n c . , Santa C l a r a , CA, U.S.A.) was used as a guard column and p l a c e d p r i o r t o the S p h e r i s o r b ODS I I (3M) 15 cm X 4.6 mm I.D. a n a l y t i c a l column ( A l l t e c h A s s o c i a t e s , D e e r f i e l d , I L , U.S.A.). Between the i n j e c t o r and guard column, a d i r e c t connect column p r e f i l t e r ( A l l t e c h A s s o c i a t e s , D e e r f i e l d , I L , U.S.A.) was used as an i n l i n e f i l t e r . 2.2.2 E l e c t r o c h e m i c a l D e t e c t o r The Coulochem d u a l e l e c t r o d e HPLC e l e c t r o c h e m i c a l d e t e c t o r model 5100A, guard c e l l model 5020, d u a l e l e c t r o d e a n a l y t i c a l c e l l model 5010 and s c r e e n e d w a l l j e t c e l l model 5012 w i t h a g o l d e l e c t r o d e were p u r c h a s e d from ESA, I n c . ( B e d f o r d , MA, U.S.A.). The guard c e l l e l e c t r o d e was p l a c e d b e f o r e t h e i n j e c t o r and used t o e l e c t r o c h e m i c a l l y c l e a n the m o b i l e phase b e f o r e the i n t r o d u c t i o n of samples. The a n a l y t i c a l c e l l (model 5010 or model 5012) was c o n n e c t e d d i r e c t l y a f t e r the a n a l y t i c a l HPLC column as shown i n F i g u r e 4. 55 1 V O L T 1 0 0 , 0 0 0 O H M -AAA-1 0 0 , 0 0 0 O H M -wv + 4 7 0 M I C R O F A R A D / 2 5 V O L T E L E C T R O L Y T I C C A P A C I T O R S 2 7 0 0 O H M F i g u r e 3. Schematic of Noise Reducer 56 2.2.3 F l u o r e s c e n c e D e t e c t o r Flow C e l l A l l s t a i n l e s s s t e e l f i t t i n g s i n the f l u o r o m e t e r f l o w c e l l were removed. A 40 mm q u a r t z tube (1 mm I.D.) was p o s i t i o n e d i n the f l o w c e l l b l o c k , h e l d i n p l a c e w i t h epoxy g l u e and j o i n e d t o the PTFE t u b i n g v i a a c i d f l e x t u b i n g ( p a r t number 116-0538-09, T e c h n i c o n I n s t r u m e n t s Corp., T a r r y t o w n , NY, U.S.A.). 2.2.4 Post-Column R e a c t o r and P r e s s u r e Chamber A sch e m a t i c diagram of the f i n a l HPLC post-column (HPLC-PC) f l u o r o g e n i c system i s g i v e n i n F i g u r e 5. A Beckman model 110 A s i n g l e p i s t o n m e t e r i n g system (Beckman I n s t r u m e n t , I n c . , F u l l e r t o n , CA, U.S.A.) was used t o pump hexane i n t o the p r e s s u r e v e s s e l t o d i s p l a c e c o n c e n t r a t e d HC1 from a s t e e l or PVC p r e s s u r e chamber ( s e c t i o n 2.1.4) t o the post-column r e a c t o r which c o n s i s t e d of k n i t t e d PTFE t u b i n g (0.3 mm I.D.). The r e a c t o r was m a i n t a i n e d a t 79°C by a r e a c t i o n b a t h t h e r m o s t a t e d u s i n g a Haake model D1 c o n s t a n t t e m p e r a t u r e c i r c u l a t o r ( F i s h e r S c i e n t i f i c Co., F a i r Lawn, N J , U.S.A.). 2.2.5 E x t r a c t i o n Equipment A V o r t e x - G e n i e ( F i s h e r S c i e n t i f i c Co., F a i r Lawn, NJ, U.S.A.), a Labquake Shaker ( L a b i n d u s t r i e s , B e r k e l e y , CA, U.S.A.) and an IEC HN-SII C e n t r i f u g e (Damon/IEC D i v i s i o n , Western S c i e n t i f i c , Vancouver, BC, Canada) were used i n the e x t r a c t i o n of b i o l o g i c a l samples. 57 GUARD CELL HPLC PUMP o INJECTOR u HPLC COLUMN HPLC MOBILE PHASE RECORDER ELECTROCHEMICAL DETECTOR ANALYTICAL FLOW CELL TO WASTE F i g u r e 4. Schematic of HPLC-EC System 58 0.4 mL/min TO WASTE Q INJECTOR HPLC COLUMN RECORDER u HPLC MOBILE PHASE THREE WAY VALVE REACTOR (20 m) FLUOROMETER (360/425 nm) HEATER WATER BATH (79° C) PVC PRESSURE VESSEL CONCENTRATED HCI HPLC HEXANE F i g u r e 5 . Schematic of HPLC-PC F l u o r o g e n i c System 59 2.2.6 Radioimmunoassay Equipment A N u c l e a r - C h i c a g o 1185 s e r i e s a u t o m a t i c gamma c o u n t e r ( S e a r l e Co., Des P l a i n e s , I L , U.S.A.) was used f o r d e t e r m i n i n g i o d i n e - 1 2 5 decay w i t h the comme r c i a l RIA k i t s . 2.3 Pre-column 3 , 5 - D i n i t r o b e n z o y l C h l o r i d e D e r i v a t i z a t i o n P r o c e d u r e s 2.3.1 For Gram Q u a n t i t i e s of D i g o x i n A s o l u t i o n "of 3,5-DNBCl (3.2 g i n 30 mL d r y p y r i d i n e ) was added t o a s o l u t i o n of d i g o x i n (0.5 g) i n 20 mL d r y p y r i d i n e (molar r a t i o of 3,5-DNBCl/digoxin ( 2 2 / 1 ) ) . The m i x t u r e was s t i r r e d f o r 25 minutes a t 50°C, poured i n t o 60 mL c o l d d i l u t e HC1 ( 1 % of c o n c e n t r a t e d ) and the p r e c i p i t a t e was f i l t e r e d and washed w i t h w a t e r . The crud e p r o d u c t was d i s s o l v e d i n 1 L of e t h y l a c e t a t e / h e x a n e ( 1 / 1 ) , washed t w i c e w i t h 250 mL of 5% sodium b i c a r b o n a t e w i t h 4-DMAP and t h r e e t i m e s w i t h HPLC water (300 mL). A f t e r d r y i n g the o r g a n i c l a y e r w i t h anhydrous sodium s u l f a t e and e v a p o r a t i o n in vacuo, the r e s i d u e was p u r i f i e d by double r e c r y s t a l l i z a t i o n i n methanol (reagent grade) (mp 199-204°C). 2.3.2 For M i l l i g r a m Q u a n t i t i e s of C a r d i a c G l y c o s i d e s 3,5-DNBCl (15 mg) was added t o a s o l u t i o n of c a r d i a c g l y c o s i d e (3.0 mg) i n 0.2 mL of d r y p y r i d i n e and the m i x t u r e was v o r t e x e d f o r 20 seconds (molar r a t i o of 3,5-D N B C l / d i g o x i n ( 2 0 / 1 ) ) . A f t e r r e a c t i n g f o r 3 hours a t room 60 t e m p e r a t u r e , the sample was p l a c e d i n a 37°C water b a t h and the p y r i d i n e was e v a p o r a t e d under n i t r o g e n . The r e s i d u e was d i s s o l v e d i n HPLC grade e t h y l a c e t a t e (1.5 mL) and washed f o u r t i m e s w i t h 1 mL of a 5% s o l u t i o n of sodium b i c a r b o n a t e w i t h 4-DMAP. The o r g a n i c l a y e r was then washed f o u r t i m e s w i t h 1 mL of 1% of c o n c e n t r a t e d HC1 and then f o u r t i m e s w i t h HPLC grade water (1 mL). A f t e r e v a p o r a t i o n of the e t h y l a c e t a t e w i t h n i t r o g e n gas, the d e r i v a t i v e s were r e c o n s t i t u t e d w i t h the m o b i l e phase and a n a l y z e d by HPLC. 2.3.3 For Nanogram Q u a n t i t i e s of D i g o x i n To 0.5 t o 10 ng of d i g o x i n i n 0.5 mL d r y p y r i d i n e , 100 ng of 3,5-DNBCl i n 10 ML of p y r i d i n e was added (molar r a t i o of 3,5-DNBCl/digoxin from 34/1 t o 677/1). To 50 t o 80 ng of d i g o x i n , 800 ng of 3,5-DNBCl i n 80 txL was added (molar r a t i o of 3,5-DNBCl/digoxin from 34/1 t o 54/1). The samples were a l l o w e d t o r e a c t a t room t e m p e r a t u r e f o r 2 hours a f t e r which the p y r i d i n e was e v a p o r a t e d u s i n g a 37°C water b a t h and n i t r o g e n gas. A f t e r r e c o n s t i t u t i o n of t h e r e s i d u e w i t h e t h y l a c e t a t e / h e x a n e ( 1 / 1 ) , t h e o r g a n i c l a y e r was washed f o u r t i m e s w i t h 2 mL of 5% sodium b i c a r b o n a t e w i t h 4-DMAP, 1% of c o n c e n t r a t e d HC1 and HPLC grade water j u s t p r i o r t o e v a p o r a t i o n under n i t r o g e n . The d e r i v a t i v e was then r e c o n s t i t u t e d w i t h m o b i l e phase and a n a l y s e d by HPLC. 2.4 P r e p a r a t i o n of S o l u t i o n s 61 2.4.1 Reagents For 3,5-DNBCl D e r i v a t i z a t i o n 2.4.1.1 3,5-DNBCl i n P y r i d i n e 3,5-DNBCl (1 mg) was d i s s o l v e d i n and taken t o a f i n a l volume of 100 mL w i t h d r y p y r i d i n e (10 ng/uL). The s o l u t i o n was s t i r r e d f o r 20 minutes p r i o r t o use. 2.4.1.2 Sodium B i c a r b o n a t e (5%) w i t h 4-DMAP Sodium b i c a r b o n a t e (5 g) and 4-DMAP (250 mg) were d i s s o l v e d i n HPLC grade water and the s o l u t i o n was taken t o a f i n a l volume of 100 mL w i t h HPLC grade w a t e r . The s o l u t i o n was s t i r r e d f o r 20 m i n u t e s . 2.4.1.3 D i l u t e H y d r o c h l o r i c A c i d C o n c e n t r a t e d HC1 (10 mL) was added t o 600 mL HPLC grade w a t e r , t a k e n t o a f i n a l volume of 1 L and s t i r r e d f o r 20 mi n u t e s . 2.4.2 Reagents For F l u o r o g e n i c D e r i v a t i z a t i o n 2.4.2.1 A s c o r b i c A c i d S o l u t i o n L - A s c o r b i c a c i d (250 mg) was weighed o u t , d i s s o l v e d , t a k e n t o a f i n a l volume of 500 mL w i t h HPLC grade w a t e r , and s t i r r e d f o r 20 minutes p r i o r t o use. 62 2.4.2.2 D i l u t e Hydrogen P e r o x i d e S o l u t i o n Hydrogen p e r o x i d e (1 mL of 30%) was d i l u t e d and taken t o 200 mL w i t h HPLC grade w a t e r , s t i r r e d f o r 20 minutes and used i m m e d i a t e l y . 2.4.2.3 D e h y d r o a s c o r b i c A c i d D i l u t e hydrogen p e r o x i d e (12.5 mL) was added t o the a s c o r b i c a c i d s o l u t i o n (500 mL) and s t i r r e d f o r 2 h o u r s . The d e h y d r o a s c o r b i c a c i d was s t o r e d i n a r e f r i g e r a t o r u n t i l use. New d e h y d r o a s c o r b i c a c i d was p r e p a r e d e v e r y week. 2.4.2.4 Hydrogen P e r o x i d e w i t h P h o s p h o r i c A c i d Hydrogen p e r o x i d e (40 uL of 30%) was d i l u t e d and taken t o 5 mL w i t h HPLC grade w a t e r . C o n c e n t r a t e d p h o s p h o r i c a c i d was added t o t h e hydrogen p e r o x i d e i n a 1/1 r a t i o . T h i s m i x t u r e was p r e p a r e d d a i l y . 2.4.3 Sodium A c e t a t e B u f f e r (0.1 M, pH 4.6) Sodium a c e t a t e t r i h y d r a t e (6.8 g) and 3 mL g l a c i a l a c e t i c a c i d were d i s s o l v e d i n 500 mL HPLC water. The s o l u t i o n was ta k e n t o a f i n a l volume of 1 L w i t h HPLC grade w a t e r , s t i r r e d f o r 20 minutes and s t o r e d i n a r e f r i g e r a t o r u n t i l use. New b u f f e r s o l u t i o n was p r e p a r e d e v e r y week. 2.4.4 M o b i l e Phase The HPLC m o b i l e phase was p r e p a r e d by m i x i n g the i n d i v i d u a l l y measured s o l v e n t s and then d e g a s s i n g the 63 m i x t u r e by s t i r r i n g r a p i d l y f o r 30 minutes and was f i l t e r e d p r i o r t o use. The m o b i l e phases used a r e d e s c r i b e d i n s e c t i o n s 3.3 and 3.4. 2.4.5 S t a n d a r d S o l u t i o n s 2.4.5.1 D i g o x i n i n E t h a n o l A s t o c k s o l u t i o n of d i g o x i n was p r e p a r e d i n e t h a n o l (1 mg/100 mL). The s t o c k s o l u t i o n was f u r t h e r d i l u t e d t o g i v e f i n a l c o n c e n t r a t i o n s of 5, 7 and 10 ng/10 uL (0.5, 0.7 and 1 mL of s t o c k s o l u t i o n taken t o 10 mL w i t h e t h a n o l r e s p e c t i v e l y ) . A 0.5 ng/yL s o l u t i o n (1.25 mL s t o c k s o l u t i o n d i l u t e d t o 25 mL w i t h e t h a n o l ) was used t o p r e p a r e 1.5, 2 and 3 ng/10 uL s o l u t i o n s ( 3 , 4 and 6 mL of 0.5 n g / j i L s o l u t i o n d i l u t e d t o 10 mL w i t h e t h a n o l ) . C a l i b r a t i o n c u r v e serum samples were s p i k e d w i t h 10 uL of the f i n a l d i g o x i n s o l u t i o n s (1.5, 2, 3, 5, 7 and 10 ng d i g o x i n / 3 mL serum). 2.4.5.2 3 , 5 - D i n i t r o b e n z o y l D i g o x i n i n Methanol A s t o c k s o l u t i o n of 3 , 5 - d i n i t r o b e n z o y l d i g o x i n i n methanol (1 mg/100 mL or 100 ng/10 uD was f u r t h e r d i l u t e d t o g i v e a f i n a l c o n c e n t r a t i o n of 5 ng/10 uL (0.5 mL of s t o c k s o l u t i o n t a k e n t o 10 mL w i t h m e t h a n o l ) . 2.4.5.2 D i q i t o x i g e n i n i n E t h a n o l For the f l u o r o g e n i c HPLC a s s a y , d i g i t o x i g e n i n was used as i n t e r n a l s t a n d a r d . A s t o c k s o l u t i o n of d i g i t o x i g e n i n was 64 p r e p a r e d i n e t h a n o l (4 mg/100 mL). The s t o c k s o l u t i o n was f u r t h e r d i l u t e d t o g i v e a f i n a l c o n c e n t r a t i o n of 80 ng/10 uL (10 mL s t o c k d i l u t e d t o 50 mL w i t h e t h a n o l ) . Serum samples were s p i k e d w i t h 20 uL of t h i s i n t e r n a l s t a n d a r d s o l u t i o n (160 n g ) . 2.4.5.3 D i h y d r o d i g o x i n i n E t h a n o l One m i l l i g r a m of d i h y d r o d i g o x i n was a c c u r a t e l y weighed and made up t o 100 mL w i t h e t h a n o l . An a l i q u o t of t h i s s o l u t i o n (0.5 mL) was d i l u t e d t o 10 mL w i t h e t h a n o l (5 ng/10 ML). To t e s t the s p e c i f i c i t y of the HPLC post-column f l u o r o g e n i c a s s a y , 10 uL of the f i n a l s o l u t i o n was as s a y e d . 2.4.5.4 S t e r o i d Samples i n Methanol One m i l l i g r a m of each of the f o l l o w i n g s t e r o i d s was weighed a c c u r a t e l y , s e p a r a t e l y made up t o 100 mL i n methanol and the i n d i c a t e d amount was a s s a y e d by the post-column f l u o r o g e n i c a s s a y t o e v a l u a t e the s p e c i f i c i t y of the f l u o r o g e n i c a s s a y : n o r e t h i n d r o n e (680 n g ) , 17 a - e t h y n y l e s t r a d i o l (18 n g ) , 1 7 a - e s t r a d i o l (96 n g ) , 1 7 0 - e s t r a d i o l (37 n g ) , e s t r o n e (55 n g ) , 6 a - m e t h y l - 1 7 a - h y d r o x y l p r o g e s t e r o n e a c e t a t e (25 n g ) , e s t r i o l (55 n g ) , e s t r o n e - 3 - s u l f a t e (30 n g ) , t e s t o s t e r o n e (133 n g ) , 1 9 - n o r t e s t o s t e r o n e (67 ng) and 17a-m e t h y l t e s t o s t e r o n e (60 n g ) . For the f o l l o w i n g samples, one m i l l i g r a m of each was weighed a c c u r a t e l y , and made up t o 10 mL i n methanol and a p p r o x i m a t e l y 500 ng of each was e v a l u a t e d u s i n g the HPLC-PC a s s a y : a n d r e n o s t e r o n e , 5a-65 a n d r o s t a n e - 3 , 1 7 - d i o n e , A 4 - a n d r o s t e n e - 3 , 1 7 - d i o n e , A 4-androst e n - 1 1 /3-ol-3,1 7-dione, d e o x y c o r t i c o s t e r o n e , 21-d e o x y c o r t i s o n e , e s t r i o l , h y d r o c o r t i s o n e , 17a-hydroxypregnendone, 1 7 a - h y d r o x y p r o g e s t e r o n e , A^-pregnene-3 / 3 , 2 0 a - d i o l , 5-pregnen-3/3-ol-20-one, c o r t i s o n e , d e h y d r o i s o a n d r o s t e r o n e , d e h y d r o i s o a n d r o s t e r o n e , d e h y d r o i d o a n d r o s t e r o n e - 3 - s u l f a t e , 5/3-pregnene-3a, 2 0 a - d i o l , p r o g e s t e r o n e and R e i c h s t e i n ' s Substance S. 2.4.5.5 P r e p a r a t i o n of M e x i l e t i n e H y d r o c h l o r i d e  i n 50% E t h a n o l One m i l l i g r a m of R , S - m e x i l e t i n e h y d r o c h l o r i d e was weighed a c c u r a t e l y , d i s s o l v e d i n 50% e t h a n o l i n HPLC grade water and taken t o a f i n a l volume of 1 mL. T h i s s o l u t i o n was then e v a l u a t e d by the HPLC-PC f l u o r o g e n i c a s s a y . 