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Metoclopramide : fused-silica capillary GLC-ECD; placental transfer in humans and sheep Riggs, Kenneth Wayne 1982

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Metocloprami.de: F u s e d - S t l i c a C a p i l l a r y GLC-ECD; Pl a c e n t a l Transfer i n Humans and Sheep by KENNETH WAYNE RIGGS B.Sc. (Pharm.).University of B r i t i s h Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE OF , MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Facu l t y of Pharmaceutical Sciences ( D i v i s i o n of Pharmaceutics) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December 1982 © KENNETH WAYNE RIGGS In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PHARMACEUTICAL SCIENCES (DIVISION OF PHARMACEUTICS) The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DECEMBER 1982 i i ABSTRACT Metoclopramide (MCP), a procainamide d e r i v a t i v e , i s a potent antiemetic and g a s t r i c m o t i l i t y s t i m u l a t o r . I t i s used c l i n i c a l l y to f a c i l i t a t e c e r t a i n g a s t r o i n t e s t i n a l d i a g n o s t i c procedures, to t r e a t a v a r i e t y of g a s t r o i n t e s t i n a l d i s o r d e r s and as an antiemetic i n the t r e a t -ment of nausea and vomiting of varying e t i o l o g i e s . MCP i s also used p a r e n t e r a l l y p r i o r to general anaesthesia i n labour where i t s antiemetic p r o p e r t i e s and a b i l i t y to promote g a s t r i c emptying help to reduce the inci d e n c e of a s p i r a t i o n of stomach contents and death due to Mendel son's Syndrome. This t h e s i s reports the m o d i f i c a t i o n of an e x i s t i n g packed column elec t r o n - c a p t u r e g a s - l i q u i d chromatographic (GLC-ECD) assay f o r MCP i n human plasma (Tam et a l _ . , 1979). The reasons f o r changes to the method are discussed and i l l u s t r a t e d . Components i n t e r f e r i n g with MCP estima-t i o n could not be resolved using packed column technology; a fused s i l i c a c a p i l l a r y GLC-ECD procedure was developed to overcome t h i s problem. The method in v o l v e s the e x t r a c t i o n of MCP and m a p r o t i l i n e (MAP), the i n t e r n a l standard, from a l k a l i n i z e d plasma, followed by an acid back-e x t r a c t i o n i n t o benzene. The benzene e x t r a c t i s d r i e d under a gentle stream of nitrogen and r e c o n s t i t u t e d with toluene. MCP and MAP are d e r i v a t i z e d at 55°C with h e p t a f l u o r o b u t y r i c anhydride (HFBA) i n the presence of the c a t a l y s t , t r i e t h y l a m i n e (0.05 M). Excess HFBA i s removed by h y d r o l y s i s with water and n e u t r a l i z a t i o n with 4% ammonium hydroxide. The d e r i v a t i z e d organic l a y e r i s immediately removed from the aqueous i i 1 phase f o r n e u t r a l i z a t i o n and 2 yL a l i q u o t s i n j e c t e d i n t o a reporting gas-l i q u i d chromatograph equipped with a ^ N i electron-capture d e t e c t o r . The samples may be i n j e c t e d on the day of preparation or can be stored at -20°C f o r up to 4 days f o r subsequent a n a l y s i s . Quantitation of MCP i n t e s t samples i s made from a c a l i b r a t i o n curve prepared from plasma e x t r a c t s c o n t a i n i n g known concentrations of MCP and MAP. A 25 m x 0.31 mm I.D. c r o s s - l i n k e d fused s i l i c a c a p i l l a r y column was used f o r a l l q u a n t i t a t i v e plasma MCP analyses. L i n e a r i t y was observ-ed i n the range of 4-40 ng of MCP base.mL - 1 f o l l o w i n g the e x t r a c t i o n of 0.25-0.5 mL of plasma. This represents from =0.9-9 pg at the detector employing the s p l i t mode of sample i n j e c t i o n ( s p l i t r a t i o , 30:1) and an i n j e c t i o n volume of 2 yL. The developed c a p i l l a r y assay method i s s e n s i t i v e , s p e c i f i c and s e l e c t i v e . It has been found to be s u i t a b l e f o r small volume plasma a n a l y s i s i n humans and sheep, has been applied to patient samples i n the c l i n i c a l s e t t i n g without i n t e r f e r e n c e from the other drugs used i n the study and has shown pharmacokinetic a p p l i c a b i l i t y to the study of MCP p l a c e n t a l t r a n s f e r i n sheep. MCP was observed to undergo p l a c e n t a l t r a n s f e r i n both humans and sheep f o l l o w i n g i . v . maternal a d m i n i s t r a t i o n . Twenty-three p a t i e n t s undergoing general anaesthesia f o r Caesarian s e c t i o n f o r healthy term pregnancies were included i n the human study. The patients received e i t h e r i . v . MCP or a normal s a l i n e placebo on a double-blind b a s i s . Maternal and f e t a l ( u m b i l i c a l cord) plasma concentrations were measured and an average f e t a l r m a t e r n a l MCP c o n c e n t r a t i o n r a t i o (F/M) of i v 0.60 ± 0.17 determined. Apgar t e s t i n g and thorough chart reviews were conducted to attempt e v a l u a t i o n of neonatal response to MCP. There were no s i g n i f i c a n t d i f f e r e n c e s between the treated and untreated groups of neonates. The i n utero pl a c e n t a l t r a n s f e r of MCP to the fetus of a c h r o n i c a l l y c a t h e t e r i z e d pregnant ewe was also studied. Measurable con-c e n t r a t i o n s appeared i n the f e t a l c i r c u l a t i o n w i t h i n 1 minute, peaked at 20 minutes and exceeded maternal l e v e l s at about 90 minutes. Maternal plasma MCP concentrations were observed to f o l l o w a biexponent!al decay. Terminal e l i m i n a t i o n h a l f - l i v e s of 40 and 54 minutes were c a l c u l a t e d f o r MCP i n the ewe and f e t u s , r e s p e c t i v e l y . V TABLE OF CONTENTS Chapter ; P a 9 e ABSTRACT 1 1 LIST OF TABLES i x LIST OF FIGURES x LIST OF SCHEMES x i i LIST OF STRUCTURES AND DIAGRAMS x i 1 1 ABBREVIATIONS x i v ACKNOWLEDGEMENTS x v i 1 INTRODUCTION 1 Rationale 2 1.1. A n a l y t i c a l Methods For MCP In B i o l o g i c a l F l u i d s 8 1.2. Basic MCP Pharmacokinetics 10 1.3. MCP's Use In Pregnancy 13 1.4. Factors A f f e c t i n g Maternal-Fetal Drug Transfer 15 1.4.1. Pharmacokinetics of P l a c e n t a l Drug Transfer 17 1.4.2. Studies of P l a c e n t a l Drug Transfer 18 1.5. Sheep As A Model For P l a c e n t a l Drug Transfer 20 1.6. C a p i l l a r y Column Gas Chromatography: Fused S i l i c a Columns 23 2. EXPERIMENTAL 28 2.1. M a t e r i a l s And Supplies 28 2.1.1. Chemicals 28 2.1.2. Reagents 28 2.1.3. Solvents 29 2.2. Equipment 29 2.3. Preparation Of Drug Stock S o l u t i o n 30 2.3.1. Metoclopramide. HC1.H20 30 2.4. Preparation Of I n t e r n a l Standard S o l u t i o n s 30 v i Chapter 2.4.1. M a p r o t f l i n e , KC1 2.4.2. Diazepam 2.4.3. Prazepam 2.5. Plasma E x t r a c t i o n Procedure 2.6. Chemistry 2.6.1. D e r i v a t i v e Formation A. Packed Column GLC-ECD B. Fused S i l i c a C a p i l l a r y Column GLC-ECD 2.6.2. E f f e c t of Base Strength (NH40H) And C a t a l y s t (TEA, P y r i d i n e ) On D e r i v a t i v e S t a b i l i t y (area r a t i o , HFB-MCP/diazepam v a r i a b i l i t y ) 2.6.3. S i l a n i z a t i o n Of Glassware To Test For P o s s i b l e MCP Adsorption Losses Onto Glassware 2.6.4. Optimization Of Metoclopramide And M a p r o t i l i n e D e r i v a t i z a t i o n Reaction Time 2.7. Standard Curve Preparation For Fused S i l i c a C a p i l l a r y Column A n a l y s i s 2.8. GLC-ECD 2.8.1. Packed Column Studies 2.8.1.1. Commercially Prepared Column Packings 2.8.1.2. In-lab Coated Column Packings 2.8.1.3. S o l i d Supports 2.8.2. General Procedure For In-lab S o l i d Support Coating 2.8.3. General Chromatographic Conditions For Packed Column Studies 2.8.4. Fused S i l i c a C a p i l l a r y Column Studies: Assay Development And Optimization Of GLC-ECD Parameters. 