2.4.5.6 P r e p a r a t i o n of Plasma Samples f o r RIA A n a l y s i s Samples were p r e p a r e d by d i s s o l v i n g d i g o x i n or the i n d i v i d u a l m e t a b o l i t e s of d i g o x i n i n 20% e t h a n o l - w a t e r . S e r i a l d i l u t i o n s of the s t o c k s o l u t i o n (1 mg/100 mL) were made and added t o plasma from a h e a l t h y , n o n - d i g i t a l i z e d v o l u n t e e r , t o g i v e f i n a l c o n c e n t r a t i o n s of 0, 0.5, 1.0, 2.0 and 3.0 ng/mL i n 2 mL of plasma. For d i g o x i n , 5.0 ng/mL samples were a l s o p r e p a r e d . 66 2.4.5.7 P r e p a r a t i o n of Serum Samples S p i k e d w i t h  D i g o x i n M e t a b o l i t e s f o r FPIA A n a l y s i s Samples were p r e p a r e d by w e i g h i n g the i n d i v i d u a l m e t a b o l i t e s of d i g o x i n (1 mg), d i s s o l v i n g them i n e t h a n o l and t a k i n g the volume t o 100 mL w i t h e t h a n o l . D i l u t i o n s of t h i s s t o c k s o l u t i o n were p r e p a r e d so t h a t the f i n a l c o n c e n t r a t i o n was 1 ng/10 ML. Blank serum samples (1 mL) were i n d i v i d u a l l y s p i k e d w i t h one m e t a b o l i t e (from 5 t o 10 ML of the d i l u t e s o l u t i o n s ) and the serum samples were as s a y e d by the ACU FPIA. 2.5 P r e p a r a t i o n of S o l v e n t s f o r Serum E x t r a c t i o n 2.5.1 I s o o c t a n e / D i c h l o r o m e t h a n e (20/5) D i c h l o r o m e t h a n e (5 mL) was added t o i s o o c t a n e (20 mL) and the m i x t u r e was s t i r r e d f o r 20 m i n u t e s . 2.5.2 E x t r a c t i o n S o l v e n t P r o p a n - 1 - o l (2 mL) and d i c h l o r o m e t h a n e (98 mL) were s t i r r e d f o r 20 minutes j u s t p r i o r t o use. 2.5.3 P r e p a r a t i o n of P r o t e i n P r e c i p i t a t i o n Reagents 2.5.3.1 Z i n c S u l f a t e H e p t a h y d r a t e (5%) Z i n c s u l f a t e h e p t a h y d r a t e (5 g) was weighed, d i s s o l v e d and taken t o a f i n a l volume of 100 mL w i t h d i s t i l l e d w a t e r . 67 2.5.3.2 Barium H y d r o x i d e O c t a h y d r a t e (0.3 N) Barium h y d r o x i d e o c t a h y d r a t e (23.7 g) was weighed, d i s s o l v e d and taken t o a f i n a l volume of 50 mL w i t h d i s t i l l e d w ater. 2.5.3.3 C u p r i c S u l f a t e P e n t a h y d r a t e (10%) C u p r i c s u l f a t e p e n t a h y d r a t e (10 g) was weighed, d i s s o l v e d i n and t a k e n t o a f i n a l volume of 100 mL w i t h d i s t i l l e d w a t e r . 2.5.3.4 Sodium H y d r o x i d e (12%) Sodium h y d r o x i d e (12 g) was weighed, d i s s o l v e d i n and t a k e n t o a f i n a l volume of 100 mL w i t h d i s t i l l e d w a t e r . 2.6 RIA A n a l y s i s and C r o s s - R e a c t i v i t y C a l c u l a t i o n s A l l samples were assay e d i n d u p l i c a t e u s i n g one l o t of each of the s i x RIA k i t s . The p r e s e n c e of e t h a n o l i n the samples was e v a l u a t e d f o r i n t e r f e r e n c e w i t h the RIA p r o c e d u r e s . A b l a n k plasma sample was a l s o a s s a y e d t o de t e r m i n e i f any c r o s s - r e a c t i n g components were p r e s e n t i n the plasma used. Each of the f o u r m e t a b o l i t e - s p i k e d plasma samples was a s s a y e d i n d u p l i c a t e . C r o s s - r e a c t i v i t y w i t h t h e D i g i - T a b RIA was c a l c u l a t e d as the r a t i o between the q u a n t i t y of d i g o x i n and the q u a n t i t y of m e t a b o l i t e which caused 50% d i s p l a c e m e n t of the d i g o x i n t r a c e r . For a l l o t h e r RIA k i t s , c r o s s - r e a c t i v i t y was c a l c u l a t e d as the r a t i o of t h e d i g o x i n 68 c o n c e n t r a t i o n t o the m e t a b o l i t e c o n c e n t r a t i o n a t 50% i n h i b i t i o n of maximum b i n d i n g . S i n c e the RIA assay p r o c e d u r e i s d e s i g n e d f o r c o m p e t i t i v e b i n d i n g on a molar b a s i s and t h e s e m e t a b o l i t e s d i f f e r g r e a t l y i n t h e i r m o l e c u l a r w e i g h t , the p e r c e n t c r o s s - r e a c t i v i t y was d e t e r m i n e d on a molar b a s i s . 2 .7 HPLC W i t h E l e c t r o c h e m i c a l D e t e c t i o n 2.7.1 O p t i m i z a t i o n of E l e c t r o c h e m i c a l C o n d i t i o n s U s i n g the HPLC system shown i n F i g u r e 4 w i t h the 5020 guard c e l l e l e c t r o d e b e f o r e the i n j e c t o r and the 5012 s c r e e n e d w a l l j e t c e l l as t h e a n a l y t i c a l e l e c t r o d e , 3,5-d i n i t r o b e n z o y l d e r i v a t i v e s of d i g o x i n and d i h y d r o d i g o x i n were a s s a y e d . W i t h a m o b i l e phase of a c e t o n i t r i l e / a c e t a t e b u f f e r ( 2 0 / 7 ) , the l o c a t i o n of d e r i v a t i z e d d i g o x i n was c o n f i r m e d u s i n g u l t r a v i o l e t d e t e c t i o n a t 254 nm (Beckman model 153 d e t e c t o r ) . R e c o r d i n g from d e t e c t o r 1 ( g l a s s y c a r b o n e l e c t r o d e from the 5012 c e l l ) , g r a d u a l l y d e c r e a s i n g t h e v o l t a g e from 0 t o -0.85 V i n d i c a t e d -0.80 V gave the g r e a t e s t peak h e i g h t and t h e r e f o r e was used f o r f u r t h e r e x p e r i m e n t s . 69 2.7.2 M o b i l e Phases f o r HPLC w i t h E l e c t r o c h e m i c a l  D e t e c t i o n The l i m i t of d e t e c t i o n f o r d i g o x i n and r e s o l u t i o n of d i h y d r o d i g o x i n from d i g o x i n was d e t e r m i n e d f o r a v a r i e t y of m o b i l e phases ( s e c t i o n 3.2.1) u s i n g -0.80 V f o r d e t e c t i o n . 2.8 HPLC Post-Column D e r i v a t i z a t i o n A s s a y 2.8.1 Assembly of S t e e l and PVC P r e s s u r e V e s s e l s The s t e e l weld neck f l a n g e s were welded t o g e t h e r back t o back. T e f l o n j o i n t s e a l a n t was a p p l i e d t o t h e exposed end of b o t h neck f l a n g e s and the b l i n d f l a n g e s were mounted w i t h 1.9 cm (3/4 i n . ) b o l t s ( N a t i o n a l Coarse Thread, 316 s t a i n l e s s s t e e l ) ( I n d u f a s t F a s t n e r s L t d . , Vancouver, B.C.). The ends of the PVC p i p e s e c t i o n and the s o c k e t s of the PVC f l a n g e s were p r e p a r e d by l i g h t s a n d i n g . PVC c l e a n i n g s o l v e n t f o l l o w e d by PVC g l u e ( S c e p t e r Mfg. Co., L t d . , Vancouver, B.C., Canada) were a p p l i e d t o a l l s u r f a c e s t o be g l u e d . The socked weld f l a n g e s were p r e s s f i t t e d onto the ends of the p i p e and s e c u r e d u n t i l t he g l u e was s e t . Excess g l u e was wiped c l e a n from the i n t e r n a l and e x t e r n a l s u r f a c e s . Hypalon g a s k e t s were p l a c e d between the p i p e s e c t i o n f l a n g e s and the b l i n d f l a n g e s and 1.3 cm (1/2 i n . ) b o l t s ( N a t i o n a l Coarse Thread, 316 s t a i n l e s s s t e e l ) ( I n d u f a s t F a s t n e r s L t d . , Vancouver, B.C.) were used t o s e c u r e the f l a n g e s t o g e t h e r . 70 On b o t h th e s t e e l and PVC p r e s s u r e v e s s e l s , the b l i n d f l a n g e s ( t o be the top) were d r i l l e d and t h r e a d e d t o a c c e p t 3/8 i n . N a t i o n a l p i p e t h r e a d . Swagelok f i t t i n g s (Swagelok Tube F i t t i n g s , Columbia V a l v e and F i t t i n g Co., L t d . , N o r t h Vancouver, B.C.) were used t o produce the i n l e t f o r hexane and o u t l e t f o r HC1 as shown i n F i g u r e 6. 2.8.2 O p t i m i z a t i o n of HPLC Post-Column D e r i v a t i z a t i o n U s i n g the Waters U6K i n j e c t o r , the S p h e r i s o r b ODS I I (3 Mm) a n a l y t i c a l column w i t h an ODS guard column and m o b i l e phase of m e t h a n o l / e t h a n o l / i s o p r o p a n o l / a q u e o u s (52/3/1/45) at a f l o w r a t e of 0.4 t o 0.5 mL/min., the c a r d i a c g l y c o s i d e samples were e v a l u a t e d . The HPLC e f f l u e n t was combined w i t h c o n c e n t r a t e d HC1 d e l i v e r e d from a s t e e l or PVC p r e s s u r e v e s s e l . Hexane was added t o the p r e s s u r e v e s s e l v i a an HPLC pump a t a f l o w r a t e of 0.5 t o 1.0 mL/min. U s i n g 10 m and 20 m r e a c t o r s , the post-column d e r i v a t i z a t i o n was o p t i m i z e d by comparing peak h e i g h t s from 9.1 ng of d i g o x i n under the v a r i o u s c o n d i t i o n s . 2.8.2.1 F l u o r o g e n i c D e r i v a t i z a t i o n w i t h Hydrogen  P e r o x i d e , P h o s p h o r i c A c i d and C o n c e n t r a t e d HC1 U s i n g the 10 m r e a c t o r and the aqueous p o r t i o n of the m o b i l e phase as d e h y d r o a s c o r b i c a c i d and hydrogen p e r o x i d e w i t h p h o s p h o r i c a c i d , peak h e i g h t was d e t e r m i n e d f o r 9.1 ng of d i g o x i n ( u s i n g 360 nm/425 nm f i l t e r s ) . 71 2.8.2.2 E v a l u a t i o n of the F l u o r e s c e n c e E m i s s i o n  F i l t e r s w i t h D e h y d r o a s c o r b i c A c i d and HCl D e r i v a t i z a t i o n W i t h a 360 nm e x c i t a t i o n f i l t e r and t h e m o b i l e phase d e s c r i b e d i n s e c t i o n 2.7.3 w i t h a l l of the aqueous p o r t i o n as d e h y d r o a s c o r b i c a c i d , e m i s s i o n f i l t e r s (425, 440 and 460 nm) were e v a l u a t e d by comparing peak h e i g h t s from 13 ng i n j e c t i o n s of d i g o x i n . 2.8.3 F i n a l HPLC-PC Assay P r o c e d u r e A s c h e m a t i c of the HPLC-PC as s a y p r o c e d u r e i s shown i n F i g u r e 5. The f i n a l a s say used an HPLC f l o w r a t e of 0.4 mL/min. w i t h a hexane f l o w r a t e of 0.5 mL/min. and 360 nm e x c i t a t i o n and 425 nm e m i s s i o n f i l t e r s w i t h the f l u o r o m e t e r . 2.9 Serum E x t r a c t i o n P r o c e d u r e 2.9.1 P r o t e i n P r e c i p i t a t i o n Methods Blank serum samples (3 mL) were p r e c i p i t a t e d u s i n g a c e t o n e (3 mL and 6 mL), z i n c / b a r i u m (0.6 mL of 5% z i n c s u l f a t e p l u s 0.6 mL of 0.3 N barium h y d r o x i d e ) and c u p r i c s u l f a t e (1 mL of 10% c u p r i c s u l f a t e p l u s 1 mL of 12% sodium h y d r o x i d e ) f o l l o w e d by e x t r a c t i o n (Kwong 1986a, 1986b). 72 Concentrated HC1 1 cm (3/8 in.) X 0.16 cm (1/16 in.) Stainless Steel Reducing Union (1 of 2). Hexane 1 cm (3/8 in.) NPT X 1 cm (3/8 in.) Stainless Steel Tube Union (1 of 2). 5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Blind Flange (1 of 2). Top only d r i l l e d and tapped with 3/8 i n . NPT. 1.3 cm (1/2 in.) X 6.4 cm (2 1/2 in.) 316 Stainless Steel UNC Bolt complete with nut and two 1.3 cm (1/2 in.) Diameter X 0.16 cm (1/16 in.) 316 Stainless F l a t Washers (1 sets ) . 0.16 cm (1/16 i n . ) PTFE Tubing (0.3 mm I.D.). One continuous length to bottom of pressure vess e l . 1 cm (3/8 in.) Stainless Steel Union Tee. 1 cm (3/8 in.) Diameter X 3.8 cm (1 1/2 in.) Stainless Steel Tube (1 of 3). 5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Socket Weld Flange (1 of 2). 5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Pipe (36.8 cm long). 0.32 cm (1/8 in.) Thick X 5.1 cm (2 in.) Diameter Fu l l Face Hypalon Gasket (1 of 2). F i g u r e 6. Diagram o f HPLC-PC Pressure V e s s e l With F i t t i n g s 73 2.9.2 C e n t r i f r e e and C e n t r i f l o F i l t r a t i o n Serum samples were f i l t e r e d by the C e n t r i f r e e (1 mL serum c e n t r i f u g e d a t 1000 r . c . f . f o r 30 m i n u t e s ) and the C e n t r i f l o (3 mL serum c e n t r i f u g e d a t 1000 r . c . f . f o r 40 m i n u t e s ) systems f o l l o w e d by an i s o o c t a n e wash and double e x t r a c t i o n (Kwong 1986a). Both serum and water samples were s p i k e d w i t h 9.1 ng of d i g o x i n and p r e p a r e d u s i n g the C e n t r i f r e e and C e n t r i f l o systems as d e s c r i b e d above. These samples were e v a l u a t e d u s i n g the f i n a l HPLC-PC assay p r o c e d u r e . 2.9.3 S o l v e n t - s o l v e n t E x t r a c t i o n U s i n g 2 mL of i s o o c t a n e and i s o o c t a n e / d i c h l o r o m e t h a n e (20/5) as s o l v e n t wash, water samples (3 mL) s p i k e d w i t h 9.1 ng of d i g o x i n were e x t r a c t e d and e v a l u a t e d u s i n g the HPLC-PC a s s a y . Serum (3 mL) c o n t a i n i n g 1.5 ng of d i g o x i n and b l a n k serum samples were e x t r a c t e d (Kwong 1986a, 1986b) f o l l o w i n g an i s o o c t a n e / d i c h l o r o m e t h a n e s o l v e n t wash (2 mL of 20/5) and a s s a y e d u s i n g the HPLC-PC a s s a y . 2.9.4 F i n a l P r o c e d u r e f o r E x t r a c t i o n of D i g o x i n i n Serum The f r o z e n serum samples were thawed a t room t e m p e r a t u r e j u s t p r i o r t o a n a l y s i s . For s t a n d a r d c u r v e samples, 3 mL of b l a n k serum was s p i k e d w i t h 10 uL of d i g o x i n i n e t h a n o l (1.5 t o 10 ng/10 ML). For a l l s t a n d a r d c u r v e and p a t i e n t samples, 20 ML of i n t e r n a l s t a n d a r d 74 s o l u t i o n (160 ng) was added t o the serum. A f t e r a d d i t i o n of 3 mL a c e t o n e , the sample was v o r t e x e d 20 seconds, c e n t r i f u g e d 5 minutes a t 1500 r . c . f . and the aqueous/acetone s u p e r n a t a n t l a y e r was t r a n s f e r r e d t o a c l e a n t u b e . T h i s s o l u t i o n was then washed w i t h 2 mL of i s o o c t a n e / d i c h l o r o m e t h a n e ( 2 0 / 5 ) , v o r t e x e d 60 seconds, c e n t r i f u g e d 5 minutes a t 1500 r . c . f . and the aqueous/acetone l a y e r was p a r t i a l l y d r i e d under n i t r o g e n u s i n g a 37°C water b a t h f o r 20 mi n u t e s . The r e m a i n i n g aqueous l a y e r was e x t r a c t e d t w i c e w i t h 10 mL of e x t r a c t i o n s o l v e n t ( d i c h l o r o m e t h a n e / p r o p a n - 1 - o l 98/2) and the combined o r g a n i c phases were f i l t e r e d (Nylon 66 membrane) and d r i e d under n i t r o g e n . The r e s i d u e was resuspended i n 100 uL of methanol/water (50/50). I n j e c t i o n s i n t o the HPLC-PC were performed u s i n g a 100 uL H a m i l t o n s y r i n g e ( H a m i l t o n Co., Reno, NV, U.S.A.). A f l o w diagram of t h i s e x t r a c t i o n p r o c e d u r e i s g i v e n i n F i g u r e 7. 2.10 Recovery and P r e c i s i o n of E x t r a c t i o n The r e c o v e r y of d i g o x i n from serum u s i n g the f i n a l e x t r a c t i o n p r o c e d u r e was e v a l u a t e d . Serum s p i k e d w i t h 1.5, 3 and 10 ng of d i g o x i n were e x t r a c t e d and 10 uL of e x t e r n a l s t a n d a r d s o l u t i o n ( d i g i t o x i g e n i n i n e t h a n o l ) was added j u s t p r i o r t o a n a l y s i s . Recovery was c a l c u l a t e d by comparing the peak h e i g h t r a t i o ( d i g o x i n t o e x t e r n a l s t a n d a r d ) f o r e x t r a c t e d serum samples t o t h a t f o r u n e x t r a c t e d samples. 75 3 mL Serum 1 3 mL Acetone I Vortex 20 seconds. Centrifuge 5 minutes (1500 r . c . f . ) . Wash with 2 mL isooctane/dichloromethane (20/5) Vortex 60 seconds. Centrifuge 5 minutes (1500 r . c . f . ) . Isooctane layer ^ _ (discard) Acetone/aqueous layer I Evaporate (20 minutes) Add 10 mL Extraction solvent (Dichloromethane/ propan-l-ol 98/2) Mix 10 minutes. Centrifuge 5 minutes 1X (1500 r . c . f . ) Aqueous layer* Organic layer 1 Filter . Evaporate. Reconstitute in 0.1 mL methanol/water (50/50). F i g u r e 7. Flow Diagram f o r Serum E x t r a c t i o n P r o c e d u r e 76 P r e c i s i o n of the e x t r a c t i o n p r o c e d u r e was d e t e r m i n e d by r e p e a t e d e x t r a c t i o n of f i v e b l a n k serum samples (3 mL) s p i k e d w i t h 3 ng of d i g o x i n and 20 vL of i n t e r n a l s t a n d a r d f o r f i v e c o n s e c u t i v e days. The c o e f f i c i e n t of v a r i a t i o n f o r w i t h i n and between days was c a l c u l a t e d . 2.11 C a l i b r a t i o n Curve Serum samples (3 mL) were s p i k e d w i t h d i g o x i n i n e t h a n o l ( s e c t i o n 2.4.5.1), e x t r a c t e d and a n a l y z e d u s i n g the HPLC-PC f l u o r o g e n i c a s s a y . The peak h e i g h t and c o n c e n t r a t i o n r a t i o s were then c a l c u l a t e d . 2.12 S p e c i f i c i t y 2.12.1 S t e r o i d s The s t e r o i d samples p r e p a r e d i n s e c t i o n 2.4.5.4 were a s s a y e d u s i n g the post-column f l u o r o g e n i c HPLC a s s a y . 2.12.2 C o - A d m i n i s t e r e d Drugs The R , S - m e x i l e t i n e sample ( s e c t i o n 2.4.5.5) was as s a y e d by the HPLC-PC method t o e s t a b l i s h a s s a y s p e c i f i c i t y i n the p r e s e n c e of t h i s d r u g . 77 2.13 Q u a l i t y C o n t r o l Procedure Blank serum samples (4 mL) were s p i k e d w i t h 13 uL of the d i g o x i n i n e t h a n o l s o l u t i o n s ( s e c t i o n 2.4.5.1). A f t e r m i x i n g , 3 mL samples were removed, e x t r a c t e d and a n a l y z e d u s i n g the HPLC-PC f l u o r o g e n i c a s s a y . The r e m a i n i n g sample was then a n a l y s e d w i t h the FPIA f o r d i g o x i n a t the ACU L a b o r a t o r y . 2.14 A n a l y s i s of D i g i t a l i z e d P a t i e n t Serum Samples Serum samples from 25 U.B.C. H e a l t h S c i e n c e s C e n t r e Acute Care U n i t H o s p i t a l (Vancouver, B.C.) p a t i e n t s who r e c e i v e d d i g o x i n t h e r a p e u t i c a l l y were assa y e d by both HPLC-PC and FPIA methods. The r e s u l t s from the two a n a l y t i c a l methods were then compared. 2.15 A n a l y s i s of Serum from U n d i g i t a l i z e d P a t i e n t Groups Where High DLIS L e v e l s have been Reported 2.15.1 H y p e r t e n s i o n Serum samples from 5 u n d i g i t a l i z e d h y p e r t e n s i v e p a t i e n t s were o b t a i n e d (U.B.C. H e a l t h S c i e n c e s C e n t r e Acute Care U n i t H o s p i t a l , Vancouver, B.C.) and e v a l u a t e d by both t h e HPLC-PC and FPIA methods. 2.15.2 Re n a l F a i l u r e Serum samples from 20 u n d i g i t a l i z e d r e n a l f a i l u r e p a t i e n t s who were on d i a l y s i s were o b t a i n e d from the W i l l o w 78 D i a l y s i s U n i t (Vancouver G e n e r a l H o s p i t a l , Vancouver, B.C.) and a s s a y e d by both the HPLC-PC and FPIA f o r d i g o x i n . 2.15.3 H e p a t i c F a i l u r e Serum samples from 8 u n d i g i t a l i z e d h e p a t i c f a i l u r e p a t i e n t s and two samples from one d i g i t a l i z e d h e p a t i c f a i l u r e p a t i e n t were o b t a i n e d from U.B.C. H e a l t h S c i e n c e s C e n t r e Acute Care U n i t H o s p i t a l (Vancouver, B.C.) and St P a u l ' s H o s p i t a l (Vancouver, B.C.) and have been e v a l u a t e d by bo t h HPLC-PC and FPIA methods. 2.15.4 U m b i l i c a l Cord B l o o d Samples A t o t a l of 17 mixed c o r d b l o o d samples from 11 p a t i e n t s were o b t a i n e d from C h i l d r e n ' s H o s p i t a l (Vancouver, B.C.) and were a s s a y e d by the HPLC-PC and FPIA methods. 79 3. RESULTS AND DISCUSSIONS 3.1 Radioimmunoassay of Di g o x i n and I t s M e t a b o l i t e s Samples of d i g o x i n and i t s m e t a b o l i t e s e x h i b i t e d c r o s s -r e a c t i v i t y v a l u e s of v a r y i n g magnitudes, c a l c u l a t e d on a molar b a s i s , u s i n g the radioimmunoassay k i t s as shown i n T a b l e I I . C r o s s - r e a c t i v i t y of d i h y d r o d i g o x i n was found t o be a p p r o x i m a t e l y 46% f o r the D i g i - T a b RIA k i t but was too low t o be de t e r m i n e d f o r the o t h e r f i v e k i t s u s i n g t h i s c o n c e n t r a t i o n range. However, the s e r e s u l t s i n d i c a t e t h a t d i h y d r o d i g o x i n , a t c o n c e n t r a t i o n s w i t h i n t h e t h e r a p e u t i c range f o r d i g o x i n , i n t e r f e r e s t o a g r e a t e r e x t e n t than r e p o r t e d by t h i s m a n u f a c t u r e r ( c r o s s - r e a c t i v i t y of 1.8%). Other i n v e s t i g a t o r s have found t h a t t h i s m e t a b o l i t e has a c r o s s - r e a c t i v i t y w i t h d i g o x i n a n t i b o d i e s r a n g i n g from v e r y low v a l u e s ( M a l i n i 1982) t o as h i g h as 30% ( G a u l t 1979; Kramer 1976, 1978; Oge 1978). The c r o s s - r e a c t i v i t y o bserved here f o r d i h y d r o d i g o x i n was g r e a t e r than t h a t i n d i c a t e d p r e v i o u s l y i n the l i t e r a t u r e . D i h y d r o d i g o x i g e n i n , i f p r e s e n t , r e p r e s e n t s a minor f r a c t i o n of the m e t a b o l i t e s of d i g o x i n . However, l a r g e q u a n t i t i e s of d i h y d r o d i g o x i g e n i n have been o b s e r v e d i n u r i n e samples from p a t i e n t s w i t h i n c r e a s e d d i g o x i n r e q u i r e m e n t s ( L u c h i 1968). C o n s i d e r a b l e i n t e r f e r e n c e ( a p p r o x i m a t e l y 22% c r o s s - r e a c t i v i t y ) was ob s e r v e d when t h i s m e t a b o l i t e was as s a y e d u s i n g the D i g i - T a b k i t . The c r o s s - r e a c t i v i t y of d i h y d r o d i g o x i g e n i n was too low t o be e s t i m a t e d w i t h the 80 o t h e r k i t s a t th e s e c o n c e n t r a t i o n s . For d i h y d r o d i g o x i g e n i n , a c r o s s - r e a c t i v i t y of l e s s than 12% has been r e p o r t e d w i t h d i g o x i n RIA methods ( G a u l t 1982). The i n t e r f e r e n c e found here f o r d i h y d r o d i g o x i g e n i n was g r e a t e r than t h a t r e p o r t e d i n the l i t e r a t u r e . Table I I . Percent C r o s s - R e a c t i v i t y on a Molar B a s i s f o r Digoxin M e t a b o l i t e s With Commercial Digoxin RIA K i t s . RIA K i t s Used A B C D E F D i g o x i n 100 100 100 100 100 100 D i g o x i g e n i n 60 250 95 103 74 95 D i g o x i g e n i n M o n o d i g i t o x o s i d e 133 152 95 116 113 95 D i g o x i g e n i n B i s d i g i t o x o s i d e 208 159 89 102 103 104 D i h y d r o d i g o x i n 46 D i h y d r o d i g o x i g e n i n 22 - -RIA K i t s Used: A D i g i - T a b RIA B B i o - R I A 1-125 D i g o x i n C Amerlex D i g o x i n RIA K i t D D i g o x i n S o l i d Phase RIA (Becton D i c k i n s o n ) E GammaCoat D i g o x i n RIA F D i g o x i n 1-125 Imusay For d i g o x i g e n i n , d i g o x i g e n i n mono- and b i s -d i g i t o x o s i d e , the average c r o s s - r e a c t i v i t y was 120% (range 60% t o 250%). These m e t a b o l i t e s e x h i b i t e d t h e g r e a t e s t range i n c r o s s - r e a c t i v i t y f o r the D i g i - T a b RIA (A) and B i o -RIA (B) k i t s . The o t h e r f o u r RIA p r o c e d u r e s showed a s m a l l e r range and an average c r o s s - r e a c t i v i t y of 99% (range 81 74% t o 116%) f o r t h e s e t h r e e d i g i t o x o s e m e t a b o l i t e s . These r e s u l t s agree w i t h p r e v i o u s r e p o r t s on c r o s s - r e a c t i v i t y of t h e s e m e t a b o l i t e s w i t h c ommercial i o d i n e - 1 2 5 d i g o x i n RIA k i t s ( V a l d e s 1984). The r e s u l t s r e p o r t e d here emphasize the l a c k of s p e c i f i c i t y of the p o l y c l o n a l a n t i b o d i e s used i n c ommercial i o d i n e - 1 2 5 RIA k i t s f o r d i g o x i n a n a l y s i s . V a r i a b i l i t y i n the c r o s s - r e a c t i v i t y was o b s e r v e d between the RIA k i t s and between the m e t a b o l i t e s of d i g o x i n . The presence of an i n t a c t l a c t o n e r i n g t o i n t e r a c t w i t h the d i g o x i n a n t i b o d y appears t o be n e c e s s a r y f o r h i g h c r o s s - r e a c t i v i t y . However, removal of the d i g i t o x o s e s u g ars does not g r e a t l y a f f e c t the c r o s s - r e a c t i v i t y . D i g o x i n p o l y c l o n a l a n t i b o d i e s a r e formed a g a i n s t an immunogenic d i g o x i n - p r o t e i n c o n j u g a t e w i t h an i n t a c t a g l y c o n e ( B u t l e r 1978), t h e r e f o r e , most d i g o x i n a n t i s e r a w i l l c r o s s - r e a c t e x t e n s i v e l y t o the d i g o x i g e n i n m o i e ty as was o b s e r v e d . A l o n g w i t h t h i s l a c k of s p e c i f i c i t y , i n t e r p r e t a t i o n of RIA r e s u l t s are f u r t h e r c o m p l i c a t e d s i n c e the m e t a b o l i t e s of d i g o x i n do not have the same potency as d i g o x i n (Aronson 1980; I i s a l o 1977) and t h e r e i s a l a r g e i n t e r p a t i e n t v a r i a b i l i t y i n d i g o x i n m e t a b o l i s m (Aronson 1980; C l a r k 1974; G a u l t 1976; I i s a l o 1977). I n a p p r o p r i a t e changes i n p a t i e n t d i g o x i n t h e r a p y may be i n i t i a t e d due t o the l a c k of s p e c i f i c i t y of the RIA used. 82 3.2 F l u o r e s c e n c e P o l a r i z a t i o n Immunoassay of Digoxin M e t a b o l i t e s i n Serum The FPIA r e q u i r e s serum p r o t e i n s t o be p r e c i p i t a t e d w i t h 5 - s u l f o s a l i c y l i c a c i d (Skogen 1987) which has been s u g g e s t e d t o r e s u l t i n the t r a n s f o r m a t i o n of d i g o x i n and i t s m e t a b o l i t e s t o d i g o x i g e n i n and/or d i h y d r o d i g o x i g e n i n ( G a u l t 1977; Sonobe 1980; S t e r n s o n 1978). T h e r e f o r e i t i s a n t i c i p a t e d t h a t d i g o x i n w i l l not be d i f f e r e n t i a t e d from i t s m e t a b o l i t e s which may be p r e s e n t i n p a t i e n t samples. Serum samples c o n t a i n i n g a p p r o x i m a t e l y 1.3 nmol/L of each m e t a b o l i t e were assay e d by the FPIA. The r e s u l t s from the FPIA shown i n T a b l e I I I ranged from 0.7 t o 4.8 nmol/L a l t h o u g h no d i g o x i n was p r e s e n t i n t h e s e samples. I n t e r f e r e n c e from d i g o x i n m e t a b o l i t e s i s t h e r e f o r e a s i g n i f i c a n t problem w i t h the FPIA a s s a y . The c r o s s -r e a c t i v i t y v a l u e s r e p o r t e d w i t h the FPIA f o r d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e a r e as g r e a t or g r e a t e r than th o s e o b s e r v e d f o r the s i x RIA k i t s e v a l u a t e d . For d i h y d r o d i g o x i g e n i n , the r e p o r t e d c r o s s -r e a c t i v i t y f o r the FPIA (91%) was c o n s i d e r a b l y g r e a t e r than t h a t found w i t h the D i g i - T a b RIA ( 2 2 % ) . The c r o s s -r e a c t i v i t y of the FPIA method w i t h the d i g o x i n m e t a b o l i t e s i s as g r e a t or g r e a t e r than t h a t o b s e r v e d w i t h the RIA p r o c e d u r e . The FPIA assay i n d i c a t e d i n c r e a s e d a p p a r e n t d i g o x i n l e v e l s i n the presence of DLIS ( B i a n c h i 1986; F r y e 1987; 83 Table I I I . E v a l u a t i o n of Di g o x i n M e t a b o l i t e s i n Serum Using the FPIA f o r Digoxin A n a l y s i s M e t a b o l i t e A c t u a l C o n c e n t r a t i o n (nmol/L) Bl a n k Serum none D i g o x i g e n i n 1.41 D i g o x i g e n i n M o n o d i g i t o x o s i d e 1.48 D i g o x i g e n i n B i s d i g i t o x o s i d e 1.17 D i h y d r o d i g o x i n 1.27 D i h y d r o -d i g o x i g e n i n 1 .70 FPIA C r o s s - R e a c t i v i t y Assay R e p o r t e d by FPIA R e s u l t s M a n u f a c t u r e r (nmol/L) (%) none 1.8 >100 4.8 >100 3.6 >100 0.8 0.7 91 Kanan 1987; Skogen 1987; S o l d i n 1986b; Weiner 1987), however l e s s i n t e r f e r e n c e from DLIS was r e p o r t e d w i t h the FPIA than the RIA method ( Y a t s c o f f 1984). For e v a l u a t i o n of d i g o x i n i n p a t i e n t samples, the FPIA may t h e r e f o r e be b e t t e r than the RIA when DLIS i s p r e s e n t . The c l i n i c a l s i g n i f i c a n c e of h i g h c r o s s - r e a c t i v i t y of bo t h the RIA and FPIA methods w i t h d i g o x i g e n i n b i s d i g i t o x o s i d e and d i g o x i g e n i n m o n o d i g i t o x o s i d e may be l o s t s i n c e t h e s e m e t a b o l i t e s r e t a i n some potency (Keys 1980). D i g o x i g e n i n , d i h y d r o d i g o x i n and d i h y d r o d i g o x i g e n i n have s u b s t a n t i a l l y lower potency than d i g o x i n so h i g h c r o s s -r e a c t i v i t y w i t h the RIA and FPIA methods i s more s i g n i f i c a n t . C o n s i d e r i n g the l a r g e i n t e r p a t i e n t v a r i a b i l i t y i n d i g o x i n m e t a b o l i s m (Aronson 1980) and t h a t from 57 t o 60% 84 of d i g o x i n may be e x c r e t e d as m e t a b o l i c p r o d u c t , m a i n l y d i h y d r o d i g o x i n ( C l a r k 1974; L u c h i 1968), t h e p o s s i b i l i t y of p a t i e n t samples c o n t a i n i n g h i g h l e v e l s of t h e s e m e t a b o l i t e s w i t h low potency and h i g h c r o s s - r e a c t i v i t y e x i s t s . S i n c e t h e p o t e n c i e s of these m e t a b o l i t e s a r e v e r y low w i t h r e s p e c t t o d i g o x i n , t h e i r i n t e r f e r e n c e w i t h the RIA and FPIA methods i s c l i n i c a l l y s i g n i f i c a n t . O v e r - e s t i m a t i o n of d i g o x i n due t o i n t e r f e r e n c e from i t s m e t a b o l i t e s may l e a d t o i n a p p r o p r i a t e changes i n p a t i e n t d i g o x i n t h e r a p y . 