2.8.4.1. Sample Preparation 2.8.4.2. Optimization Of GLC-ECD Parameters 2.9. Fused S i l i c a C a p i l l a r y Column GLC-ECD: Assay A p p l i c a t i o n To The A n a l y s i s Of Human And Sheep Plasma 2.9.1. Human (Pharmacokinetics) Studies: MCP P l a c e n t a l Transfer 30 31 31 31 32 32 33 35 36 38 38 39 40 40 40 41 41 42 42 42 43 44 2.9.1.1. Experimental Protocol 2.9.1.2. Q u a n t i t a t i v e Plasma A n a l y s i s 46 46 v i i Chapter Page 2.9.2, Sheep Studies. 47 2.9.2.1. Animal Care And S u r g i c a l Preparation 47 2.9.2.2. Experimental Protocol 48 2.9.2.3. Q u a n t i t a t i v e Plasma A n a l y s i s 50 3 RESULTS 51 3.1. Packed Column GLC-ECD 51 3.1.1. S t a t i o n a r y Phases 51 3.1.2. D e r i v a t i z i n g Agents: HFBA, HFBI, PFPA, TFAA,PFBC 55 3.1.3. S o l i d Supports 56 3.1.4. S i l a n i z a t i o n Of Glassware 58 3.1.5. D e r i v a t i v e S t a b i l i t y : E f f e c t Of NH^OH, TEA, 58 Py r i d i n e 3.2. Fused S i l i c a C a p i l l a r y Column GLC-ECD 62 3.2.1. Column S e l e c t i o n 62 3.2.2. Optimization Of GLC-ECD Conditions 63 3.2.2.1. I n j e c t i o n Temperature 63 3.2.2.2. Column Temperature 63 3.2.2.3. C a r r i e r Gas: Helium (He), Hydrogen (H ?) 66 3.2.2.4. C a r r i e r Gas (H ?) Flow Rate 66 3.2.2.5. Make-up Gas (Argon-Methane 95:5) Flow 69 Rate 3.2.2.6. S p l i t L i n e r s : Jennings Tube, Fused 72 S i l i c a I n s e r t 3.2.2.7. I n j e c t i o n Mode: S p l i t , S p l i t l . e s s 74 3.2.2.8. I n j e c t i o n Solvent 76 3.2.2.9. S e l e c t i o n Of Inte r n a l Standard 78 3.2.2.10. Optimization Of HFBA D e r i v a t i z a t i o n 79 Time For MCP and MAP 3.3. A p p l i c a t i o n Of The Fused S i l i c a C a p i l l a r y GLC-ECD 79 Assay: Q u a n t i t a t i v e Plasma (Human, Sheep) MCP An a l y s i s 3.3.1. Human Pl a c e n t a l Transfer Studies: Data C o l l e c t i o n 79 3.3.2. Human Pl a c e n t a l Transfer: Plasma MCP Determination 86 3.3.3. Sheep P l a c e n t a l Transfer Study: Plasma MCP 91 Determination v i i i Chapter Page 4 DISCUSSION 95 4.1. Packed Column GLC-ECD 95 4.1.1. S t a t i o n a r y Phase S e l e c t i o n 95 4.1.2. D e r i v a t i z i n g Agent S e l e c t i o n 96 4.1.3. I n v e s t i g a t i o n s Of (HFB-MCP/Diazepam, HFB-MAP) 97 Area Ratio V a r i a b i l i t y 4.2. Fused S i l i c a C a p i l l a r y Column GLC-ECD 100 4.2.1. Column S e l e c t i o n 100 4.2.2. Optimization of GLC-ECD Conditions 102 4.2.2.1. I n j e c t i o n Temperature: S p l i t Mode 102 Of Sample I n j e c t i o n 4.2.2.2. S e l e c t i o n Of Column Temperature 104 4.2.2.3. S e l e c t i o n Of C a r r i e r Gas And Flow 104 Rate 4.2.2.4. S e l e c t i o n Of Make-Up Gas Flow Rate 107 4.2.2.5. S p l i t L i n e r s : Jennings Tube, Fused 109 Si 1ica I n s e r t . 4.2.2.6. I n j e c t i o n Mode: S p l i t , S p l i t l e s s 114 4.2.2.7. S e l e c t i o n Of I n j e c t i o n Solvent 115 4.2.2.8. S e l e c t i o n Of Inte r n a l Standard 116 4.2.2.9. Optimization Of HFBA D e r i v a t i z a t i o n 117 Reaction Time For MCP And MAP 4.3. Human Pl a c e n t a l Transfer Study 119 4.4. Sheep P l a c e n t a l Transfer Study 122 5 SUMMARY AND CONCLUSIONS 124 6 REFERENCES 127 6 i x LIST OF TABLES Table Page I S t a t i o n a r y Phases 53 II S o l i d Supports 57 I I I E f f e c t of Ammonium Hydroxide Concentration And 60 C a t a l y s t On D e r i v a t i v e S t a b i l i t y IV Maternal Data 84 V Infant Data 85 VI C o e f f i c i e n t Of V a r i a t i o n Study 88 VII Plasma A n a l y s i s Data 90 X LIST OF FIGURES Figure Page 1 A r e p r e s e n t a t i v e chromatogram obtained from a MCP-spiked 52 plasma e x t r a c t on 3% 0V-225. 2 Representative chromatograms obtained from blank (a.) 54 and MCP-spiked (b.) plasma e x t r a c t s on 3% S i l a r - 9CP. 3 E f f e c t s of i n j e c t i o n port temperature on HFB-MCP and 64 HFB-MAP response. 4 E f f e c t of column temperature on the separation of 65 HFB-MCP from endogenous plasma components, a.235°, b.240OC, c. 2450C. 5 E f f e c t s of helium (a.) and hydrogen (b.) on HFB-MCP 67 separation and a n a l y s i s time. 6. E f f e c t of c a r r i e r gas (H 2) f l o w r a t e on HFB-MCP 68 response. a. s p l i t vent f l o w , 30mL.min-l ( s p l i t r a t i o , 30:1). b. s p l i t vent f l o w , 60mL.min -l ( s p l i t r a t i o , 60:1). 7 Influence of make-up gas (Argon-Methane, 95:5) flow r a t e 70 on HFB-MCP response. 8 Influence of make-up gas (Argon-Methane, 95:5) flow r a t e 71 on b a s e l i n e ( s i g n a l to noise) p l o t s . a.20mL.min-l b. 40mL.min - i , c.60mL.min-1 9 E f f e c t of a 2% 0V-1 packed Jennings s p l i t l i n e r on MCP, 7 3 MAP and prazepam peak shape, a. with 2% 0V-1 packing, b. without packing. 10. Comparison of standard curve parameters obtained using an 75 unpacked Jennings tube ( ), (y = 0.024 x + 0.0086, r = 0.9996) and a fused s i l i c a i n s e r t packed with a " t i g h t " s i l a n i z e d g l a s s wool plug ( ), (y = 0.0243 x + 0.004, r = 0.9997). 11. Optimization of HFBA d e r i v a t i z a t i o n r e a c t i o n time at 55°C 80 f o r MCP and MAP i n the presence of 0.05M TEA. 12. MCP Data C o l l e c t i o n Sheet 82 i x i Figure Page 13 A r e p r e s e n t a t i v e standard curve of human plasma e x t r a c t s 87 obtained by p l o t t i n g the area r a t i o of HFB-MCP/HFB-MAP versus MCP conc e n t r a t i o n . 14. Representative chromatograms obtained from blank Ca-) 92 and MCP-spiked (b.) plasma e x t r a c t s on the 25m SE-54 fused s i l i c a column. 15 A semi-log p l o t of the plasma p r o f i l e of MCP i n a 93 pregnant ewe f o l l o w i n g a lOmg I.V. dose. (•—•) maternal plasma l e v e l s , (±-A) f e t a l plasma l e v e l s . x i i LIST OF SCHEMES Scheme page I E x t r a c t i o n And D e r t v a t i z a t i o n Procedure For Fused S i l i c a C a p i l l a r y GLC-ECD Assay 81 x i i i LIST OF STRUCTURES AND DIAGRAMS Page S p l i t L i n e r C o n f i g u r a t i o n s : Jennings Tube, Fused 45 S i l i c a I n s e r t Chemical S t r u c t u r e s Of Metoclopramide And M a p r o t i l i n e 117 x i v ABBREVIATIONS A. R. area r a t i o b.p. b o i l i n g point B. P. blood pressure C V . c o e f f i c i e n t of v a r i a t i o n DMCS d i m e t h y l c h i o r o s i l a n e ECD elec t r o n - c a p t u r e detector F/M feta l : m a t e r n a l r a t i o G a c c e l e r a t i o n due to g r a v i t y (cm.s - 2) GC-MS gas chromatographic mass spectrometric GLC g a s - l i q u i d chromatography GLC-ECD electron-capture g a s - l i q u i d chromatographic h height e q u i v a l e n t to a t h e o r e t i c a l p l a t e HFB he p t a f l u o r o b u t y r y l HFB-MAP he p t a f l u o r o b u t y r y l d e r i v a t i v e of m a p r o t i l i n e HFB-MCP h e p t a f l u o r o b u t y r y l d e r i v a t i v e of metoclopramide HFBA h e p t a f l u o r o b u t y r i c anhydride HFBI h e p t a f l u o r o b u t y r y l imidazole H. R. heart r a t e (beats per minute) I. D. i n s i d e diameter ( i n t e r n a l diameter) im. intramuscular I.V. or i . v . intravenous MAP m a p r o t i l i n e MAP.HC1 m a p r o t i l i n e monohydrochloride XV MCP metoclopramide MCP.HC1 metoclopramide monohydrochloride MCP.HCl.H2O metoclopramide