3.3 Pre-Column 3 , 5 - D i n i t r o b e n z o y l D e r i v a t i z a t i o n of C a r d i a c G l y c o s i d e s w i t h HPLC-EC A n a l y s i s 3.3.1 U l t r a v i o l e t D e t e c t i o n W i th the 3,5-DNB d e r i v a t i z a t i o n p r o c e d u r e r e p o r t e d by F u j i i (1983), t h e s t r u c t u r e of d e r i v a t i z e d d i g o x i n formed i s shown i n F i g u r e 8. Tra c e amounts of d i g o x i n , d i h y d r o d i g o x i n , d i g o x i g e n i n , d i h y d r o d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e as w e l l as d i g i t o x i g e n i n and g i t o x i n were d e r i v a t i z e d . The r e t e n t i o n t i m e s of 3,5-DNB d i g o x i n and i t s d e r i v a t i z e d m e t a b o l i t e s a r e shown i n T a b l e IV. The chromatogram of d i g o x i n d e r i v a t i z e d by t h i s p r o c e d u r e ( F i g u r e 9) i n d i c a t e s t h a t s m a l l amounts of the d e r i v a t i z e d m e t a b o l i t e s [ d i g o x i g e n i n (5.96 m i n u t e s ) , d i g o x i g e n i n m o n o d i g i t o x o s i d e (9.82 minutes) and p o s s i b l y d i g o x i g e n i n b i s d i g i t o x o s i d e (17.77 m i n u t e s ) ] a r e p r e s e n t . 85 Table IV. HPLC R e t e n t i o n Times of 3,5-DNB D e r i v a t i v e s G l y c o s i d e R e t e n t i o n time ( m i n u t e s ) D i g o x i n 37.23 D i g o x i g e n i n 5.64 D i h y d r o d i g o x i g e n i n 5.63 D i g o x i g e n i n M o n o d i g i t o x o s i d e 9.63 D i g o x i g e n i n B i s d i g i t o x o s i d e 16.90 D i h y d r o d i g o x i n 35.81 Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3Mm HPLC (4.6 mm x 15 cm); f l o w r a t e : 1.0 mL/min.; m o b i l e phase: a c e t o n i t r i l e / w a t e r ( 2 0 / 7 ) ; u l t r a v i o l e t d e t e c t i o n w a v e l e n g t h : 254 nm. F i g u r e 8. S t r u c t u r e of 3,5-DNB Digo x i n 86 1 i 1 r-0 2 0 4 0 TIME (MIN) F i g u r e 9. Chromatogram of 3,5-DNB Dig o x i n D e r i v a t i z e d i n M i l l i g r a m Q u a n t i t i e s C h r o m a t o g r a p h i c c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3/xm HPLC (4.6 mm x 15 cm); f l o w r a t e : 1.0 mL/min.; m o b i l e phase: a c e t o n i t r i l e / w a t e r ( 2 0 / 7 ) ; u l t r a v i o l e t d e t e c t i o n w a v e l e n g t h : 254 nm; range: 0.005; i n j e c t i o n volume: 10 uL; c h a r t speed: 0.2 cm/min. Peak i d e n t i t y : 1, 3,5-DNB d i g o x i n . 87 Comparison t o the chromatogram o b t a i n e d from the bl a n k sample c o n f i r m s t h a t t h e s e peaks a r e due t o breakdown p r o d u c t s or c o n t a m i n a n t s of the d i g o x i n used. D i g o x i n was d e r i v a t i z e d on a l a r g e s c a l e w i t h 3,5-d i n i t r o b e n z o y l c h l o r i d e as d e s c r i b e d i n s e c t i o n 2.3.1 and p u r i f i e d by double r e c r y s t a l l i z a t i o n from methanol. The m e l t i n g p o i n t ( o b s e r v e d = 203 °C; e x p e c t e d = 203-205 °C ( F u j i i 1 9 8 3 ) ) , HPLC r e t e n t i o n t ime w i t h a c e t o n i t r i l e / w a t e r (20/7) m o b i l e phase and u l t r a v i o l e t d e t e c t i o n f o r 3,5-DNB d i g o x i n ( o b s e r v e d = 37.23 m i n u t e s ; e x p e c t e d = 36 minutes ( F u j i i 1 9 8 3 ) ) , and t h e o r d e r of e l u t i o n of 3,5-DNB d i g o x i n m e t a b o l i t e s , 3,5-DNB g i t o x i n and 3,5-DNB d i g i t o x i g e n i n ( s e c t i o n 2.3.2) c o n f i r m e d the i d e n t i t y of the r e c r y s t a l l i z e d 3,5-DNB d i g o x i n . 3.3.2 D e t e c t i o n W i t h a S i n g l e G l a s s y Carbon E l e c t r o d e P r e l i m i n a r y r e s u l t s w i t h an a c e t o n i t r i l e / a c e t a t e b u f f e r system (85/15) and r e d u c t i o n u s i n g the s i n g l e g l a s s y carbon e l e c t r o d e of the 5012 sc r e e n e d w a l l j e t c e l l a t p o t e n t i a l s from 0 t o -0.85 V i n d i c a t e d t h a t a p o t e n t i a l of -0.80 V w i t h 0.1 M a c e t a t e b u f f e r gave the g r e a t e s t s e n s i t i v i t y . D e c r e a s i n g the b u f f e r c o n c e n t r a t i o n l e a d t o a l o s s i n peak h e i g h t . C o n c e n t r a t i o n s s i g n i f i c a n t l y g r e a t e r than 0.1 M a r e not recommended f o r use w i t h t h i s d e t e c t o r (ESA M a n u a l ) . With 0.1 M sodium a c e t a t e / a c e t i c a c i d b u f f e r (pH 4.6), the l i m i t of d e t e c t i o n f o r 3,5-DNB d i g o x i n and r e s o l u t i o n of 3,5-DNB d i g o x i n from 3,5-DNB d i h y d r o d i g o x i n 88 f o r v a r i o u s m o b i l e phases i s shown i n T a b l e V. The l i m i t of d e t e c t i o n was found t o be 2.2 ng of 3,5-DNB d i g o x i n (0.98 ng d i g o x i n ) u s i n g the r e c r y s t a l l i z e d sample. W i t h s i m i l a r s e n s i t i v i t y , b e t t e r r e s o l u t i o n between 3,5-DNB d i g o x i n and 3,5-DNB d i h y d r o d i g o x i n was o b t a i n e d w i t h a m o b i l e phase of m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / i s o p r o p a n o l / b u f f e r (40/3/60/2/ 2 2 ) . F i g u r e 10 shows a chromatogram of the 3,5-DNB d e r i v a t i v e s of d i g o x i n and i t s m e t a b o l i t e s u s i n g t h i s m o b i l e phase. T a b l e V. R e s o l u t i o n of 3,5-DNB D i g o x i n and 3,5-DNB D i h y d r o d i g o x i n U s i n g HPLC-EC w i t h V a r i o u s M o b i l e Phases M o b i l e Phase C o m p o s i t i o n P o l a r i t y R e s o l u t i o n L i m i t of MeOH EtOH ACN I PA B u f f e r of 3,5-DNB D e t e c t i o n D i g o x i n and (3,5-DNB 3,5-DNB d i g o x i n ) D i h y d r o d i g o x i n 40 3 40 3 1 5 6.08 none 40 3 40 3 17 6.16 none 40 3 40 3 18 6.19 0.86 38 3 40 3 18 6. 194 0.80 40 3 40 2.75 18 6.20 0.70 40 3 40 3 18.4 6.21 0.67 8.8 40 3 50 3 20 6.229 0.60 40 3 60 3 22 6.258 0.50 2.8 40 3 60 2 22 6.276 0.75 2.8 40 3 60 1 22 6.295 0.70 2.2 MeOH HPLC methanol EtOH a b s o l u t e e t h a n o l ACN HPLC a c e t o n i t r i l e I PA HPLC i s o p r o p a n o l B u f f e r 0.1 M sodium a c e t a t e / a c e t i c a c i d b u f f e r (pH 4.6) Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3 urn HPLC (4.6 mm x 15 cm); f l o w r a t e : 1.0 mL/min.; guard e l e c t r o d e : 5020 guard c e l l e l e c t r o d e a t -0.85 V p r e -i n j e c t o r ; d e t e c t i o n : 5012 s c r e e n e d w a l l j e t c e l l r e c o r d i n g g l a s s y carbon e l e c t r o d e a t -0.80 V. 89 1 0 5 10 15 TIME (MIN) F i g u r e 10. Chromatogram of D i g o x i n and i t s M e t a b o l i t e s as T h e i r 3,5-DNB D e r i v a t i v e s u s i n g E l e c t r o c h e m i c a l D e t e c t i o n Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS II 3 p HPLC (4.6 mm x 15 cm); flow r a t e : 1.0 mL/min.; mobile phase: m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / i s o p r o p a n o l / a c e t a t e b u f f e r (pH 4.6, 0.1 M) (40/3/60/2/22); guard e l e c t r o d e : 5020 guard c e l l e l e c t r o d e at -0.85 V p r e - i n j e c t o r ; d e t e c t i o n : 5012 screened w a l l j e t c e l l r e c o r d i n g g l a s s y carbon e l e c t r o d e a t -0.80 V. Peak i d e n t i t y : 1, 3,5-DNB d i g o x i g e n i n ; 2, 3,5-DNB d i g o x i g e n i n monodigitoxoside; 3, 3,5-DNB d i g o x i g e n i n b i s d i g i t o x o s i d e ; 4, 3,5-DNB d i h y d r o d i g o x i n ; 5, 3,5-DNB d i g o x i n . 90 3.3.3 Dual E l e c t r o d e D e t e c t i o n To improve the s t a b i l i t y and s e n s i t i v i t y of e l e c t r o c h e m i c a l d e t e c t i o n , a 5010 f l o w c e l l , which has two g l a s s y carbon e l e c t r o d e s , was used i n the redox mode. The 3,5-DNB d i g o x i n was reduced a t the f i r s t e l e c t r o d e (-0.80 V) and then t h e reduced p r o d u c t s were o x i d i z e d a t the second e l e c t r o d e (+0.80 V ) . R e c o r d i n g from the o x i d a t i v e e l e c t r o d e gave a maximum s e n s i t i v i t y of 0.883 ng of r e c r y s t a l l i z e d 3,5-DNB d i g o x i n (0.394 ng d i g o x i n ) ( m o b i l e phase: m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / i s o p r o p a n o l / a c e t a t e b u f f e r 40/3/60/2/22). R e c o r d i n g the o x i d a t i v e e l e c t r o d e a l o n e d e c r e a s e d the b a s e l i n e n o i s e seen i n the r e d u c t i v e mode, and l o w e r e d the e q u i l i b r i u m time f o r the system. D e r i v a t i z a t i o n of m i l l i g r a m q u a n t i t i e s of d i g o x i n ( s e c t i o n 2.3.2) f o l l o w e d by HPLC a n a l y s i s d e s c r i b e d above i n d i c a t e d 3,5-DNB d i g o x i n was formed. HPLC a n a l y s i s of the r e s i d u e from t r a c e l e v e l d e r i v a t i z a t i o n of d i g o x i n ( s e c t i o n 2.3.3) i n d i c a t e d no 3,5-DNB d i g o x i n was p r e s e n t . S i l a n i z a t i o n of a l l g l a s s w a r e w i t h d i m e t h y l c h l o r o s i l a n e i n t o l u e n e , f o l l o w e d by d e r i v a t i z a t i o n and a n a l y s i s of 1 t o 80 ng samples of d i g o x i n by the same p r o c e d u r e ( s e c t i o n 2.3.3) showed 3,5-DNB d i g o x i n was formed i n the l a r g e r samples. There i s a s i g n i f i c a n t l o s s i n s e n s i t i v i t y when ng samples a r e d e r i v a t i z e d as compared t o a n a l y s i s of ng q u a n t i t i e s of the r e c r y s t a l l i z e d d e r i v a t i v e ( s e c t i o n 2.3.1) as shown by comparison of F i g u r e s 11 and 12. F i g u r e 12 a l s o shows the 91 h i g h l e v e l s of d e r i v a t i z e d m e t a b o l i t e s formed when s i l a n i z e d g l a s s w a r e was used (3,5-DNB d i g o x i g e n i n b i s d i g i t o x o s i d e a t 7.61 minutes and 3,5-DNB d i g o x i g e n i n m o n o d i g i t o x o s i d e a t 5.51 m i n u t e s ) . The amount of m e t a b o l i t e f o r m a t i o n d u r i n g d e r i v a t i z a t i o n was much g r e a t e r than p r e v i o u s l y i n d i c a t e d ( F u j i i 1983). 3,5-DNB d i g o x i n and the d e r i v a t i z e d d i g o x i n m e t a b o l i t e s appear t o b i n d t o the u n s i l a n i z e d g l a s s , and when ng q u a n t i t i e s of d i g o x i n a r e d e r i v a t i z e d t h i s a d s o r p t i o n becomes s i g n i f i c a n t . A l s o , d e r i v a t i z a t i o n was not as complete as e x p e c t e d ( F u j i i 1983), but t h e y i e l d was not d e t e r m i n e d s i n c e the s e n s i t i v i t y of the HPLC-EC assay was not s u f f i c i e n t f o r a q u a n t i t a t i v e serum d i g o x i n a s s a y . In a c i d i c aqueous s o l u t i o n s a r o m a t i c n i t r o groups a re e l e c t r o c h e m i c a l l y reduced i n two s u c c e s s i v e s t e p s c o r r e s p o n d i n g t o 4- and 2 - e l e c t r o n t r a n s f e r s r e s p e c t i v e l y (Mousty 1986). The i n t e r m e d i a t e h y d r o x y l a m i n e formed by the 4 - e l e c t r o n t r a n s f e r can then be f u r t h e r reduced t o the amine (Mousty 1986). In n e u t r a l or b a s i c media i n t e r m e d i a t e s a r e formed p r i o r t o the h y d r o x y l a m i n e (Mousty 1986). With a r o m a t i c 3 , 5 - d i n i t r o g roups, the n i t r o groups a r e e i t h e r i n d i v i d u a l l y reduced t o amines i n 6 - e l e c t r o n t r a n s f e r s or s i m u l t a n e o u s l y reduced i n one 1 2 - e l e c t r o n t r a n s f e r (Lund 1983). In a l k a l i n e s o l u t i o n s e l e c t i v e r e d u c t i o n of one n i t r o group of 3 , 5 - d i n i t r o a r o m a t i c compounds was d i f f i c u l t (Lund 1983). 92 I I G 10 TIME (MIN) F i g u r e 11. Chromatogram of 125 ng of 3,5-DNB Digoxin P u r i f i e d by Double R e c r y s t a l l i z a t i o n Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3 um HPLC (4.6 mm x 15 cm); f l o w r a t e : 1.0 mL/min.; m o b i l e phase: m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / a c e t a t e b u f f e r (pH 4.6, 0.1 M) (40/3/50/3/20); guard e l e c t r o d e : 5020 guard c e l l e l e c t r o d e a t -0.85 V p r e - i n j e c t o r ; d e t e c t i o n : 5010 a n a l y t i c a l c e l l w i t h e l e c t r o d e 1 a t -0.80 V and e l e c t r o d e 2 a t + 0.80 V ( r e c o r d i n g e l e c t r o d e 2 ) . Peak i d e n t i t y : 1 , 3,5-DNB d i g o x i n . 93 0 10 TIME (MIN) F i g u r e 12. Chromatogram of 80 ng of D i g o x i n D e r i v a t i z e d i n M i l l i g r a m Q u a n t i t i e s Chromatographic c o n d i t i o n s : Same as f o r F i g u r e 11. Peak i d e n t i t y : 1, 3,5-DNB d i g o x i n . 94 W i t h the HPLC-EC system d e v e l o p e d f o r 3,5-DNB d i g o x i n , r e d u c t i o n o c c u r r e d i n the m i x t u r e of o r g a n i c s o l v e n t s and a c e t a t e b u f f e r (pH 4.6) used f o r the HPLC m o b i l e phase. Under t h e s e c o n d i t i o n s , i t i s a n t i c i p a t e d t h a t b o t h n i t r o groups a r e reduced s i m u l t a n e o u s l y t o amines. Whether r e d u c t i o n of one or both n i t r o groups w i t h t h i s HPLC-EC system o c c u r r e d was not d e t e r m i n e d . The p o s s i b i l i t y of h y d r o x y l a m i n e f o r m a t i o n was a l s o not e v a l u a t e d . Amperometric d e t e c t o r s a p p l y a c o n s t a n t p o t e n t i a l t o the d e t e c t i n g e l e c t r o d e and m o n i t o r the r e s u l t a n t c u r r e n t s produced as a n a l y t e s f l o w p a s t the e l e c t r o d e s . T y p i c a l l y , between 1 and 5% of the a n a l y t e i s e l e c t r o l y z e d . As the f r a c t i o n of sample e l e c t r o l y z e d approaches u n i t y , the d e t e c t o r i s s a i d t o be c o u l o m e t r i c . The h i g h s u r f a c e a r e a of the g l a s s y carbon e l e c t r o d e s used here a l l o w s them t o f u n c t i o n c o u l o m e t r i c a l l y . Assuming a l l the n i t r o groups on 3,5-DNB d i g o x i n were a v a i l a b l e f o r r e d u c t i o n , the number of e l e c t r o n s t r a n s f e r r e d per mole and t h e r e f o r e the e x t e n t of r e d u c t i o n , c o u l d be e s t i m a t e d u s i n g the peak a r e a o b t a i n e d from i n j e c t i o n of a known q u a n t i t y of 3,5-DNB d i g o x i n . 