monohydrochloride monohydrate MV maternal vein (maternal venous) n t h e o r e t i c a l p l a t e number PFBC pentafluorobenzoyl c h l o r i d e PFPA pe n t a f l u o r o p r o p i o n i c anhydride p.o. o r a l l y (oral route) R r e s o l u t i o n r.p.m. r o t a t i o n s per minute R.T. r e t e n t i o n time S.D. ± one standard d e v i a t i o n S/N s i g n a l to noise r a t i o t u h a l f - l i f e " 2 tv R^ r e t e n t i o n time of a s p e c i f i e d s o l u t e peak t m hold-up time ( r e t e n t i o n time of an unretained peak) TEA t r i e t h y l amine TFAA t r i f l u o r o a c e t i c anhydride y average l i n e a r c a r r i e r gas v e l o c i t y y Q p t optimum l i n e a r c a r r i e r gas v e l o c i t y UA u m b i l i c a l a r t e r y ( u m b i l i c a l a r t e r i a l ) UV u m b i l i c a l v e i n ( u m b i l i c a l venous) WCOT wall coated open t u b u l a r X mean x v i ACKNOWLEDGEMENT The author would l i k e to s i n c e r e l y thank Dr. James Axel son f o r h i s s u p e r v i s i o n , encouragement and f r i e n d s h i p during the course of t h i s p r o j e c t . The author expresses s i n c e r e thanks to Barbara McErlane and Ram Kapil f o r t h e i r a s s i s t a n c e , encouragement and support throughout t h i s p r o j e c t . Thanks to Dr. Frank Abbott, Dr. Keith McErlane, Dr. John McNeill and Dr. James Orr f o r t h e i r suggestions, a s s i s t a n c e and support. The author i s deeply indebted to Dr. Dan Rurak, Nancy Gruber and C e c i l i a Cooper of the Department of O b s t e t r i c s and Gynaecology, U.B.C, without whom i t would not have been p o s s i b l e to c a r r y out the sheep experimentation - thank you f o r your expert t e c h n i c a l a s s i s t a n c e , a p p r e c i a t i o n of the problem and encouragement. S i m i l a r l y s i n c e r e thanks to Dr. Margaret Bylsma-Howell and Dr. Graham McMorland of the Department of Anaesthesia, the Grace H o s p i t a l , f o r making the human aspects of t h i s study p o s s i b l e and f o r t h e i r patience and a p p r e c i a t i o n of the a n a l y t i c a l problems. The author wishes to thank Dave Hasman of the Hewlett-Packard Co., f o r h i s a s s i s t a n c e , advice and donation of i n i t i a l fused s i l i c a c a p i l l a r y columns. Special thanks to C e l i n e Gunawardene and Tracy Lakevold f o r the preparation of t h i s manuscript. This p r o j e c t was made p o s s i b l e by the Medical Research Council and the B r i t i s h Columbia Medical Services Foundation. XVI 1 To memory of my f a t h e r to Mom and Len 1 1. INTRODUCTION Metoclopramide (MCP), a potent antiemetic and g a s t r i c m o t i l i t y s t i m u l a t o r , i s a chlorbenzamide d e r i v a t i v e chemically known as 4-amino-5-chloro-2-methoxy-N(2-diethyl ami noethyl) benzamide. Although s t r u c t u r a l l y r e l a t e d to procainamide, MCP has n e g l i g i b l e cardiac e f f e c t s and poor l o c a l anaesthetic a c t i v i t y . Introduced f o r c l i n i c a l use i n Europe i n 1964 (Justin-Besancon, et a l _ . , 1964) and i n Canada about 10 years l a t e r , MCP has been used e x t e n s i v e l y f o r a wide v a r i e t y of i n d i c a t i o n s . Metoclopramide's pharma-c o l o g i c a l a c t i o n s , i n d i c a t i o n s f o r use, side e f f e c t s , and mechanisms of a c t i o n have been the subject of several comprehensive reviews (Robinson, 1973; Pinder et a l _ . , 1976; Smith and S a l t e r , 1980; Ponte and Nappi, 1981; Schulze-Del r i e u , 1981). B r i e f l y , i t i s used c l i n i c a l l y to f a c i l i t a t e c e r t a i n d i a g n o s t i c procedures ( r a d i o l o g i c , endoscopic) of the upper gas-t r o i n t e s t i n a l t r a c t and small i n t e s t i n e , to t r e a t a v a r i e t y of f u n c t i o n a l and organic g a s t r o i n t e s t i n a l d i s o r d e r s (e.g. g a s t r i c s t a s i s , r e f l u x e s o p h a g i t i s ) and as an antiemetic i n the treatment of nausea and vomiting of varying e t i o l o g i e s (e.g. r a d i a t i o n - and drug-induced, pre- and post-o p e r a t i v e ) . Metoclopramide i s also used p a r e n t e r a l l y p r i o r to general anaesthesia i n labour (McMorland and By!sma-Howell, 1982; Schulze-D e l r i e u , 1981). In t h i s instance the drug's antiemetic a c t i v i t y (McGarry, 1971) as well as i t s a b i l i t y to promote g a s t r i c emptying (Howard and Sharp, 1973), help to reduce the incidence of a s p i r a t i o n of stomach contents and death due to Mendel son's Syndrome. 2 In general the incidence of side e f f e c t s associated with MCP therapy i n adults i s low and they are u s u a l l y m i l d and t r a n s i e n t i n nature (Robinson, 1973). There are, however, reports of an increased frequency and s e v e r i t y of adverse e f f e c t s when t h i s drug i s used i n i n f a n t s and c h i l d r e n (Pinder et ^ 1_., 1976; Low and Goel, 1980). R a t i o n a l e A s p i r a t i o n of stomach contents during o b s t e t r i c a l anaesthesia i s s t i l l the most common cause of maternal m o r t a l i t y due to anaesthesia; i t i s the fo u r t h o v e r a l l cause of maternal death a f t e r hemorrhage, toxemia and s e p s i s (Schnider and Levinson, 1979). Ever since Mendelson, 1946, f i r s t described the syndrome of pulmonary a s p i r a t i o n i n o b s t e t r i c p a t i e n t s , there have been studies to e l u c i d a t e the mechanisms i n v o l v e d . G a s t r i c volume and pH are important c o n t r i b u t i n g f a c t o r s so attempts are made to c o n t r o l these two parameters. Some of the commonly employed preventative measures aimed at re-ducing the incidence of Mendelson's Syndrome c o n s i s t of f a s t i n g p a t i e n t s during labour, and p r i o r to e l e c t i v e Caesarian s e c t i o n to reduce g a s t r i c volume, rapid anaesthetic i n d u c t i o n , and the use of antacids to r a i s e the pH above the deemed c r i t i c a l value of 2.5 (Roberts and S h i r l e y , 1974). Both the oral n o n - p a r t i c u l a t e a n t a c i d , sodium c i t r a t e (0.3M) and the histamine r ^ - a n t a g o n i s t , c i m e t i d i n e , have been shown to e f f e c t i v e l y r a i s e the g a s t r i c pH above 2.5 (Foulkes and Jenkins, 1981). The use of antacid therapy to increase maternal g a s t r i c pH i s not considered to be r e l i a b l e (Howe et a l _ . , 1981) although i t does help to reduce the r i s k of a s p i r a t i o n considerably (Roberts and S h i r l e y , 1974). Fasting the patie n t s 3 f o r 8-12 hours p r i o r to d e l i v e r y by Caesarian s e c t i o n also does not guarantee an empty stomach (Roberts and S h i r l e y , 1974). A more recent method of reducing a s p i r a t i o n r i s k involves the use of the potent antiemetic and g a s t r i c m o t i l i t y m o d i f i e r MCP. Metoclopra-mide has been shown to s i g n i f i c a n t l y diminish the frequency of vomiting during labour (McGarry, 1971) as well as to increase the g a s t r i c emptying r a t e (Howard and Sharp, 1973) and tone of the lower esophageal s p h i n c t e r (Brock-Utne et a l _ . , 1978), both of which are reduced i n labour. These p o s i t i v e actions of MCP have led to i t s more frequent use i n o b s t e t r i c a l anaesthesia i n Canada (Bylsma-Howell and McMorland, 1982) as well as i n Europe and Japan ( S c h u l z e - D e l r i e u , 1981). Metoclopramide causes few adverse r e a c t i o n s when administered i n the usual therapeutic doses. Side e f f e c t s such as l a s s i t u d e , drowsiness, t r a n s i e n t f e e l i n g s of a g i t a t i o n or a n x i e t y , dry mouth, c o n s t i p a t i o n or d i a r r h e a (Robinson, 1973; Pinder et a\_., 1976) have been reported to occur i n about 5-10% of p a t i e n t s . These e f f e c t s are normally m i l d , t r a n -s i e n t and r e v e r s i b l e upon withdrawal of the drug. MCP does cross the blood-brain b a r r i e r (Smith and S a l t e r , 1980) and as a r e s u l t can cause alarming extrapyramidal r e a c t i o n s . These e f f e c t s are g e n e r a l l y only seen i n about 1% of p a t i e n t s at t h e r a p e u t i c dose l e v e l s . Infants and c h i l -dren, however, are more s u s c e p t i b l e to the t o x i c e f f e c t s of MCP with an increased incidence of extrapyramidal r e a c t i o n s ( a g i t a t i o n , i r r i t a b i l i t y , r i g i d i t y , dystonia) being reported i n t h i s population (Pinder et a l . , 1976; Casteels-van Deele et aj_., 1970; Low and Goel, 1980). I t should be noted that these e f f e c t s are g e n e r a l l y rare and have occurred i n t h i s 4 population l a r g e l y because the recommended dose (maximum, 0.5 mg.kg~ 1.day~ 1) had been exceeded (Pi nder et a l _ . , 1976; Low and Goel, 1980). Although there i s no c l i n i c a l evidence to suggest t o x i c i t y i n the neonate (McGarry, 1971; Howard and Sharp, 1973) a thorough evaluation of t h i s aspect has not been performed. Metoclopramide with i t s low molecular weight and r e l a t i v e l y high l i p i d s o l u b i l i t y has been postulated to be t r a n s f e r r e d across the placen-t a (Smith and S a l t e r , 1980; S c h u l z e - D e l r i e u , 1981), however, there are no reports on t h i s t o p i c i n the l i t e r a t u r e . The a b i l i t y of MCP to cross the blood-brain b a r r i e r (Smith and S a l t e r , 1980) and i t s e x c r e t i o n i n human breast milk (Lewis et a]_., 1980) as well as i t s low degree of plasma p r o t e i n binding (13-22%) are c h a r a c t e r i s t i c s which suggest that such t r a n s f e r i s p o s s i b l e . I t i s well known that drugs administered to the pregnant p a t i e n t may exert d e l e t e r i o u s e f f e c t s on the fetus and newborn. Early i n preg-nancy the primary concern i s d i r e c t e d toward morphologic a b n o r m a l i t i e s , with thalidomide and i t s r e s u l t a n t gross b i r t h defects serving as the best example. L a t e r i n pregnancy, at the time of d e l i v e r y , f u n c t i o n a l c o n s i d e r a t i o n s are of more s i g n i f i c a n c e . The newborn now i s faced with the e l i m i n a t i o n of drugs received i n utero and as a r e s u l t of renal and hepatic immaturity may experience p h y s i o l o g i c a l e f f e c t s such as r e s p i r a -t o r y and cardiac depression. In view of the increased incidence of side e f f e c t s i n i n f a n t s and c h i l d r e n undergoing MCP therapy an attempt w i l l be made to evaluate i t s e f f e c t s , i f any, on the neonate by means of measure-5 merit of heart rate (HR), blood pressure (BP) and Apgar scores, as well as a thorough chart review of attending p h y s i c i a n and nurses' comments. The Apgar scoring system d i r e c t s a t t e n t i o n to f i v e v i t a l s i g n s : heart r a t e , r e s p i r a t o r y e f f o r t , muscle tone, r e f l e x i r r i t a b i l i t y and c o l o r over the f i r s t few minutes of l i f e . Each category receives a r a t i n g ranging from 0-2 depending upon the degree of response, with the i n d i v i d u a l categories being summed to give a maximum score of 10. The i n f a n t i s assessed at 1 and 5 minutes a f t e r d e l i v e r y . On the basis of these 1 and 5 minute scores, the neonate i s categorized i n t o one of three groups which r e l a t e to the degree of depression and, thus, to the need f o r a c t i v e r e s u s c i t a t i o n . Infants with scores of 0-2 are severely depressed (and merit immediate a c t i v e i n t e r v e n t i o n ) , those with scores of 3-7 are considered to be moderately depressed (and gen e r a l l y require only the a d m i n i s t r a t i o n of oxygen to improve) while neonates s c o r i n g 8-10 are considered healthy and requi r e no r e s p i r a t o r y assistance (Cohen, 1979). The Apgar then, serves as a guide to i d e n t i f y i n g and t r e a t i n g the depressed neonate. It has been c r i t i c i z e d f o r i t s r e l a t i v e crudeness of measurement since i t looks only at the v i t a l functions necessary to sus-t a i n l i f e and continues these observations f o r only a very b r i e f period. A l s o , the i n f a n t has to be considerably a f f e c t e d to s i g n i f i c a n t l y lower the score, t h e r e f o r e , s u b t l e e f f e c t s of maternal drug a d m i n i s t r a t i o n may be missed e n t i r e l y . Since drug e f f e c t s may not appear i n the i n f a n t (unless severe) u n t i l well a f t e r the neonate has l e f t the d e l i v e r y room an assessment of charts w i l l a lso be made f o r the purposes of follow-up. 6 The method of placental t r a n s p o r t study i n humans most f r e q u e n t l y invol v e s s i n g l e - p o i n t f e t a l and maternal blood sampling. The sampling of maternal venous blood and u m b i l i c a l a r t e r i a l and venous blood makes pos-s i b l e the determination of fetal/maternal concentration r a t i o s (La Du et a l . , 1971). Since only one f e t a l sample per d e l i v e r y i s obtainable by t h i s technique, sampling of a l a r g e number of p a t i e n t s i s required i n order to o b t a i n a curve of f e t a l blood c o n c e n t r a t i o n with respect to time. The k i n e t i c c h a r a c t e r i z a t i o n of placental transport i s then, most o f t e n , based on a composite of data obtained from d i f f e r e n t subjects at d i f f e r e n t times. Because of the t e c h n i c a l problems and e t h i c a l issues involved i n the study of the human f e t u s e s , animal models have been very important i n the study of placental drug t r a n s f e r . Although there are species d i f f e r -ences i n placental s t r u c t u r e and t r a n s f e r c h a r a c t e r i s t i c s , p l a c e n t a l p e r m e a b i l i t y to l i p i d s o l u b l e components, l i k e most drugs, e x h i b i t s much l e s s species v a r i a t i o n than the p e r m e a b i l i t y to h y d r o p h i l i c molecules. Thus data obtained i n m a t e r n a l - f e t a l drug t r a n s f e r from animal studies are u s u a l l y relevant to human pregnancy and labour. Over the past 15 y e a r s , h i g h l y s o p h i s t i c a t e d animal preparations (e.g. sheep, goat, cow, monkey) have been developed to study the fetus i n  utero. A s e p t i c s u r g i c a l techniques are used to implant c a t h e t e r s , e l e c -trodes and other devices i n the fetus and mother. A f t e r allowing time f o r recovery from surgery, the animals can then be studied f o r the dura-t i o n of pregnancy with the fetus undisturbed, i n utero. The preparations allow f o r s e r i a l samplings of blood, urine and various other f e t a l f l u i d s 7 and f o r the monitoring of several f e t a l p h y s i o l o g i c a l v a r i a b l e s . They are very useful f o r the study of mate r n a l - f e t a l drug t r a n s f e r , since determination of the time course of a drug i n the mother and