3,5-DNB d i g o x i n and 3,5-DNB d i h y d r o d i g o x i n were p a r t i a l l y r e s o l v e d . Some breakdown of d i g o x i n t o i t s m e t a b o l i t e s d u r i n g d e r i v a t i z a t i o n and the maximum s e n s i t i v i t y o b t a i n e d made i t i m p r a c t i c a l t o d e v e l o p an assay f o r t h e r a p e u t i c m o n i t o r i n g of d i g o x i n u s i n g t h i s HPLC-EC a s s a y . T h e r e f o r e , the number of e l e c t r o n s t r a n s f e r r e d and the p r o d u c t formed by r e d u c t i o n of 3,5-DNB d i g o x i n were not d e t e r m i n e d . 95 Unexamined a l t e r a t i o n s t o t h e HPLC-EC as s a y f o r 3,5-DNB d i g o x i n t h a t may have l e d t o an i n c r e a s e d s e n s i t i v i t y were m o d i f y i n g the s u r f a c e of the d e t e c t i n g e l e c t r o d e , i n c r e a s i n g t h e time spent i n the a n a l y t i c a l f l o w c e l l and i n c r e a s i n g t h e aqueous p o r t i o n of the m o b i l e phase. E l e c t r o d e s w i t h c h e m i c a l l y m o d i f i e d s u r f a c e s have been d e v e l o p e d (Lund 1983) t o a l t e r b o t h s e l e c t i v i t y and s e n s i t i v i t y . No m o d i f i c a t i o n s have been s u g g e s t e d f o r enhancing s e n s i t i v i t y i n the r e d u c t i o n of n i t r o groups. A l s o , the f l o w - t h r o u g h g l a s s y c arbon e l e c t r o d e may d e v e l o p f l o w r e l a t e d problems i f s u r f a c e m o d i f y i n g m a t e r i a l s were added, so t h i s was not e v a l u a t e d . W i t h o u t m o d i f y i n g the geometry of t h e f l o w c e l l , t he time spent i n the d e t e c t o r c o u l d be i n c r e a s e d by d e c r e a s i n g the m o b i l e phase f l o w r a t e . Improved s e n s i t i v i t y may r e s u l t i f the r e d u c t i o n was not complete due t o i n s u f f i c i e n t t i me spent i n the f l o w c e l l . T h i s was not at t e m p t e d s i n c e the de c r e a s e i n c h r o m a t o g r a p h i c e f f i c i e n c y produced may r e s u l t i n a d e c r e a s e i n r e s o l u t i o n between 3,5-DNB d i g o x i n and 3,5-DNB d i h y d r o d i g o x i n . I n c r e a s i n g the aqueous p o r t i o n of the m o b i l e phase would a l l o w a d d i t i o n of c h e l a t i n g a gents and p o s s i b l y a more s t a b l e b a s e l i n e would be formed. T h i s was not e v a l u a t e d due t o t h e i n c r e a s e i n ch r o m a t o g r a p h i c r e t e n t i o n time and p o s s i b l e d e c r e a s e i n r e s o l u t i o n between 3,5-DNB d i g o x i n and 3,5-DNB d i h y d r o d i g o x i n . Other d e r i v a t i z a t i o n methods f o r a d d i n g n i t r o groups t o d i g o x i n have been r e p o r t e d ( F l a s c h 1981; Nachtmann 1976a, 96 1976b). The c a r b o n y l f u n c t i o n of the l a c t o n e r i n g of d i g o x i n has been d e r i v a t i z e d w i t h d i n i t r o b e n z o i c a c i d , t e t r a n i t r o b i p h e n y l and s e v e r a l o t h e r d i n i t r o a r o m a t i c compounds ( F l a s c h 1981). The t o t a l number of n i t r o groups added t o d i g o x i n by t h e s e r e a c t i o n s i s c o n s i d e r a b l y l e s s than w i t h 3,5-DNBCl. D e r i v a t i z a t i o n w i t h 4 - n i t r o b e n z o y l c h l o r i d e (Nachtmann 1976a, 1976b) would a l s o r e s u l t i n fewer n i t r o groups added t o d i g o x i n than w i t h 3,5-DNBCl. The s e n s i t i v i t y of d e t e c t i o n i s p r o p o r t i o n a l t o the t o t a l number of n i t r o groups per m o l e c u l e of d e r i v a t i z e d d i g o x i n so t h e s e methods were not e v a l u a t e d . D e r i v a t i z a t i o n of d i g o x i n w i t h o t h e r e l e c t r o a c t i v e a g e n t s has not been r e p o r t e d i n the l i t e r a t u r e . K a d i s h (1975) d e s c r i b e s the use of p o l a r o g r a p h y f o r the e l e c t r o c h e m i c a l d e t e r m i n a t i o n of both d i g o x i n and d i g i t o x i n . The peak r e d u c t i v e p o t e n t i a l f o r d i g o x i n was -2.285 V i n i s o p r o p a n o l , w i t h tetrabutylammonium i o d i d e (0.01 M) as e l e c t r o l y t e . Carbon e l e c t r o d e s have a w o r k i n g range from +1.0 V t o -0.8 V w h i c h can be extended a few t e n t h s of a v o l t by u s i n g the s c r e e n i n g e l e c t r o d e t o d e c r e a s e the background n o i s e . T h i s f l o w t h r o u g h d e t e c t o r would not be f u n c t i o n a l a t -2.285 V and so r e d u c t i o n of u n d e r i v a t i z e d d i g o x i n was not p o s s i b l e . 97 3.4 Post-Column F l u o r o g e n i c HPLC Assay 3.4.1 P r e s s u r e V e s s e l Improvements t o p r e v i o u s l y r e p o r t e d HPLC-PC f l u o r e s c e n c e a s s a y methods (Desta 1987; G f e l l e r 1977; Kwong 1986a, 1986b) a r e based on a l t e r i n g the method of d e l i v e r i n g t h e post-column r e a g e n t s . U s i n g a p r e s s u r e chamber (Reh 1985) f o r h o l d i n g the c o n c e n t r a t e d HC1 and pumping hexane i n t o the v e s s e l by an HPLC pump t h a t i s c a p a b l e of o p e r a t i n g a g a i n s t the r e s i s t a n c e from the post-column r e a c t o r , g r e a t l y reduces the c h r o m a t o g r a p h i c time [from 1 hr (Kwong 1986a, 1986b) t o 25 m i n u t e s ] . I n i t i a l l y s t e e l was used f o r the p r e s s u r e v e s s e l . When hexane was added t o the v e s s e l , the HC1 was d i s p l a c e d and v e n t e d from the v e s s e l v i a a t e f l o n tube t h a t went from the beaker ( i n the bottom of p r e s s u r e v e s s e l ) t o t h e v a l v e where i t j o i n e d the HPLC e f f l u e n t and e n t e r e d the r e a c t o r . Problems o c c u r r e d w i t h c o r r o s i o n of the s t e e l and t h e r e was the p o s s i b i l i t y of c o n t a m i n a t i n g the HC1 w i t h i r o n o x i d e s . U s i n g PVC f o r the p r e s s u r e v e s s e l e l i m i n a t e d r u s t and the a s s o c i a t e d problems. HCl does not c o r r o d e PVC so a beaker or s e p a r a t e c o n t a i n e r i n s i d e was not r e q u i r e d . A l s o , the PVC p r e s s u r e v e s s e l was l i g h t weight w h i c h a l l o w e d f o r more c o n v e n i e n t c l e a n i n g and f i l l i n g w i t h t h e a c i d . To d a t e , d e g r a d a t i o n of the PVC or H y p a l o n T M g a s k e t m a t e r i a l ( c h l o r s u l f o n a t e d p o l y e t h y l e n e ) by the hexane or HCl has not been n o t e d . 98 3.4.2 O p t i m i z a t i o n of Post-Column R e a c t o r C o n d i t i o n s C o n c e n t r a t e d HC1, p h o s p h o r i c a c i d and t r i c h l o r o a c e t i c a c i d have been used f o r d e r i v a t i z a t i o n of d i g o x i n t o f l u o r e s c e n t p r o d u c t s ( F l a s c h 1981). Post-column f l u o r o g e n i c HPLC a s s a y s r e p o r t e d f o r d i g o x i n use c o n c e n t r a t e d HC1 d e r i v a t i z a t i o n and d e h y d r o a s c o r b i c a c i d (Desta 1987; G f e l l e r 1977; Kwong 1986a) or hydrogen p e r o x i d e s o l u t i o n s (Reh 1985) f o r f l u o r e s c e n c e enhancement. The f o l l o w i n g sequence of r e a c t i o n s f o r d i g o x i n i n c o n c e n t r a t e d HC1 was r e p o r t e d ( F l a s c h 1981): h y d r o l y s i s of the g l y c o s i d e t o the g e n i n , f o r m a t i o n of the 14-a n h y d r o g e n i n , r e a c t i o n t o the c o r r e s p o n d i n g 14-anhydro-16-c h l o r o d e r i v a t i v e and d e h a l o g e n a t i o n t o the 14,16-d i a n h y d r o g e n i n . A l t h o u g h p a r t of t h e r e a c t i o n sequence has been r e p o r t e d , the e x a c t s t r u c t u r e of t h e 14,16-d i a n h y d r o g e n i n formed by the HPLC-PC assay used i s unknown. D e h y d r o a s c o r b i c a c i d was added as a post-column reagent a l o n g w i t h c o n c e n t r a t e d HC1 w i t h p r e v i o u s HPLC-PC assay p r o c e d u r e s f o r d i g o x i n a n a l y s i s (Desta 1987; G f e l l e r 1977; Kwong 1986a). The HPLC-PC assay f o r d i g o x i n r e p o r t e d by Reh and J o r k (1985) used a m o b i l e phase of m e t h a n o l / w a t e r / d i l u t e hydrogen p e r o x i d e w i t h p h o s p h o r i c a c i d and post-column a d d i t i o n of c o n c e n t r a t e d HC1 f o r d i g o x i n d e r i v a t i z a t i o n . T h i s a l l o w e d f o r s e p a r a t i o n of d i g o x i n from d i g o x i g e n i n m o n o d i g i t o x o s i d e , d i g o x i g e n i n b i s d i g i t o x o s i d e , d i g o x i g e n i n and l a n a t o s i d e C (Reh 1985). S i n c e i t i s d e s i r a b l e t o r e s o l v e d i g o x i n from d i h y d r o d i g o x i n , the 99 aqueous p o r t i o n of a m o b i l e phase p r e v i o u s l y r e p o r t e d t o p a r t i a l l y r e s o l v e d i g o x i n from d i h y d r o d i g o x i n (Kwong 1986a) was r e p l a c e d by the c o m b i n a t i o n s of d i l u t e hydrogen p e r o x i d e w i t h p h o s p h o r i c a c i d and d e h y d r o a s c o r b i c a c i d shown i n Table V I . Maximum peak h e i g h t was o b t a i n e d when 45 mL d e h y d r o a s c o r b i c a c i d was used. Table VI. E f f e c t of V a r y i n g the Aqueous P o r t i o n of the Mobile Phase on Peak Height Using HPLC-PC Fl u o r e s c e n c e Assay Aqueous P o r t i o n Peak H e i g h t Of M o b i l e Phase (cm) 30 water/15 Hydrogen p e r o x i d e / p h o s p h o r i c a c i d 1.1 25 water/20 Hydrogen p e r o x i d e / p h o s p h o r i c a c i d 1.9 45 D e h y d r o a s c o r b i c a c i d 4.9 35 D e h y d r o a s c o r b i c a c i d / 1 0 water 4.4 35 D e h y d r o a s c o r b i c a c i d /10 Hydrogen p e r o x i d e / p h o s p h o r i c a c i d 3.5 25 D e h y d r o a s c o r b i c a c i d /20 Hydrogen p e r o x i d e / p h o s p h o r i c a c i d 3.9 Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3 Mm HPLC (4.6 mm x 15 cm); m o b i l e phase: m e t h a n o l / e t h a n o l / i s o -p ropanol/aqueous (52/3/1/45); HPLC f l o w r a t e : 0.5 mL/min.; Hexane f l o w r a t e : 0.5 mL/min.; post-column r e a c t o r : 10 m i n 79 °C water b a t h ; d e t e c t i o n : f l u o r o m e t e r equipped w i t h 360 nm e x c i t a t i o n and 425 nm e m i s s i o n f i l t e r s . B oth 360 nm (Desta 1987; Kwong 1986a; Reh 1985) and 350 nm ( G f e l l e r 1977) e x c i t a t i o n f i l t e r s and e m i s s i o n f i l t e r s r a n g i n g from 415 nm ( G f e l l e r 1977) t o 480 nm (Reh 1985) were used w i t h p r e v i o u s methods f o r post-column d e r i v a t i z a t i o n of d i g o x i n . S i n c e the e x a c t s t r u c t u r e of t h e d i g o x i n d e r i v a t i v e formed when d e h y d r o a s c o r b i c a c i d was added t o the HPLC m o b i l e phase and c o n c e n t r a t e d HCl was i n t r o d u c e d as a 100 post-column reagent i s unknown, the e m i s s i o n f i l t e r w a v e l e n g t h was o p t i m i z e d f o r maximum f l u o r e s c e n c e . W i t h t h e be s t m o b i l e phase, 425, 440 and 460 nm e m i s s i o n f i l t e r s were then e v a l u a t e d u s i n g a 360 nm e x c i t a t i o n f i l t e r and the r e s u l t s a r e shown i n T a b l e V I I . Maximum peak h e i g h t was found w i t h the 425 nm e m i s s i o n f i l t e r . T a b l e V I I . E f f e c t of E m i s s i o n F i l t e r s on Peak H e i g h t of D i g o x i n U s i n g HPLC-PC F l u o r e s c e n c e Assay E m i s s i o n F i l t e r Peak H e i g h t (cm) (nm) (Average of n=4) 425 9.5 440 5.7 460 4.2 Chromatographic c o n d i t i o n s : Same as f o r Ta b l e V w i t h m o b i l e phase: m e t h a n o l / e t h a n o l / i s o p r o p a n o l / d e h y d r o a s c o r b i c a c i d (52/3/1/45); d e t e c t i o n : f l u o r o m e t e r equipped w i t h 360 nm e x c i t a t i o n f i l t e r . The p e r i s t a l t i c pumps p r e v i o u s l y used f o r d e l i v e r y of post-column r e a g e n t s (Desta 1987; G f e l l e r 1977; Kwong 1986a) s e v e r e l y l i m i t e d the c h o i c e of f l o w r a t e and r e a c t o r s i z e . These l i m i t s were e f f e c t i v e l y removed by u s i n g a second HPLC pump and hexane d i s p l a c e m e n t of HCl from a p r e s s u r e v e s s e l . The f l o w r a t e of HCl and post-column r e a c t o r l e n g t h were o p t i m i z e d t o g e t h e r s i n c e they b o t h i n f l u e n c e the time a v a i l a b l e f o r d e r i v a t i z a t i o n t o o c c u r . Changing the hexane f l o w r a t e s from 0.5 mL/min t o 1.0 mL/min, w i t h the 10 m r e a c t o r , g r a d u a l l y reduced the peak h e i g h t ( T a b l e V I I I ) . The s h o r t e r time spent i n the r e a c t o r w i t h t h e 1.0 mL/min 101 f l o w r a t e d i d not a l l o w the development of maximum f l u o r e s c e n c e . In o r d e r t o i n c r e a s e the r e a c t i o n t ime w i t h o u t r e d u c i n g the hexane f l o w r a t e below 0.5 mL/min, a 20 m r e a c t o r was used. W i t h the 20 m r e a c t o r , the b e s t s e n s i t i v i t y was o b t a i n e d w i t h a hexane f l o w r a t e of 0.5 mL/min ( d i r e c t i n j e c t i o n of 0.585 ng d i g o x i n w i t h a s i g n a l / n o i s e r a t i o of 4/1). Reduced b a s e l i n e n o i s e was a l s o o b s e r v e d w i t h the 20 m r e a c t o r . When the 20 m r e a c t o r was used, d i g o x i n e l u t e d l a t e r i n the chromatogram (17 minutes compared t o 13 minutes) a l l o w i n g the endogenous peaks from serum e l u t i n g i n the e a r l y p a r t of the chromatogram t o be w e l l s e p a r a t e d from d i g o x i n . T a b l e V I I I . E f f e c t of HC1 Flow Rate on Peak H e i g h t of D i g o x i n U s i n g HPLC-PC F l u o r e s c e n c e A s s a y HC1 (Hexane) Peak H e i g h t (cm) Flow Rate (Average of n=4) (mL/min) 0.44 (0.50) 9.3 0.68 (0.70) 7.6 0.99 (1.00) 5.4 Chromatographic c o n d i t i o n s : Same as f o r T a b l e VI w i t h d e t e c t i o n : f l u o r o m e t e r equipped w i t h 360 nm e x c i t a t i o n and 425 nm e m i s s i o n f i l t e r s . 