fetus can be c a r r i e d out concomitantly with assessment of any acute e f f e c t s of the drug on the f e t u s . A p p l i c a t i o n of an e x i s t i n g assay method f o r MCP i n human plasma (Tarn et a l _ . , 1979) to the present study revealed two i n t e r f e r i n g peaks shouldering MCP. Although the assay had been used s u c c e s s f u l l y f o r drug l e v e l s 5-10 X higher than those c u r r e n t l y being assessed, the presence of these i n t e r f e r i n g substances contributed to a s i g n i f i c a n t amount of err o r i n MCP q u a n t i t a t i o n i n the present s i t u a t i o n . Numerous attempts to r e s o l v e MCP from these two components through the use of a l t e r n a t i v e s t a t i o n a r y phases and d e r i v a t i z i n g agents were unsuccessful. S i m i l a r l y , e f f o r t s to e l i m i n a t e these peaks through the use of a l t e r n a t i v e deter-gents, s o l v e n t s , drying and c a r r i e r gases as well as e l i m i n a t i o n of p l a s -t i c s from the wash and assay set-up procedures were not t o t a l l y success-f u l . The primary goals of t h i s p r o j e c t then were: i . to modify the e x i s t i n g assay and to develop a fused s i l i c a c a p i l l a r y column g a s - l i q u i d chromatographic e l e c t r o n capture a n a l y t i c a l method s u i t a b l e f o r MCP q u a n t i t a t i o n i n human and sheep plasma. i i . to conduct a double-blind study of MCP placental t r a n s f e r i n normal healthy women undergoing e l e c t i v e Caesarian s e c t i o n and r e q u i r i n g MCP as a pre-anaesthetic medication. 8 i i i . to assess MCP's adverse e f f e c t s , i f any, on the neonate through Apgar scoring and a thorough chart review, i v . to i n i t i a t e a more comprehensive study of the k i n e t i c s of MCP's placental t r a n s f e r i n a c h r o n i c a l l y c a t h e t e r i z e d preg-nant sheep p r e p a r a t i o n . 1.1 A n a l y t i c a l Methods For MCP In B i o l o g i c a l F l u i d s A number of a n a l y t i c a l methods have been developed f o r the mea-surement of MCP i n b i o l o g i c a l f l u i d s over the l a s t 10-12 years. Many of these lack s e n s i t i v i t y and s p e c i f i c i t y and only i n the l a s t 5 years have more s p e c i f i c and s e n s i t i v e methods been reported which can be applied to pharmacokinetic s t u d i e s . A r i t a et a]_., 1970, used a TLC c o l o r i m e t r i c method to study MCP and some of i t s metabolites i n r a b b i t u r i n e . A q u a n t i t a t i v e TLC method f o r the determination of MCP i n human plasma has also been reported (Schuppan et a l _ . , 1979)., Both methods lack s p e c i f i c i t y , so i n t e r f e r e n c e from s t r u c t u r a l l y r e l a t e d metabolites l i m i t s t h e i r use. In a d d i t i o n , both techniques lack s e n s i t i v i t y i n the low nanogram range and r e q u i r e plasma volumes of one mL or more making them u n s u i t a b l e f o r s e r i a l samp-l i n g i n small animals, such as f e t a l sheep and rats and i n neonates. Two TLC-photodensitometric methods have also been reported. The procedure of Bakke and Segura, 1975, was used to measure MCP and i t s metabolites i n the plasma and urine of r a t s , r a b b i t s and dogs. The e x t r a c t i o n of 5 ml of urine or plasma provides s e n s i t i v i t y i n the range of 200-500 ng of MCP.rnL"1 again demonstrating lack of a p p l i c a b i l i t y to small animal 9 stu d i e s or to extensive k i n e t i c studies i n man. Huizing and Beckett, 1979, a l s o report a TLC-photodensitometric technique to study the metabo-l i s m of MCP i n spiked r a b b i t l i v e r homogenates, as well as i n spiked urine and plasma. S e n s i t i v i t y was reported to be i n the range of 20 ng.mL - 1 but no mention was made of the urine or plasma volumes required to achieve t h i s l e v e l . Further, the authors s t a t e the good pre-c i s i o n i n TLC-photodensitometry can be obtained "only when a l l c r i t i c a l procedures involved are r i g i d l y standardized". Again s p e c i f i c i t y i s a probl em. In the l a s t few year s , a number of high performance l i q u i d chromatographic (HPLC) methods have been reported f o r the q u a n t i t a t i o n of MCP i n human plasma and urine (Teng et a l _ . , 1977; Graffner et a l _ . , 1979; Bateman et a K , 1981; Block et a l _ . , 1981). A l l provide s e n s i t i v i t y i n the range of 5-10 ng of MCP.mL-1 of plasma. This however, requires the e x t r a c t i o n of 2-5 mL of plasma precluding t h e i r use i n i n v e s t i g a t i o n s i n v o l v i n g i n f a n t s or small animals (e.g. f e t a l sheep). They also possess considerable p o t e n t i a l f o r i n t e r f e r e n c e from other drugs used i n c l i n i c a l s t u d i e s (Bateman et a l _ . , 1981). These HPLC techniques are rapid and do provide s u f f i c i e n t s e n s i t i v i t y f o r pharmacokinetic studies i n man f o r up to 8-10 hours a f t e r a 10-20 rug oral dose of MCP. A number of electron-capture g a s - l i q u i d chromatographic (GLC-ECD) assays have also been developed f o r pharmacokinetic studies i n r a t s (Tarn and Axelson, 1978) and i n man (Tarn et a l _ . , 1979; Ross-Lee et aj_., 1980). A l l provide good s e n s i t i v i t y allowing q u a n t i t a t i o n of MCP t o 5 ng.mL"! plasma f o l l o w i n g the e x t r a c t i o n of 0.5 - 1.0 mL of human plasma. The 10 method reported by Tarn and Axel son, 1978, i s s u f f i c i e n t l y s e n s i t i v e to allow s e r i a l blood sampling (0.1-0.2 mL) f o r pharmacokinetic studies i n r a t s . Although s e n s i t i v e , these packed column GLC-ECD methods demon-s t r a t e considerable p o t e n t i a l f o r i n t e r f e r e n c e from endogenous substances when used f o r t r a c e l e v e l a n a l y s i s as well as from other drugs i n c l i n i -cal s t u d i e s . A s e n s i t i v e and s p e c i f i c assay based on s t a b l e isotope d i l u t i o n and u t i l i z i n g gas chromatography mass spectrometry (GC-MS) with selected ion monitoring has been reported by Bateman et a l _ . , 1978. The method was used f o r pharmacokinetic studies i n man and dogs. Tarn and Axel son, 1979a, also report a s e n s i t i v e GLC-ECD assay f o r the simultaneous q u a n t i t a t i o n of MCP and i t s monode-ethyl ated metabolite i n rat u r i n e . 1.2 Basic MCP Pharmacokinetics Animal ( r a t , r a b b i t , dog) experiments with MCP suggest that t h i s compound i s well absorbed, e x t e n s i v e l y metabolized and r a p i d l y excreted i n a l l species studied (Tunon et a l _ . , 1974•, Bakke and Segura, 1976; Bateman et j i l _ . , 1980; Tarn et a l _ . , 1981) with p a r t i a l metabolism by con-j u g a t i o n ( r a b b i t , dog) ( A r i t a et a l _ . , 1970; Cowan et a l _ . , 1976; Bateman et a l _ . , 1978), 0-demethyl a t i on, N-de-ethyl at i o n and amide h y d r o l y s i s ( r a t , r a b b i t , dog) ( A r i t a et a l _ . , 1970; Bakke and Segura, 1976; Cowan et a l . , 1976). The peak plasma concentration of MCP was observed w i t h i n 30-120 minutes of oral a d m i n i s t r a t i o n suggesting rapid absorption from 1 1 the g a s t r o i n t e s t i n a l t r a c t . The e l i m i n a t i o n h a l f - l i f e ( t i y g ) of MCP f o l l o w i n g intravenous ( i . v . ) i n j e c t i o n i n the r a t , r a b b i t and dog i s 20, 28 and 36 minutes, r e s p e c t i v e l y (Bakke and Segura, 1976). A s i g n i f i c a n t -l y longer t\/2 (50 minutes) has been more r e c e n t l y reported i n rats (Tarn and Axel son, 1978). I t has r e c e n t l y been shown i n the rat that MCP undergoes s a t u r -able f i r s t - p a s s metabolism at doses below 1 mg.kg-1 (Kapil et a l . , 1982) and unusual dose-dependent k i n e t i c s at high doses (>15 mg.kg-1) (Tarn et aj_., 1981) due to apparent blood flow a l t e r a t i o n s induced by MCP. A number of pharmacokinetic studies i n normal human volunteers have appeared i n recent l i t e r a t u r e (Graffner et a l _ . , 1979; Schuppan et a l _ . , 1979; Bateman et a]_., 1980; Block et a]_., 1980; Ross-Lee et a l _ . , 1981a). MCP has been found to be e x t e n s i v e l y metabolized i n man (=80% of the dose) (Teng et a l _ . , 1977). Conjugation i s the major metabolic route i n man (=50% of the dose) with a number of l e s s important minor metabo-l i t e s a lso being found i n the urine (Teng et a l _ . , 1977). S u l f a t e conju-gation has been found to be the predominant conjugation pathway f o r MCP w h i l e g l u c u r o n i d a t i o n comprised only a very small percentage of the administered dose (Bateman et a l _ . , 1980). Renal and hepatic immaturity are well documented i n the newborn (Horning et a l _ . , 1975; M o r s e l l i , 1976) and while conjugation with glucuronic acid appears to be d e f e c t i v e at b i r t h , s u l f a t e conjugation i s reported to be c l o s e to the adult pattern ( M o r s e l l i , 1976). 12 S i g n i f i c a n t f i r s t - p a s s metabolism has been reported i n humans by Bateman et a l _ . , 1980. A considerable degree of v a r i a b i l i t y was seen i n t h e i r data, p o s s i b l y due to i n t e r s u b j e c t v a r i a b i l i t y o r, i n p a r t , due to the use of a t a b l e t (which may r e s u l t i n v a r i a b l e d i s s o l u t i o n and absorp-t i o n ) r a t h e r than a s o l u t i o n dosage form i n t h e i r study. A number of other i n v e s t i g a t o r s (Graffner et a l _ . , 1979; Schuppan et a l _ . , 1979; Block e t j i l _ . , 1981) have also reported a f i r s t - p a s s e f f e c t . These studies how-ever, may be i n a p p r o p r i a t e since the comparison of b i o a v a i l a b i l i t y was made a f t e r unequal oral and i . v . doses. This l a t t e r assessment can only be made i f MCP f o l l o w s dose-independent k i n e t i c s and to date no study of MCP over a wide dose range has been conducted. P r e l i m i n a r y studies i n our l a b o r a t o r y comparing equal oral ( s o l u t i o n ) and i . v . doses seem to confirm f i r s t - p a s s metabolism of MCP. A p r e l i m i n a r y study by Bateman et a l _ . , 1981, i n p a t i e n t s with renal f a i l u r e , has shown that the t\/2 of MCP was increased f o u r -f o l d while t o t a l body clearance was decreased to a s i m i l a r extent. These observations are unexpected since only a small percentage (-20%) of an administered dose i s excreted i n the urine as i n t a c t drug. This suggests that the renal e l i m i n a t i o n pathway f o r MCP would be r e l a t i v e l y unimpor-t a n t . A s i m i l a r d i s p r o p o r t i o n a t e increase i n MCP t^/2 a n d reduction i n clearance has been observed i n rats with experimental renal dysfunc-t i o n (Tarn et a l _ . , 1981a), d e s p i t e the f a c t that only about 20% of the administered MCP dose i s excreted as i n t a c t drug i n t h i s species. These i n v e s t i g a t o r s postulated that s i g n i f i c a n t extrahepatic metabolism or diminished hepatic metabolism secondary to renal f a i l u r e might account 13 f o r these observations (Tarn et a]_., 1981a). However, more recent studies with i n v i t r o l i v e r , kidney and lung t i s s u e homogenates have ruled out extrahepatic (renal) metabolism (Kapil et aj_., 1982) and diminished hepa-t i c metabolism secondary to renal f a i l u r e seems more l i k e l y (Bateman et a l _ . , 1981; Kapil et a l . , 1982). 1.3 MCP's Use In Pregnancy There are few reports i n the l i t e r a t u r e concerning the use of MCP during the course of pregnancy or at the time of labour and d e l i v e r y . The drug i s used p a r e n t e r a l l y l o c a l l y (Byl sma-Howel 1 and McMorland, 1982) and abroad (Europe and Japan) ( S c h u l z e - D e l r i e u , 1981) f o r intrapartum and emergency anaesthesia. MCP has been shown to s i g n i f i c a n t l y reduce the emesis of e a r l y pregnancy (Pinder et a l _ . , 1976) as well as during labour (McGarry, 1971) when compared to a placebo. There have been no reports of dysmorphogenic e f f e c t s i n animals or i n women undergoing short-term (7 day) MCP therapy to control emesis during e a r l y pregnancy (<12-19 weeks) (Pinder et a l . , 1976). As with most new drugs however, the safety of MCP i n earl y preg-nancy has not been d e f i n i t e l y e s t a b l i s h e d (Pi nder et a l _ . , 1976; Smith and S a l t e r , 1980) so i t i s not recommended f o r use during the f i r s t t r i -mester. The e f f e c t of a s i n g l e dose of MCP on the g a s t r i c emptying rate of a 750 mL l i q u i d t e s t meal was measured by Howard and Sharp, 1973, i n 14 25 p a r t u r i e n t women undergoing a normal vaginal d e l i v e r y . The dye con-t a i n i n g l i q u i d meal was administered to the women during labour by means of a nasogastric tube, samples were withdrawn f o r a n a l y s i s at various times during the two hour period of study. The pa t i e n t s had been f a s t e d f o r 18 hours p r i o r to the study. The subjects received e i t h e r 10 mg of MCP or an equivalent volume of s t e r i l e water by intramuscular i n j e c t i o n on a double-blind basis at the s t a r t of the t e s t . The g a s t r i c emptying rate was found to be s i g n i f i c a n t l y increased i n those 13 women who had received MCP compared to the 12 who received the s t e r i l e water placebo. No adverse e f f e c t s associated with the use of MCP i n labour have been reported (McGarry, 1971; Howard and Sharp, 1973) i n e i t h e r the mothers or fetuses (assessed by Apgar s c o r e s ) . Brock-Utne et a l _ . , 1978, studied the e f f e c t of MCP on lower esopha-geal sphincter pressure i n pregnant p a t i e n t s at term. MCP was found to s i g n i f i c a n t l y increase the tone of the lower esophageal s p h i n c t e r , which l i k e g a s t r i c emptying, i s impaired during pregnancy and labour. 15 1.4. Factors A f f e c t i n g Maternal-Fetal Drug Transfer The placenta i s composed of l i p o p r o t e i n and as a r e s u l t the mechanisms f o r pl a c e n t a l t r a n s f e r are e s s e n t i a l l y the same as f o r any other b i o l o g i c a l membrane. Passive d i f f u s i o n i s considered to be the most important mode of tr a n s p o r t f o r drug substances. Other mechanisms such as a c t i v e t r a n s p o r t , p i n o c y t o s i s and passage through membrane pores are f e l t to be g e n e r a l l y unimportant f o r drugs although important f o r some b i o l o g i c a l m a t e r i a l s (eg. amino a c i d s , immunproteins) ( A s l i n g and Way, 1971). V i r t u a l l y a l l drugs w i l l cross the placenta,consequently,only the rate and extent of t r a n s f e r w i l l vary. Because of t h i s , considerable a t t e n t i o n has been devoted to the study of the mechanisms inv o l v e d . In a d d i t i o n , those f a c t o r s governing the rate and extent of t r a n s f e r which u l t i m a t e l y determine the drug's concentration i n the mother and subsequently i n the fetus/neonate have been thoroughly reviewed ( A s l i n g and Way, 1971; Tuchrnann-Duplessis, 1975; M i r k i n and Singh, 1976; O'Brien e t a l . , 1977; F i n s t e r and Pedersen, 1979; Reynolds,1979; Brock-Utne et a l . , 1980; Ward et a l . , 1980; D i 1 t s , 1981); these complex f a c t o r s may be b r i e f l y summarized as f o l l o w s : 1. Maternal plasma drug l e v e l s are dependent upon ( i ) the t o t a l dose, the s i t e and mode of a d m i n i s t r a t i o n (p.o., i . m . , i . v . ) , ( i i ) d i s t r i -buti.on ?which i s a f f e c t e d by haemodynamics, t i s s u e a f f i n i t y and the degree of p r o t e i n b i n d i n g , ( i i i ) rate and degree of metabolism (clear a n c e , h a l f - l i f e ) and, ( i v ) the rate of e x c r e t i o n ( i n t a c t drug, metaboli t e s ) . 