3.4.3 Maximum S e n s i t i v i t y The maximum s e n s i t i v i t y o b t a i n e d w i t h the o p t i m i z e d HPLC-PC c o n d i t i o n s g i v e n above was 0.5 ng d i g o x i n / i n j e c t i o n 102 d i r e c t l y and 1.5 ng d i g o x i n / 3 mL serum w i t h e x t r a c t i o n as d e s c r i b e d i n s e c t i o n 2.9.4. For o t h e r HPLC-PC d i g o x i n a s s a y methods, t h e maximum s e n s i t i v i t y v a l u e s r e p o r t e d were 0.1 ng d i g o x i n / i n j e c t i o n (Reh 1985), 0.5 ng d i g o x i n / i n j e c t i o n (Kwong 1986a) and 10 ng d i g o x i n / i n j e c t i o n (Desta 1987). The HPLC-PC a s s a y d e s c r i b e d here i s l e s s s e n s i t i v e than t h a t d e s c r i b e d by Reh and J o r k (1985) but i s s t i l l p r e f e r a b l e f o r e v a l u a t i n g p a t i e n t samples s i n c e p a r t i a l r e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n was o b t a i n e d . T h i s HPLC-PC method has e q u a l or b e t t e r s e n s i t i v i t y than t h e o t h e r HPLC-PC methods r e p o r t i n g s e n s i t i v i t y t o d i g o x i n (Desta 1987; Kwong 1986a). Removal of the problems a s s o c i a t e d w i t h u s i n g a p e r i s t a l t i c pump f o r post-column reagent d e l i v e r y (Desta 1987; Kwong 1986a) make the HPLC-PC system d e v e l o p e d here more a p p r o p r i a t e f o r r o u t i n e use. The s e n s i t i v i t y o b s e r v e d w i t h t h i s HPLC-PC assay may be s i g n i f i c a n t l y improved by a s s e s s i n g d i f f e r e n t f l u o r o m e t r i c d e t e c t o r s . The s e n s i t i v i t y of immunoassay methods and HPLC-RIA p r o c e d u r e s f o r d i g o x i n i s c o n s i d e r a b l y g r e a t e r than t h a t o b t a i n e d f o r HPLC-PC methods of a n a l y s i s . Immunoassay methods a l o n e l a c k the d e s i r e d s p e c i f i c i t y f o r d i g o x i n and c h r o m a t o g r a p h i c s e p a r a t i o n of d i g o x i n from d i h y d r o d i g o x i n was not d e s c r i b e d by most HPLC-RIA methods of d i g o x i n a n a l y s i s (Gibson 1980; Loo 1977, 1981; Margot 1983; Plum 1986). The HPLC-RIA assay method t h a t p a r t i a l l y s e p a r a t e s d i g o x i n from d i h y d r o d i g o x i n does not d e s c r i b e t h e r e s o l u t i o n 103 o b t a i n e d ( M o r a i s 1981; Wagner 1983). T h e r e f o r e , the HPLC-PC assay d e s c r i b e d here i s s u p e r i o r f o r d i g o x i n a n a l y s i s when adequate sample volumes (3 mL serum) a r e a v a i l a b l e . 3.4.4 E v a l u a t i o n of D i g o x i n and i t s M e t a b o l i t e s U s i n g t h e f i n a l HPLC-PC system, d i g o x i n i s b a s e l i n e r e s o l v e d from d i g o x i g e n i n , d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e as shown i n F i g u r e 13. The r e s o l u t i o n between d i g o x i n and d i h y d r o d i g o x i n (R = 0.899) i s s u f f i c i e n t f o r peak h e i g h t q u a n t i t a t i o n . S i n c e the HPLC-PC system s e p a r a t e s d i g o x i n from a l l of i t s m e t a b o l i t e s , t h e r e i s no i n t e r f e r e n c e from th e s e m e t a b o l i t e s i n the a n a l y s i s of d i g o x i n . R e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n was not d e s c r i b e d f o r HPLC-RIA methods f o r d i g o x i n a n a l y s i s (Gibson 1980; Loo 1977, 1981; Margot 1983; Plum 1986). A l o n g w i t h the l a c k of s p e c i f i c i t y of the RIA used f o r d i g o x i n i n the presence of DLIS ( V a l d e s 1985a, 1985b), i n t e r f e r e n c e from d i h y d r o d i g o x i n may o c c u r w i t h t h e s e HPLC-RIA methods. S i n c e the p o t e n t i a l f o r c r o s s - r e a c t i v i t y of d i h y d r o d i g o x i n and DLIS w i t h the RIA e x i s t s , the HPLC-PC method d e s c r i b e d i n t h i s r e p o r t i s b e t t e r than the HPLC-RIA methods w i t h r e s p e c t t o s p e c i f i c i t y f o r d i g o x i n . The one HPLC-RIA method c l a i m i n g p a r t i a l s e p a r a t i o n of d i g o x i n from d i h y d r o d i g o x i n does not d e s c r i b e the r e s o l u t i o n o b t a i n e d by t h e i r c h r o m a t o g r a p h i c system ( M o r a i s 1981; Wagner 1983). 104 1,2 3 4 6 I i 5 15 30 TIME (MIN) F i g u r e 13. Chromatogram of Di g o x i n and I t s M e t a b o l i t e s by HPLC-PC C h r o m a t o g r a p h i c c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3 Mm HPLC (4.6 mm x 15 cm); m o b i l e phase: m e t h a n o l / e t h a n o l / i s o -p r o p a n o l / d e h y d r o a s c o r b i c a c i d (52/3/1/45); HPLC f l o w r a t e : 0.4 mL/min.; Hexane f l o w r a t e : 0.5 mL/min.; post-column r e a c t o r : 20 m i n 79 °C water b a t h ; d e t e c t i o n : f l u o r o m e t e r e q u i p p e d w i t h 360 nm e x c i t a t i o n and 425 nm e m i s s i o n f i l t e r s . Peak i d e n t i t y : 1, d i g o x i g e n i n ; 2, d i h y d r o d i g o x i g e n i n ; 3, d i g o x i g e n i n m o n o d i g i t o x o s i d e ; 4, d i g o x i g e n i n b i s d i g i t o x o s i d e ; 5, d i h y d r o d i g o x i n ; 6, d i g o x i n . 105 The post-column f l u o r o g e n i c d e r i v a t i z a t i o n HPLC a s s a y s f o r d i g o x i n r e p o r t e d by G f e l l e r et al. (1977) and Reh and J o r k (1985) do not d e s c r i b e s e p a r a t i o n of d i g o x i n from d i h y d r o d i g o x i n . B a s e l i n e r e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n has been r e p o r t e d (Desta 1987) but t h e s e n s i t i v i t y of t h i s a ssay f o r d i g o x i n was not adequate f o r t h e r a p e u t i c m o n i t o r i n g . The r e s o l u t i o n of d i g o x i n from d i h y d r o d i g o x i n o b t a i n e d w i t h the HPLC-PC assay d e s c r i b e d here was s l i g h t l y l e s s than p r e v i o u s l y r e p o r t e d (R = 0.91) (Kwong 1986a). A l t h o u g h the s e n s i t i v i t y of t h i s a s s a y was s u f f i c i e n t f o r e v a l u a t i n g p a t i e n t samples (Kwong 1986a, 1986b) th e l o n g c h r o m a t o g r a p h i c time (40 m i n u t e s ) and u n r e l i a b l e d e l i v e r y of post-column r e a g e n t s make i t u n s u i t a b l e f o r r o u t i n e c l i n i c a l use. 3.5 A n a l y s i s of B i o l o g i c a l Samples U s i n g The HPLC F l u o r o g e n i c Assay 3.5.1 O p t i m i z a t i o n of Serum E x t r a c t i o n Method 3.5.1.1 P r o t e i n P r e c i p i t a t i o n Methods E m u l s i o n f o r m a t i o n of t h e aqueous phase and the e x t r a c t i o n s o l v e n t i s a problem found w i t h a p r e v i o u s l y r e p o r t e d e x t r a c t i o n p r o c e d u r e (Kwong 1986a). R e p o r t s i n t h e l i t e r a t u r e i n d i c a t e t h a t complete p r o t e i n p r e c i p i t a t i o n i s d i f f i c u l t or i m p o s s i b l e u n l e s s c o l d a c e t o n e i s used or u n l e s s the sample i s heated t o c o a g u l a t e the p r e c i p i t a t e 1 06 (Henry 1964). A f i n a l c o n c e n t r a t i o n of a t l e a s t 63% c o l d a c e tone i s r e q u i r e d f o r serum or whole b l o o d (1 volume sample p l u s 9 volumes of 70% a c e t o n e ) (Henry 1964). The e m u l s i o n formed i s due t o i n c o m p l e t e p r o t e i n p r e c i p i t a t i o n s i n c e i n c r e a s i n g the amount of acetone used i n t h i s e x t r a c t i o n p r o c e d u r e t o 6 mL reduced the amount of e m u l s i o n between the aqueous phase and t h e e x t r a c t i o n s o l v e n t . A n i o n i c p r e c i p i t a n t s a r e commonly used f o r the removal of p r o t e i n s (such as p i c r i c a c i d and t r i c h l o r o a c e t i c a c i d ) (Henry 1964) but would degrade any d i g o x i n p r e s e n t and t h e r e f o r e c o u l d not be used w i t h t h i s e x t r a c t i o n . Z i n c , z i n c p l u s b a rium, and copper p r e c i p i t a t i o n methods do not use a c i d so may be used i n the e x t r a c t i o n of d i g o x i n . Z i n c s u l f a t e w i t h barium h y d r o x i d e (Henry 1964; Somogyi 1945) and copper s u l f a t e w i t h sodium h y d r o x i d e (Henry 1964) methods were both e v a l u a t e d f o r p r o t e i n p r e c i p i t a t i o n , r e p l a c i n g the acetone i n t h i s e x t r a c t i o n p r o c e d u r e . E m u l s i o n f o r m a t i o n w i t h the e x t r a c t i o n s o l v e n t ( d i c h l o r o m e t h a n e / p r o p a n - 1 - o l 98/2), s t i l l o c c u r r e d r e s u l t i n g i n lower r e c o v e r y of d i g o x i n and a l s o made the f i l t r a t i o n s t e p more d i f f i c u l t than when acetone was used. Other a l t e r a t i o n s t o the e x t r a c t i o n method ( s e c t i o n 3.5.1.2) reduced the emulsi<on f o r m a t i o n p r e v i o u s l y o b s e r v e d . P r o t e i n p r e c i p i t a t i o n i n the f i n a l e x t r a c t i o n p r o c e d u r e was a c h i e v e d by u s i n g 3 mL a c e t o n e . 107 3.5.1.2 Removal of Endogenous I n t e r f e r e n c e U s i n g t h e HPLC-PC as s a y d e s c r i b e d i n s e c t i o n 2.8.3 and a serum e x t r a c t i o n method p r e v i o u s l y r e p o r t e d (Kwong 1986a, 1986b), serum from one h e a l t h y u n d i g i t a l i z e d female v o l u n t e e r c o n t a i n e d an i n t e r f e r i n g peak e l u t i n g c l o s e t o the r e t e n t i o n time of d i g o x i n as shown i n F i g u r e 14. A l t h o u g h the s t r u c t u r e and i m m u n o r e a c t i v i t y of t h i s i n t e r f e r i n g peak were not e v a l u a t e d , i t may be a f r a c t i o n of the DLIS observed i n normal a d u l t s u b j e c t s who never r e c e i v e d d i g o x i n ( B a l z a n 1984; C l e r i c o 1985; Diamandis 1985; Hamlyn 1982; K l i n g m u l l e r 1982; V a l d e s 1983a; V i n g e 1988). At t e m p t s t o e l i m i n a t e DLIS from serum have i n c l u d e d u l t r a f i l t r a t i o n w i t h C e n t r i f r e e m i c r o p a r t i t i o n systems ( C h r i s t e n s o n 1987; Graves 1986). T h i s removed 90% of the DLIS and a l l o w e d f o r g r e a t e r than 95% r e c o v e r y of d i g o x i n (Graves 1986). The C e n t r i f r e e u l t r a f i l t r a t i o n system has a maximum sample volume of 1.0 mL. L a r g e r serum samples (3 mL) were r e q u i r e d here so the C e n t r i f l o system (maximum sample volume of 7.0 mL) was a l s o e v a l u a t e d . U l t r a f i l t r a t i o n of serum c o n t a i n i n g t h i s i n t e r f e r i n g peak by the C e n t r i f r e e and C e n t r i f l o systems c o m p l e t e l y removed the i n t e r f e r i n g compound but d i d not a l l o w r e c o v e r y of d i g o x i n from the samples. These r e s u l t s suggest t h a t the i n t e r f e r i n g peak was r e l a t i v e l y l a r g e ( g r e a t e r than 25000 Da i n m o l e c u l a r w e i g h t ) . U l t r a f i l t r a t i o n c o u l d not be used f o r sample p u r i f i c a t i o n s i n c e d i g o x i n was not r e c o v e r e d . 108 1,2 3 A Kj/v^ B 15 TIME (MIN) 30 F i g u r e 14. Chromatograms of Dig o x i n and I t s M e t a b o l i t e s i n E t h a n o l and Blank Serum C o n t a i n i n g I n t e r f e r i n g Peak Chromatographic c o n d i t i o n s : Same as f o r F i g u r e 13. A D i g o x i n and I t s M e t a b o l i t e s i n Ethanol B Blank Serum C o n t a i n i n g I n t e r f e r i n g Peak Peak i d e n t i t y : 1 f d i g o x i g e n i n ; 2, d i h y d r o d i g o x i g e n i n ; 3, d i g o x i g e n i n monodigitoxoside; 4, d i g o x i g e n i n b i s d i g i t o x o s i d e ; 5, d i h y d r o d i g o x i n ; 6, d i g o x i n ; 7, i n t e r f e r i n g peak. 109 Removal of the i n t e r f e r i n g peak by m o d i f y i n g the i s o o c t a n e s o l v e n t wash was then e v a l u a t e d . Recovery of d i g o x i n from water a f t e r an i s o o c t a n e / d i c h l o r o m e t h a n e (20/5) s o l v e n t wash and e x t r a c t i o n was 8% lower than when i s o o c t a n e a l o n e was used. B l a n k serum (3 mL) which c o n t a i n e d the i n t e r f e r i n g peak and a serum sample (3 mL) s p i k e d w i t h d i g o x i n (1.5 ng) and i n t e r n a l s t a n d a r d (160 ng d i g i t o x i g e n i n ) were washed w i t h 2 mL of i s o o c t a n e / d i c h l o r o m e t h a n e (20/5) and e x t r a c t e d . The chromatogram o b t a i n e d from HPLC-PC a n a l y s i s (see F i g u r e 15) i n d i c a t e d t h a t the i n t e r f e r i n g peak was removed and d i g o x i n was r e c o v e r e d when d i c h l o r o m e t h a n e was added t o the s o l v e n t wash. Serum p r o t e i n p r e c i p i t a t i o n w i t h 3 mL acetone f o l l o w e d by a 2 mL i s o o c t a n e / d i c h l o r o m e t h a n e s o l v e n t wash r e s u l t e d i n l i t t l e or no e m u l s i o n formed w i t h the e x t r a c t i o n s o l v e n t . The f i n a l e x t r a c t i o n p r o c e d u r e ( s e c t i o n 2.9.4) employed 3 mL acetone f o r p r o t e i n p r e c i p i t a t i o n and a 2 mL i s o o c t a n e / d i c h l o r o m e t h a n e (20/5) s o l v e n t wash f o r removal of endogenous compounds t h a t i n t e r f e r e w i t h d i g o x i n a n a l y s i s . 3.5.2 Recovery and P r e c i s i o n The r e c o v e r y of d i g o x i n was d e t e r m i n e d f o r b l a n k serum s p i k e d w i t h 0.5, 3.0 and 10.0 ng of d i g o x i n . T a b l e IX shows the p e r c e n t r e c o v e r y f o r each c o n c e n t r a t i o n . 