16 2. P l a c e n t a l drug t r a n s f e r i s a f u n c t i o n of ( i ) the physico-chemical p r o p e r t i e s of the drug such as i t s l i p i d s o l u b i l i t y , molecular weight, degree of i o n i z a t i o n and p r o t e i n b i n d i n g , ( i i ) the concentration gradient across the p l a c e n t a , ( i i i ) maternal and f e t a l p l a c e n t a l blood f l o w s , ( i v ) the m a t e r n a l - f e t a l blood pH g r a d i e n t , (v) the stage of p l a c e n t a l development (which a f f e c t s i t s t h i c k n e s s , the area a v a i l a b l e f o r exchange and the degree of p e r f u s i o n ) , ( v i ) p o s s i b l e p l a c e n t a l metabolism (metabolism of drugs has been demonstrated with i n v i t r o homogenates (Juchau, 1976) but i t s s i g n i f i c a n c e i n vivo i s unknown), ( v i i ) d i f f e r e n c e s i n maternal and f e t a l p r o t e i n binding and ( v i i i ) the duration of exposure to the drug i n the maternal c i r c u l a t i o n ( v i z . , s i n g l e i . v . bolus, repeated i n j e c t i o n s or an i n f u s i o n ) . 3. Fetal drug concentrations are a f f e c t e d by ( i ) the degree of d i s t r i -bution which i s a f u n c t i o n of the f e t a l c i r c u l a t i o n p a t t e r n , t i s s u e a f f i n i t y and p r o t e i n b i n d i n g , ( i i ) hepatic metabolism (presence and c a p a c i t y of i n v i v o enzyme systems i s l a r g e l y unknown),(ii) renal e x c r e t i o n i n t o the amniotic f l u i d (may be some re-uptake of the drug i n t o the f e t a l c i r c u l a t i o n as a r e s u l t of swallowed amniotic f l u i d ) , ( i v ) u m b i l i c a l cord blood flow (e.g. cord may be compressed during l a b o u r ) , (v) d i l u t i o n of the drug i n the f e t a l c i r c u l a t i o n ( r e s u l t s i n a delay i n e q u i l i b r a t i o n between f e t a l t i s s u e s and f e t a l blood) and, ( v i ) uptake and/or metabolism by the f e t a l l i v e r ( f i r s t - p a s s e f f e c t ) . 17 4. Neonatal d r u g - l e v e l s are dependent.upon ( i ) v p h y s i o l o g i c a l changes which occur at b i r t h , (Ix) hepatic f u n c t i o n (enzyme a c t i v i t y may be low i n the f i r s t few days and weeks of l i f e , Horning et a l . ,1975; M o r s e l l i , 1976), ( i i i ) renal e x c r e t i o n (renal f u n c t i o n s such as t u b u l a r s e c r e t i o n , t u b u l a r reabsorption and glomerular f i l t r a t i o n are d e f i c i e n t at b i r t h , o f t e n t a k i n g days and weeks to mature, M o r s e l l i , 1976) and ( i v ) l o s s of the placenta as an organ of e l i m i n a t i o n ( i . e . , i n utero " f e t a l k i d ney"). 1.4.1. Pharmacokinetics Of P l a c e n t a l Drug Transfer Experimental access to the p l a c e n t a l - f e t a l u n i t i n humans i s r e s t r i c t e d f o r obvious t e c h n i c a l , e t h i c a l and l e g a l reasons. Because of t h i s most of the a v a i l a b l e data on human p l a c e n t a l drug t r a n s f e r i s based on s i n g l e - p o i n t determinations. Paired maternal and i n f a n t ( u m b i l i c a l cord) samples are drawn at b i r t h and from a l a r g e number of d i f f e r e n t p a t i e n t s maternal and neonatal concentration-time p r o f i l e s are constructed. The considerable i n t e r s u b j e c t v a r i a b i l i t y as w e l l as a r t i f a c t s introduced by data averaging severely l i m i t s the pharmacokinetic i n t e r p r e t a t i o n s which can be drawn from such composite p r o f i l e s (Krauer and Krauer, 1977; Levy, 1981; Waddell and Marlowe, 1981). In attempts to overcome these drawbacks and to more f u l l y u t i l i z e the information obtained from composite s t u d i e s , a number of i n v e s t i g a t o r s have c a r r i e d out t h e o r e t i c a l computer simulations of the m a t e r n a l - p l a c e n t a l - f e t a l u n i t (Levy and Hayton, 1973; Krauer and Krauer, 1977; Anderson et a l . , 1980; Krauer et a l . , 1980; Levy, 1981; Waddell and Marlowe, 1981). Various concentration-time p r o f i l e s have been 18 generated based on pharmacokinetic compartmental models. The f e t u s i s considered as a s i n g l e compartment and the mother as one or two. The placenta may a l s o be represented as a separate elimination-compartment. The f e t a l compartment may be viewed as e i t h e r being shallow ( r a p i d l y a c c e s s i b l e ) or deep (s l o w l y a c c e s s i b l e ) . These models are complex but have been a p p l i e d w i t h some success to inf o r m a t i o n from composite s t u d i e s (Levy and Hayton, 1973; Levy, 1981; Weddell and Marlowe, 1981). I t i s f e l t , at l e a s t at t h i s time, that such si m u l a t i o n s may best serve as an a i d to designing experimental protocols so that the information obtained from ( s i n g l e - p o i n t ) p l a c e n t a l drug t r a n s f e r studies can be maximized (Levy and Hayton, 1973; Krauer e t a l . , 1980). 1.4.2. Studies Of P l a c e n t a l Drug Transfer There are numerous p u b l i c a t i o n s d e a l i n g with the m a t e r n a l - f e t a l exchange of drugs. The m a j o r i t y of these demonstrate t r a n s f e r d i r e c t l y (e.g. A s l i n g and Way, 1971), that i s , through q u a n t i t a t i v e drug measurement while others are reviews based upon p h y s i o l o g i c a l or morphological observations i n the neonate at b i r t h (O'Brien and McManus, 1977; H i l l and S t e r n , 1979). The review of A s l i n g and Way, 1971, provides a comprehensive l i s t of drugs undergoing p l a c e n t a l t r a n s f e r . Fetal /maternal concentra-t i o n r a t i o s determined from s i n g l e - p o i n t maternal and f e t a l ( u m b i l i c a l cord) blood samples are given. Drugs from several t h e r a p e u t i c c a t e g o r i e s are i n c l u d e d , f o r example, l o c a l and general a n a e s t h e t i c s , 19 a n t i b i o t i c s , analgesics, and t r a n q u i l i z e r s , A comprehensive review of the p l a c e n t a l t r a n s f e r of l o c a l a naesthetics used i n s p i n a l anaesthesia and the f a c t o r s a f f e c t i n g t h e i r net t r a n s f e r (pH, pka, p r o t e i n - b i n d i n g ) has r e c e n t l y been published (Yurth, 1982). Review a r t i c l e s i n t h i s area are l a c k i n g , however, many reports d e a l i n g w i t h the m a t e r n a l - f e t a l t r a n s f e r ( s i n g l e - p o i n t s t u d i e s ) of i n d i v i d u a l drugs are a v a i l a b l e . In a small number of instances neonatal drug l e v e l s (plasma, urine) have been assessed immediately a f t e r b i r t h . These types of studies would provide more useful pharmacokinetic information but are r e s t r i c t e d l a r g e l y f o r e t h i c a l reasons. B u p i v i c a i n e and procainamide are two examples of drugs which have been studied i n t h i s manner; both bear some s t r u c t u r a l s i m i l a r i t y to MCP. B u p i v i c a i n e blood concentrations were determined i n s i x i n f a n t s born to mothers undergoing ep i d u r a l anaesthesia f o r a normal vaginal d e l i v e r y . Fetal blood samples were obtained during d e l i v e r y by means of scalp puncture and from the neonate at 2,6 and 24 hours a f t e r b i r t h (heel p r i c k ) . An average h a l f - l i f e {%) of 18 hours was found i n the 'I newborns compared to 1.25 hours i n the mothers. At no time d i d f e t a l or neonatal drug concentrations exceed maternal and no adverse e f f e c t s were noted i n the i n f a n t s (Caldwell et a l . , 1977). 