110 A I 1 T 0 15 3 0 TIME (MIN) F i g u r e 15. Chromatogram of Blank Serum and Serum Spiked with 1.5 ng Di g o x i n and I n t e r n a l Standard Chromatographic c o n d i t i o n s : Same as f o r F i g u r e 13. Peak i d e n t i t y : 1, d i g o x i n ; 2, i n t e r n a l s t a n d a r d ( d i g i t o x i g e n i n ) . A B l a n k Serum B S p i k e d Serum 111 T a b l e IX. Recovery of D i g o x i n from S p i k e d Serum Samples C o n c e n t r a t i o n Recovery C.V. Number (ng d i g o x i n / 3 mL serum) (%) (%) of Samples 1.5 72.0 6.4 7 3.0 78.2 7.2 8 10.0 78.8 6.1 7 B e t t e r d i g o x i n r e c o v e r y was obser v e d here than w i t h a p r e v i o u s l y r e p o r t e d HPLC-PC as s a y and s i m i l a r e x t r a c t i o n p r o c e d u r e (70%) (Kwong 1986b). C o n s i d e r a b l y g r e a t e r r e c o v e r y of d i g o x i n (99.7%) u s i n g a v e r y d i f f e r e n t e x t r a c t i o n method has been r e p o r t e d (Reh 1985) but i s not a p p r o p r i a t e f o r r o u t i n e use. The p r e c i s i o n of the HPLC-PC assay was d e t e r m i n e d u s i n g d i g o x i n i n e t h a n o l r a t h e r than r e p e a t e d i n j e c t i o n s from one e x t r a c t e d serum sample s i n c e the e n t i r e e x t r a c t e d serum sample was r e q u i r e d f o r a n a l y s i s . For the HPLC-PC as s a y the c o e f f i c i e n t of v a r i a t i o n f o r 1.5 and 10 ng i n j e c t i o n s of d i g o x i n i n e t h a n o l was 4.7% (n=4) and 3.3% (n=l0) r e s p e c t i v e l y . The p r e c i s i o n of the e x t r a c t i o n p r o c e d u r e was d e t e r m i n e d by r e p e a t e d e x t r a c t i o n of f i v e b l a n k serum samples (3 mL) s p i k e d w i t h 3 ng of d i g o x i n and 20 ML of i n t e r n a l s t a n d a r d (160 ng) f o l l o w e d by HPLC-PC a n a l y s i s and comparison of peak h e i g h t r a t i o s . The c o e f f i c i e n t of v a r i a t i o n f o r w i t h i n each day i s shown i n T a b l e X. A n a l y s i s of v a r i a n c e f o r peak h e i g h t r a t i o means ( F ( 3 tg) = 0.84 (p=0.49)) and f o r e q u a l i t y of v a r i a n c e ( F ( 3 1 g ) = 0.22 112 (p=0.88)) v e r i f i e s t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e i n the between-day r e s u l t s . A g r e a t e r i n t e r - a s s a y c o e f f i c i e n t of v a r i a t i o n (8%) was r e p o r t e d f o r a s i m i l a r e x t r a c t i o n p r o c e d u r e (Kwong 1986b). T h i s i n d i c a t e s t h a t i n comparison t o a p r e v i o u s method (Kwong 1986b) the e x t r a c t i o n p r o c e d u r e and HPLC-PC assay d e s c r i b e d h e r e has a lower s p r e a d of v a l u e s i n r e l a t i o n t o t h e mean v a l u e s . T a b l e X. P r e c i s i o n of D i g o x i n Assay Day C.V. (%) 1 4.6 2 5.7 3 6.6 4 5.3 mean 5.6 3.5.3 C a l i b r a t i o n Curve Serum samples (3 mL) were s p i k e d w i t h from 1 t o 3.3 ng d i goxin/mL and i n t e r n a l s t a n d a r d , e x t r a c t e d and a n a l y z e d u s i n g the HPLC-PC f l u o r o g e n i c a s s a y . The peak h e i g h t and c o n c e n t r a t i o n r a t i o s were then c a l c u l a t e d and the c a l i b r a t i o n c u r v e shown i n F i g u r e 16 was p r e p a r e d u s i n g average v a l u e s from f o u r s e p a r a t e d e t e r m i n a t i o n s . The c o r r e l a t i o n c o e f f i c i e n t of 0.9876 was low but a c c e p t a b l e f o r t h i s complex an assay ( F a l k n e r 1981) and the y - i n t e r c e p t (0.0273) was not s i g n i f i c a n t l y d i f f e r e n t from z e r o ( t - r a t i o = 1.23). C a l i b r a t i o n c u r v e c o r r e l a t i o n c o e f f i c i e n t s as h i g h 113 F i g u r e 16. C a l i b r a t i o n Curve f o r HPLC-PC D i g o x i n Assay 114 as 0.9999 have been p r e v i o u s l y r e p o r t e d w i t h HPLC-PC a n a l y s i s of d i g o x i n (Kwong 1986b). The c o r r e l a t i o n c o e f f i c i e n t o b s e r v e d here i n d i c a t e s t h a t 97.5% of the t o t a l v a r i a b i l i t y i n the h e i g h t and weight r a t i o s i s a c c o u n t e d f o r by mutual dependence of t h e s e r a t i o s . 3.5.4 Comparison of HPLC-PC and FPIA Methods i n S p i k e d  Serum Samples Seven b l a n k serum samples s p i k e d w i t h from 0 t o 3.4 ng of d i g o x i n per mL were p r e p a r e d and a s s a y e d by b oth methods. As shown i n F i g u r e 17, the c o r r e l a t i o n between the HPLC-PC and FPIA methods was 0.9897. T h i s i n d i c a t e s t h a t 98% of the t o t a l v a r i a b i l i t y of the two methods i s a c c o u n t e d f o r by mutual dependence. A l s o , a comparison of the i n d i v i d u a l methods w i t h the a c t u a l d i g o x i n l e v e l s shows t h a t the HPLC-PC p r o c e d u r e ( r = 0.9979) had b e t t e r c o r r e l a t i o n w i t h the a c t u a l v a l u e s than the FPIA method ( r = 0.9895). The p l o t of FPIA v e r s u s a c t u a l v a l u e s gave a s l o p e of 1.25 i n d i c a t i n g t h a t the FPIA r e s u l t s were h i g h e r than the amount of d i g o x i n added. W i t h the FPIA, no d i g o x i n was found i n t h e serum p r i o r t o s p i k i n g . A p o s s i b l e e x p l a n a t i o n f o r t h e s e h i g h e r than a c t u a l FPIA r e s u l t s i s t h a t DLIS p r e s e n t i n t h e b l a n k serum was d i s p l a c e d from t i g h t b i n d i n g s i t e s t o unbound or weak b i n d i n g s i g h t s ( V a l d e s 1985a, 1985b). The movement of DLIS from t i g h t b i n d i n g s i t e s would 115 F i g u r e 17. C o r r e l a t i o n Between HPLC-PC and FPIA Methods For D i g o x i n A n a l y s i s A HPLC-PC v e r s u s FPIA Method B FPIA v e r s u s A c t u a l L e v e l s C HPLC-PC v e r s u s A c t u a l L e v e l s 116 117 ACTUAL VALUE ng/mL 118 119 make i t a v a i l a b l e f o r d e t e c t i o n w i t h the FPIA ( V a l d e s 1985a, 1985b) and t h e r e f o r e may cause h i g h e r a p p arent d i g o x i n l e v e l s t o be o b s e r v e d . 3.5.5 S p e c i f i c i t y 3.5.5.1 S t e r o i d s Numerous s t e r o i d s have been r e p o r t e d t o c r o s s - r e a c t w i t h d i g o x i n a n t i s e r a (Diamandis 1985; L o n g e r i c h 1988; Matthewson 1988). S i n c e c r o s s - r e a c t i v i t y w i t h d i g o x i n a n t i s e r a was r e p o r t e d , the s p e c i f i c i t y of immunoassay methods u s i n g t h e s e a n t i b o d i e s i s q u e s t i o n a b l e . Samples of endogenous and s y n t h e t i c s t e r o i d s were e v a l u a t e d t o ensure t h a t they d i d not i n t e r f e r e w i t h the HPLC-PC as s a y p r o c e d u r e . No f l u o r e s c e n t peaks were o b s e r v e d a f t e r d i r e c t i n j e c t i o n of t h e s e s t e r o i d s i n methanol. The s t e r o i d s e v a l u a t e d e i t h e r do not e l u t e from the HPLC system or do not produce a f l u o r e s c e n t p r o d u c t under t h e s e c o n d i t i o n s and, t h e r e f o r e , would not i n t e r f e r e w i t h the HPLC-PC a n a l y s i s of d i g o x i n . 3.5.5.2 C o - a d m i n i s t e r e d Drugs I t i s i m p e r a t i v e t h a t drugs which may be c o -a d m i n i s t e r e d w i t h d i g o x i n be e v a l u a t e d f o r i n t e r f e r e n c e w i t h t h e a n a l y t i c a l method. U s i n g a s i m i l a r HPLC-PC assay f o r d i g o x i n the f o l l o w i n g drugs were e v a l u a t e d (Kwong 1986b): s p i r o n o l a c t o n e , f u r o s e m i d e , d i s o p y r a m i d e , c a p t o p r i l , 120 d i p y r i d a m o l , q u i n i d i n e , v e r a p a m i l , propafenone, p r o c a i n a m i d e and t r i m e t h o p r i m - s u l f a m e t h o x a z o l e . Only f u r o s e m i d e and s p i r o n o l a c t o n e y i e l d e d a f l u o r e s c e n t response and under t h e c o n d i t i o n s used were c h r o m a t o g r a p h i c a l l y s e p a r a t e d from d i g o x i n (Kwong 1986b). The newer a n t i a r r h y t h m i c a g e n t , m e x i l e t i n e , was not p r e v i o u s l y e v a l u a t e d . T h e r e f o r e , m e x i l e t i n e was a s s a y e d u s i n g the HPLC-PC as s a y d e v e l o p e d here t o d e t e r m i n e i f any f l u o r e s c e n c e was produced under t h e s e c o n d i t i o n s . No f l u o r e s c e n c e was found on i n j e c t i o n of 7.5 ng of m e x i l e t i n e which would be the maximum e x p e c t e d i n 3 mL of serum from p a t i e n t s w i t h i n the t h e r a p e u t i c range f o r t h i s drug ( T a l b o t 1973). 3.6 A n a l y s i s of D i g o x i n i n D i g i t a l i z e d P a t i e n t Serum Serum samples from 25 p a t i e n t s who r e c e i v e d d i g o x i n t h e r a p e u t i c a l l y were assay e d by b o t h HPLC-PC and FPIA methods. The r e s u l t s from b o t h methods a r e shown i n T a b l e X I . S i n c e t h e s e methods d i f f e r i n t h e i r s e n s i t i v i t y t o d i g o x i n , o n l y the samples w i t h i n range f o r both a s s a y s were used ( n = l 5 ) . The mean c o n c e n t r a t i o n o b t a i n e d u s i n g the HPLC-PC assay was 0.99 +0.56 and t h a t found w i t h the FPIA method was 1.13 + 0.73. The FPIA a s s a y gave h i g h e r average r e s u l t s which i s p o s s i b l y due t o the m e t a b o l i t e s of d i g o x i n b e i n g i n c l u d e d i n the t o t a l a s s a y v a l u e . W h i l e t h e r e were i n d i v i d u a l d i s c r e p a n c i e s between the a n a l y s e s of the sample by the two p r o c e d u r e s , i t was not p o s s i b l e t o a s s e s s i n t e r f e r e n c e w i t h the FPIA method t h a t would l e a d t o lower 121 Table XI. Comparison of the HPLC-PC and FPIA Methods f o r D igoxin A n a l y s i s i n D i g i t a l i z e d P a t i e n t s P a t i e n t D i g o x i n C o n c e n t r a t i o n ng/mL HPLC-PC/FPIA R a t i o HPLC-PC FPIA 1 1.11 1.2 0.925 2 1 .41 1 .6 0.881 3 0.69 0.8 0.863 4 0.98 1.0 0.980 5 ND 0.9 -6 1.01 1 .0 1.010 7 0.89 0.9 0.989 8 0.94 1 .1 0.855 9 1 .07 0.9 1 . 189 10 0.47 0.6 0.783 1 1 0.93 1 .1 0.845 1 2 2.80 3.6 0.778 13 ND 0.6 -1 4 0.41 0.5 -15 0.53 + 0.9 0.589 1 6 0.42 0.6 -17 0 .81 0.9 0.900 18 0.41 0.6 -19 ND 0.2 -20 0.2 -21 0.25* 0.2 22 0.41 0.5 -23 0.62 0.6 1 .033 24 0.69 0.7 0.986 25 ND 0.4 — mean+S.D. 0.99+0.56 0.86+0.66 0.91+0 (n=15) (n=25) (n=l5) * q u a n t i t i e s a r e below the a c c e p t e d s i g n a l - t o - n o i s e 4:1 and a r e not i n c l u d e d i n the mean d a t a . ND = none d e t e c t e d . l e v e l s found w i t h t h i s method f o r t h r e e of the samples. However, no i n t e r f e r e n c e has been shown w i t h the m e t a b o l i t e s of d i g o x i n or o t h e r drugs a s s e s s e d u s i n g the HPLC-PC a s s a y . 122 I n t e r f e r e n c e from endogenous s u b s t a n c e s p r e v i o u s l y o b s e r v e d w i t h t h i s a s s a y was e l i m i n a t e d by a l t e r i n g t h e e x t r a c t i o n method. The range of HPLC-PC t o FPIA v a l u e s f o r t h e s e samples was from 0.589 t o 1.189. HPLC/immunoassay r a t i o v a l u e s have been r e p o r t e d by Loo et al. (1981) (0.84 + 0.13), Gibson and N e l s o n (1980) (0.83 + 0.12 f o r r e n a l f a i l u r e d i a l y s i s p a t i e n t s ; 1.06 + 0.09 f o r p a t i e n t s w i t h r e n a l impairment) u s i n g HPLC-RIA and RIA assay methods and Kwong (1984) (1.00 + 0.34 and 0.94 + 0.30 from s e p a r a t e h o s p i t a l s ) u s i n g HPLC-PC and RIA p r o c e d u r e s . A l t h o u g h d i r e c t c o m parison of the HPLC/immunoassay r a t i o v a l u e s may not be j u s t i f i e d due t o t h e d i f f e r e n t HPLC and immunoassay methods used, the r a t i o o b t a i n e d h e r e w i t h HPLC-PC and FPIA methods i s s i m i l a r t o p r e v i o u s l y r e p o r t e d r a t i o v a l u e s . 3.7 A n a l y s i s of Serum from U n d i g i t a l i z e d P a t i e n t Groups Where H i g h L e v e l s of DLIS have been Reported 3.7.1 H y p e r t e n s i v e P a t i e n t s Serum samples from f i v e u n d i g i t a l i z e d h y p e r t e n s i v e p a t i e n t s were o b t a i n e d . These samples were e v a l u a t e d by b o t h the HPLC-PC f i n a l a s say p r o c e d u r e and the FPIA and t h e r e s u l t s a r e shown i n T a b l e X I I . 123 Table X I I . Comparison of U n d i g i t a l i z e d H y p ertensive P a t i e n t Samples E v a l u a t e d by the HPLC-PC and FPIA Methods P a t i e n t Apparent D i g o x i n C o n c e n t r a t i o n ng/mL HPLC-PC FPIA 1 ND ND 2 ND ND 3 ND 0.1 4 ND ND 5 ND ND ND = none d e t e c t e d . The h y p e r t e n s i v e p a t i e n t s were on the f o l l o w i n g m e d i c a t i o n s : a t e n o l o l , s p i r a p r i l , e n a l a p r i l , e l t r o x i n , t i m o l o l and p i l o c a r p i n e . T h e i r b l o o d p r e s s u r e ranged from 154/104 t o 174/110. Endogenous compounds t h a t c r o s s - r e a c t w i t h d i g o x i n immunoassays have been r e p o r t e d i n some p a t i e n t s w i t h h y p e r t e n s i o n . Recent r e p o r t s suggest t h a t DLIS, or a f r a c t i o n t h e r e o f , may be the n a t r i u r e t i c hormone and i n v o l v e d i n h y p e r t e n s i o n (Buckalew 1985; C l o i x 1987; De Wardener 1982a, 1982b; Grantham 1984; W i l k i n s 1985) and may a l s o be p r e s e n t i n n o r m o t e n s i v e s u b j e c t s ( C l o i x 1987; Hamlyn 1982). The r e s u l t s p r e s e n t e d i n T a b l e X I I do not show s i g n i f i c a n t l e v e l s of endogenous compounds t h a t i n t e r f e r e w i t h the FPIA a s s a y . S i n c e t h e HPLC-PC r e s u l t s a l s o show no i n t e r f e r e n c e from endogenous compounds, i t appears t h a t the HPLC-PC i s as u n a f f e c t e d by endogenous compounds i n t h i s 124 p a t i e n t group as the FPIA i n t h e s e samples. DLIS i n t e r f e r e n c e w i t h the FPIA method i s c o n s i d e r a b l y l e s s than w i t h RIA methods (Greenway 1985; N a n j i 1985). A n a l y s i s of t h e s e samples by an RIA method may have r e s u l t e d i n g r e a t e r a p p a rent d i g o x i n l e v e l s but the l i m i t e d sample s i z e p r o h i b i t e d d u p l i c a t e d e t e r m i n a t i o n by immunoassay. A l a r g e r number of p a t i e n t s may have r e v e a l e d h i g h e r a p p arent d i g o x i n l e v e l s w i t h the FPIA, however, t h e s e a d d i t i o n a l p a t i e n t s were not a v a i l a b l e d u r i n g the c o u r s e of t h i s i n v e s t i g a t i o n . 3.7.2 Renal F a i l u r e P a t i e n t s Serum samples from 20 u n d i g i t a l i z e d r e n a l f a i l u r e p a t i e n t s whc were on d i a l y s i s a t t h e W i l l o w D i a l y s i s U n i t were o b t a i n e d . A l l the p a t i e n t s were on the f o l l o w i n g m e d i c a t i o n s : f o l i c a c i d , Z-Bec ( m u l t i v i t a m i n s w i t h z i n c ) , a n t a c i d s ( R o b a l a t e , Amphojel, and Turns), B a s a l j e l (aluminum h y d r o x i d e f o r b i n d i n g phosphate i n the g a s t r o i n t e s t i n a l t r a c t ) , Imferon ( i r o n d e x t r a n ) , and v i t a m i n D supplements. A l o n g w i t h t h e s e , some p a t i e n t s r e q u i r e d o t h e r m e d i c a t i o n s ( m e t o p r o l o l , p r o p r a n o l o l , s u l f i s o x a z o l e , p h e n y t o i n , i n d o m e t h a c i n , a l l o p u r i n o l , a c e t y l s a l i c y l i c a c i d , d ocusate sodium, i s o s o r b i d e d i n i t r a t e , i n s u l i n , t r i a z o l a m , p r e d n i s o n e , c a p t o p r i l , and h y d r o x y z i n e ) . The r e n a l f a i l u r e p a t i e n t serum samples were e v a l u a t e d by b o t h the HPLC-PC f i n a l a s s a y p r o c e d u r e and the FPIA and d i g o x i n was not d e t e c t e d i n any of the serum samples by e i t h e r method. T h i s i n d i c a t e s t h a t endogenous s u b s t a n c e s 125 were not p r e s e n t i n s u f f i c i e n t q u a n t i t i e s t o i n t e r f e r e w i t h e i t h e r of the a s s a y methods. The HPLC-PC assay i s a t l e a s t as s p e c i f i c as the FPIA f o r d i g o x i n i n t h i s p a t i e n t group as r e p r e s e n t e d by t h e s e samples. Endogenous compounds (DLIS) t h a t i n t e r f e r e - w i t h RIA methods have been obse r v e d w i t h t h i s p a t i e n t group ( B o u r g o i g n i e 1972; C r a v e r 1983; D'Arcy 1984; G i b s o n 1980; Graves 1983a, 1983b; Kramer 1985b). DLIS has a l s o been o b s e r v e d u s i n g FPIA assay methods i n r e n a l f a i l u r e p a t i e n t s ( O l d f i e l d 1985; Y a t s c o f f 1984) but the degree of i n t e r f e r e n c e i s g e n e r a l l y l e s s than t h a t found w i t h RIA methods. I t i s p o s s i b l e t h a t the r e n a l f a i l u r e p a t i e n t samples e v a l u a t e d here c o n t a i n e d DLIS a t l e v e l s t o o low t o be d e t e r m i n e d u s i n g the FPIA. I t was not p o s s i b l e t o f u r t h e r e v a l u a t e t h e s e samples w i t h an RIA p r o c e d u r e due t o the l i m i t e d sample s i z e . A n a l y s i s of t h e s e samples by RIA may have i n d i c a t e d s i g n i f i c a n t l e v e l s of DLIS. E v a l u a t i o n of a l a r g e r number of r e n a l f a i l u r e p a t i e n t serum samples w i t h the FPIA may have p r o v i d e d samples w i t h h i g h e r a p p arent d i g o x i n l e v e l s . 3.7.3 H e p a t i c F a i l u r e P a t i e n t s Ten serum samples from n i n e h e p a t i c f a i l u r e p a t i e n t s were e v a l u a t e d by both HPLC-PC and FPIA methods. The r e s u l t s a r e g i v e n i n T a b l e X I I I . The h e p a t i c f a i l u r e p a t i e n t s were on the f o l l o w i n g m e d i c a t i o n s : f u r o s e m i d e , s p i r o n o l a c t o n e , n i f e d i p i n e , c h o l e s t y r a m i n e , oxazepam, c o - t r i m o x a z o l e , r a n i t i d i n e , 126 c e p h a l e x i n , c o d e i n e phosphate, demerol, domperidone, h a l o p e r i d o l , p r o c a i n a m i d e , camphorated opium t i n c t u r e , b i s a c o d y l , v a l i u m , c l i n d a m y c i n , gentamycin, c e f o x i t i n , v i t a m i n E, v i t a m i n K, p a n c r e l i p a s e , a t r o p i n e , h e p a r i n , l a c t u l o s e , t h i a m i n e , s a l b u t a m o l , p o t a s s i u m c h l o r i d e , c a l c i u m g l u c o n a t e , f o l i c a c i d and n o r f l o x a c i n . As r e q u i r e d , t y l e n o l , d i m e n h y d r i n a t e , p e n t a z o c i n e , lorazepam and p r o c h l o r p e r a z i n e were a d m i n i s t e r e d . P a t i e n t n i n e was the o n l y d i g i t a l i z e d p a t i e n t (0.125 mg d a i l y ) . The p a t i e n t s were a l l d i a g n o s e d as h a v i n g a l c o h o l i c c i r r h o s i s . T a b l e X I I I . H e p a t i c F a i l u r e P a t i e n t Samples E v a l u a t e d by the HPLC-PC and FPIA Methods P a t i e n t 1 2 3 4 5 6 7 8 Apparent D i g o x i n C o n c e n t r a t i o n ng/mL HPLC-PC FPIA ND ND ND ND ND ND ND ND 0.62 1 .06 D i g i t a l i z e d ND = none d e t e c t e d . L i v e r F u n c t i o n T e s t (normal range) Serum B i l i r u b i n T o t a l (2-23 wmole/L) A l k a l i n e Phosphatase (30-110 U/L) A s p a r t a t e A m i n o t r a n s f e r a s e (5-47 U/L) 7 -Glutamyl T r a n s p e p t i d a s e (5-55 U/L) 0.6 0.2 0.6 0.5 ND ND 0.2 ND 1 .2 1.3 P a t i e n t v a l u e s normal - 344 normal - 610 63 - 223 122 - 393 (one p a t i e n t a t 1856) 127 U s i n g RIA methods, DLIS has been r e p o r t e d i n h e p a t i c f a i l u r e p a t i e n t serum samples ( D i P i r o 1980; Greenway 1985; N a n j i 1985, 1986; Yang 1988). As p r e v i o u s l y d e s c r i b e d , DLIS i n t e r f e r e n c e w i t h the FPIA method i s l e s s than w i t h RIA methods i n h e p a t i c f a i l u r e p a t i e n t samples (Greenway 1985; N a n j i 1985). For t h e u n d i g i t a l i z e d p a t i e n t s , the HPLC-PC as s a y was c o n s i s t e n t l y blank w h i l e the FPIA i n d i c a t e d a p parent d i g o x i n l e v e l s as h i g h as 0.6 ng/mL. T h i s i n d i c a t e s t h a t whatever gave the f a l s e p o s i t i v e v a l u e s w i t h the FPIA d i d not a f f e c t the HPLC-PC assay and t h a t the HPLC-PC method would more a c c u r a t e l y e s t i m a t e d i g o x i n l e v e l s i n the s e p a t i e n t s . In h e p a t i c f a i l u r e p a t i e n t s , N a n j i and Greenway (1985) r e p o r t e d a p p arent d i g o x i n l e v e l s of 0.2 ng/mL or l e s s w i t h the FPIA w h i l e RIA l e v e l s as g r e a t as 1.1 ng/mL were o b s e r v e d . The FPIA v a l u e s of 0.6 ng/mL r e p o r t e d here may t h e r e f o r e r e p r e s e n t s i g n i f i c a n t l y h i g h l e v e l s of DLIS. A n a l y s i s of th e s e samples by an RIA method was not p o s s i b l e due t o the l i m i t e d sample s i z e . For t h e d i g i t a l i z e d p a t i e n t , the FPIA r e s u l t s were h i g h e r than th o s e f o r t h e HPLC-PC a s s a y . T h i s d i f f e r e n c e i s p o s s i b l y due t o i n t e r f e r e n c e from m e t a b o l i t e s or endogenous compounds w i t h the FPIA. The HPLC-PC as s a y i s u n a f f e c t e d by the m e t a b o l i t e s of d i g o x i n and endogenous compounds t h a t were p r e s e n t i n the u n d i g i t a l i z e d h e p a t i c f a i l u r e p a t i e n t samples. T h e r e f o r e , t h e HPLC-PC r e s u l t s p r o b a b l y r e p r e s e n t the t r u e d i g o x i n c o n c e n t r a t i o n i n t h e s e samples. 128 3.7.4 U m b i l i c a l Cord B l o o d A t o t a l of 17 mixed u m b i l i c a l c o r d b l o o d samples from 11 p a t i e n t s were o b t a i n e d and a s s a y e d by b o t h HPLC-PC and FPIA methods. The r e s u l t s a r e shown i n T a b l e XIV. T a b l e XIV. Mixed U m b i l i c a l Cord B l o o d Samples E v a l u a t e d by the HPLC-PC and FPIA Methods P a t i e n t Apparent D i g o x i n C o n c e n t r a t i o n ng/mL HPLC-PC FPIA 1 ND 0.4 ND 0.3 2 ND 0.3 ND 0.4 ND 0.4 3 ND ND ND 0.2 4 ND 0.2 5 ND 0.2-ND 0.2 6 ND 0.3 7 ND 0.4 8 ND 0.2 9 ND ND ND 0.6 10 ND 0.3 1 1 ND 0.2 ND = none d e t e c t e d . The m e d i c a t i o n p r o f i l e s f o r t h e p a t i e n t s i n v o l v e d i n t h i s p a r t of the c l i n i c a l s t udy were not a v a i l a b l e . D i g o x i n - l i k e immunoreactive s u b s t a n c e s have been found i n u m b i l i c a l c o r d b l o o d (Besch 1976; Diamandis 1985; K e l l y 1981; Ng 1985; Pudek 1983a, 1983b; Scherrmann 1986a, 1986b; Y a t s c o f f 1984). R e p o r t s i n the l i t e r a t u r e ( G o n z a l e z 1987; Koren 1988) suggest t h a t DLIS l e v e l s i n u m b i l i c a l c o r d b l o o d 129 ( b o t h venous and a r t e r i a l b l o o d ) were s i g n i f i c a n t l y g r e a t e r than i n m a t e r n a l venous b l o o d . For t h e 17 c o r d b l o o d samples a s s a y e d , none i n d i c a t e d d i g o x i n was p r e s e n t a t l e v e l s above the s e n s i t i v i t y of the HPLC-PC as s a y (0.5 ng/mL). A l l but one sample f o r p a t i e n t 9 gave FPIA r e s u l t s l e s s than 0.5 ng/mL. The FPIA has been r e p o r t e d t o g i v e lower apparent d i g o x i n l e v e l s than the RIA i n the p r e s e n c e of DLIS (Greenway 1985; N a n j i 1985). Comparison of FPIA and RIA l e v e l s r e p o r t e d ( N a n j i 1985) suggest t h a t even when low DLIS l e v e l s a r e found w i t h the FPIA (0.2 ng/mL or l e s s ) t h a t s i g n i f i c a n t i n t e r f e r e n c e w i t h the RIA may r e s u l t (up t o 1.1 ng/mL). The low apparent d i g o x i n FPIA v a l u e s o b s e r v e d here may c o r r e s p o n d t o r e l a t i v e l y h i g h l e v e l s w i t h the RIA. I t was not p o s s i b l e t o e v a l u a t e t h e s e samples by b o t h the FPIA and RIA methods due t o the l i m i t e d sample s i z e a v a i l a b l e . S i n c e no i n t e r f e r e n c e was found w i t h the HPLC-PC a s s a y , i t i s a t l e a s t as u n a f f e c t e d by endogenous compounds as the FPIA i n t h i s p a t i e n t group as r e p r e s e n t e d by t h e s e samples. 130 4. SUMMARY AND CONCLUSIONS The r e s u l t s o b t a i n e d f o r c r o s s - r e a c t i v i t y of the m e t a b o l i t e s of d i g o x i n w i t h s i x RIA k i t s ( T a b l e I I ) show t h e r e i s e x t e n s i v e c r o s s - r e a c t i v i t y t o the d i g o x i g e n i n m o i e t y as was e x p e c t e d ( B u t l e r 1978; V a l d e s 1984). H i g h e r c r o s s - r e a c t i v i t y was demonstrated w i t h one RIA t o d i h y d r o d i g o x i n (46%) and d i h y d r o d i g o x i g e n i n (22%) than p r e v i o u s l y r e p o r t e d . F u r t h e r m o r e , i n t e r f e r e n c e from d i g o x i n m e t a b o l i t e s w i t h the FPIA ( T a b l e I I I ) was shown and may be p a r t i a l l y due t o the p r o t e i n p r e c i p i t a t i o n s t e p ( E r i c k s o n 1984; P o r t e r 1984; Skogen 1987) which w i l l cause the d i g i t o x o s e sugars t o be removed from d i g o x i n and i t s m e t a b o l i t e s p r i o r t o a n a l y s i s . The c r o s s - r e a c t i v i t y of d i h y d r o d i g o x i g e n i n r e p o r t e d w i t h the FPIA i s s i g n i f i c a n t l y g r e a t e r than t h a t o b s e r v e d w i t h t h e RIA methods. Pre-column d e r i v a t i z a t i o n of d i g o x i n and i t s m e t a b o l i t e s w i t h 3 , 5 - d i n i t r o b e n z o y l c h l o r i d e ( F u j i i 1983) f o l l o w e d by HPLC a n a l y s i s w i t h e l e c t r o c h e m i c a l d e t e c t i o n was dem o n s t r a t e d ( s e c t i o n 3.3). To d a t e , a n a l y s i s of 3,5-DNB d i g o x i n u s i n g HPLC w i t h e l e c t r o c h e m i c a l d e t e c t i o n has not been r e p o r t e d i n the l i t e r a t u r e . A maximum s e n s i t i v i t y of 0.394 ng d i g o x i n was ob s e r v e d u s i n g d u a l e l e c t r o d e d e t e c t i o n i n the redox mode. P a r t i a l r e s o l u t i o n between 3,5-DNB d i g o x i n and 3,5-DNB d i h y d r o d i g o x i n was a l s o r e p o r t e d . Problems e n c o u n t e r e d w i t h d e r i v a t i z a t i o n of s m a l l (ng) q u a n t i t i e s of d i g o x i n r e s u l t e d i n a s i g n i f i c a n t l o s s i n 131 s e n s i t i v i t y making t h i s method i m p r a c t i c a l f o r use w i t h c l i n i c a l samples. A post-column f l u o r o g e n i c HPLC assay u s i n g c o n c e n t r a t e d HC1 and d e h y d r o a s c o r b i c a c i d d e r i v a t i z a t i o n was d e v e l o p e d . Hexane, d e l i v e r e d by a second HPLC pump, was used t o d i s p l a c e the c o n c e n t r a t e d HC1 from a p r e s s u r e v e s s e l a l l o w i n g f o r r e l i a b l e and r e l a t i v e l y p u l s e - f r e e f l o w of a c i d i n t o the post-column r e a c t o r . D e h y d r o a s c o r b i c a c i d was added t o the aqueous p o r t i o n of t h e HPLC m o b i l e phase. The c h r o m a t o g r a p h i c column s e p a r a t e d d i g o x i n from i t s m e t a b o l i t e s p r i o r t o d e r i v a t i z a t i o n ( F i g u r e 13) a l l o w i n g f o r q u a n t i t a t i o n of d i g o x i n i n d i g i t a l i z e d p a t i e n t samples where m e t a b o l i t e s may be p r e s e n t . Numerous s t e r o i d s have been r e p o r t e d t o c r o s s - r e a c t w i t h d i g o x i n a n t i s e r a (Diamandis 1985; L o n g e r i c h 1988; Matthewson 1988). S t e r o i d samples e v a l u a t e d by the HPLC-PC ass a y i n t h i s s tudy e i t h e r do not e l u t e from the HPLC system or do not produce a f l u o r e s c e n t p r o d u c t under t h e s e c o n d i t i o n s . T h i s i n d i c a t e s t h a t the HPLC-PC as s a y d e v e l o p e d here would be a b l e t o q u a n t i t a t e d i g o x i n i n the p r e s e n c e of the s t e r o i d s t e s t e d . Comparison of the HPLC-PC and FPIA methods f o r d i g i t a l i z e d p a t i e n t samples shows h i g h e r mean d i g o x i n l e v e l s by the FPIA assay which i s p o s s i b l y due t o the i n c l u s i o n of d i g o x i n m e t a b o l i t e s i n the t o t a l a s s a y v a l u e . E v a l u a t i o n of u n d i g i t a l i z e d p a t i e n t samples from groups where h i g h DLIS l e v e l s have been r e p o r t e d shows t h a t the 132 HPLC-PC as s a y g i v e s fewer f a l s e p o s i t i v e r e s u l t s than the FPIA. In t h e pr e s e n c e of DLIS, t h e FPIA assay has been r e p o r t e d t o g i v e c o n s i d e r a b l y lower a p parent d i g o x i n l e v e l s t han the RIA (Greenway 1985; N a n j i 1985). For a number of the h e p a t i c f a i l u r e and u m b i l i c a l c o r d b l o o d samples, a p p a r e n t d i g o x i n v a l u e s r a n g i n g from 0.4 t o 0.6 ng/mL were o b t a i n e d w i t h the FPIA. T a k i n g i n t o c o n s i d e r a t i o n the d i f f e r e n c e i n s e n s i t i v i t y t o DLIS r e p o r t e d f o r the FPIA and RIA methods, the f a l s e p o s i t i v e v a l u e s o b t a i n e d here u s i n g the FPIA may r e p r e s e n t r e l a t i v e l y h i g h l e v e l s of i n t e r f e r i n g compounds. The HPLC-PC a s s a y was u n a f f e c t e d by the endogenous compounds r e s p o n s i b l e f o r the f a l s e p o s i t i v e FPIA v a l u e s o b t a i n e d h e r e . T h i s i n d i c a t e s t h a t t h e HPLC-PC as s a y d e v e l o p e d here i s u n a f f e c t e d by the endogenous compounds g i v i n g the f a l s e p o s i t i v e FPIA v a l u e s and t h e r e f o r e would be a b l e t o s p e c i f i c a l l y q u a n t i t a t e d i g o x i n i n t h e s e p a t i e n t samples. 133 5 . R E F E R E N C E S A b e l R.M., L u n c h i R.L., P e s k i n G.W., Conn H.L., M i l l e r C D . /. Pharmacol. Exp. Ther. 1 50:463 ( 1965). 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