20 The time course of procainamide, a cl o s e s t r u c t u r a l analogue of MCP, and i t s N-acetyl metabolite were stud i e d i n a s i n g l e neonate f o l l o w i n g a normal vaginal d e l i v e r y . The i n f a n t ' s mother had received procainamide f o r c o n t r o l of v e n t r i c u l a r arrhythmias (375 mg every 4 hours) f o r 3 days p r i o r to d e l i v e r y . Complete urine c o l l e c t i o n s were obtained from the newborn during f i v e consecutive 8 hour i n t e r v a l s a f t e r b i r t h and a s i n g l e blood sample was obtained a t 8 hours. The tx f o r procainamide was 13.5 hours and that of N-acetylprocainamide 19.5 hours. The average h a l f - l i v e s i n a d u l t s are 3 and 6 hours, r e s p e c t i v e l y . Both the parent drug and i t s metabolite undergo a c t i v e t u b u l a r s e c r e t i o n i n a d u l t s ; t h i s s e c r e t o r y mechanism i s f r e q u e n t l y immature i n the neonate ( M o r s e l l i , 1976) and l i k e l y accounts f o r the prolonged h a l f - l i v e s observed i n the i n f a n t . The concentrations of procainamide and N-acetylprocainamide i n the neonate's blood at 8 hours a f t e r b i r t h exceeded those found i n both cord and maternal blood. T h i s may be due to an i o n - t r a p p i n g e f f e c t based on the m a t e r n a l - f e t a l pH gradient (-7.4 and -7.3, r e s p e c t i v e l y ) and the pka . of procainamide (9.4) and N-acetylprocainamide (9.8) which could r e s u l t i n a net t r a n s f e r to the fetus, i n utero. No adverse effects; were reported i n the neonate (Lima et a l . , 1978). MCP with i t s s i m i l a r pka (9.3) and low degree of p r o t e i n binding (- 13-22%) may a l s o undergo a s i m i l a r net t r a n s f e r to the fetus, i n utero. 1.5. Sheep As a Model For P l a c e n t a l Drug Transfer A number of animals have been used to study the p l a c e n t a l t r a n s f e r of biochemical compounds and/or drugs i n c l u d i n g r a t s , guinea p i g s , p i g s , s e a l s , goats, cows, horses, sheep and monkeys. Experiments 21 i n v o l v i n g s e r i a l blood sampling however are l i m i t e d to l a r g e r animals, such as monkeys, sheep and goats, The s i z e of these animals a l l o w s the c h r o n i c i m p l a n t a t i o n of catheters i n t o various f e t a l and maternal blood v e s s e l s and the attachment of e l e c t r o n i c devices to monitor various p h y s i o l o g i c a l parameters (e.g. heart r a t e , blood pressure). The s u r g i c a l p r e p a r a t i o n , advantages and disadvantages of some of the common animal model species (sheep, monkey, horse, pig) employed as models i n f e t a l medicine has been thoroughly reviewed ( N a t h a n i e l s z , 1980). The monkey has a reproductive system very s i m i l a r t o man's both a n a t o m i c a l l y and p h y s i o l o g i c a l l y (Ramsey, 1975; Novy, 1980) so i t may be the most r e p r e s e n t a t i v e model f o r studying p l a c e n t a l drug t r a n s f e r . They are however, expensive, d i f f i c u l t to handle and prepare s u r g i c a l l y ( v i z . , small f e t a l s i z e ) and need to be r e s t r a i n e d f o l l o w i n g surgery (Novy, 1980). The l a t t e r aspect a l s o renders the preparation somewhat abnormal p h y s i o l o g i c a l l y (Novy, 1980). In a d d i t i o n s u r g i c a l manipulation of the t h i c k , muscular uterus of the primates often r e s u l t s i n spontaneous u t e r i n e c o n t r a c t i o n and a b o r t i o n . For these reasons they are not g e n e r a l l y used f o r drug t r a n s f e r studies, although there are a few rare reports i n the l i t e r a t u r e . Sheep appear to be the most commonly used chronic preparation (model) f o r i n v e s t i g a t i o n s i n t o f e t a l biochemistry, and physiology (Comlitie. and S i l v e r , 1974; Szeto et a l . , 1978; Van Petten et a l . , 1978). They are of moderate s i z e , are r e l a t i v e l y easy to handle s u r g i c a l l y ( v i z . , f e t a l s i z e ) are d o c i l e and g e n e r a l l y stand q u i e t l y during blood 22 sampling^ and monitoring. Although used l a r g e l y f o r studies of i n utero f e t a l physiology (e.g. oxygen consumption, glucose metabolism) they have been recognized as valuable model f o r pharmacological i n v e s t i g a t i o n s (Van Petten e t ; a l . ,1978). This i s s u b s t a n t i a t e d by the appearance of more frequent reports of t h e i r use i n p l a c e n t a l drug t r a n s f e r studies e s p e c i a l l y w i t h i n the l a s t 5 years. Sheep do possess a d i f f e r e n t placenta (syndesmochorial:5 t i s s u e l a y e r s ; 3 f e t a l , 2 maternal) from man (hemochorial:3 f e t a l t i s s u e l a y e r s ) and there are p h y s i o l o g i c a l d i f f e r e n c e s . As with a l l animal models e x t r a p o l a t i o n s to the human s i t u a t i o n must be cautious. They do,however, provide valuable information on the pharmacokinetics of drugs i n the ewe as w e l l as i n the fetus i n utero. A few examples of the use of c h r o n i c a l l y c a t h e t e r i z e d sheep i n p l a c e n t a l drug t r a n s f e r studies i n c l u d e : meperidine (Szeto, et a l . , 1978), indomethacin (Anderson et a l . , 1980a),acetyl s a l i c y l i c a c i d (Anderson, et a l . , 1980b) and diazepam (Conk! i n - e t a l . , 1980) 23 1.6 C a p i l l a r y Column Gas Chromatography: Fused S i l i c a Columns Open t u b u l a r or c a p i l l a r y columns were f i r s t introduced i n 1957 by M.J.E. Golay. These columns provide a dramatic increase i n separation power over conventional packed columns and t h e i r development i s regarded as a major t e c h n o l o g i c a l advance i n the f i e l d of gas chromatography. C a p i l l a r y columns are e s s e n t i a l l y lengths of c a p i l l a r y tubing ( u s u a l l y glass) the i n s i d e wall of which has been coated with a t h i n , uniform f i l m (0.1-0.4 um) of a s t a t i o n a r y phase, hence the name Wall Coated Open Tubular columns (WCOT). Unlike conventional packed columns, c a p i l l a r y columns have a high s p e c i f i c gas permeability due to t h e i r "openess" and a very small amount of s t a t i o n a r y phase. These two f a c t o r s coupled with small i n s i d e diameters (0.2-0.7 mm) and increased length (5-100 m) c o n t r i b u t e to the high r e s o l v i n g power of the WCOT column. In the l a s t 3 or 4 years fused s i l i c a has been introduced as an a l t e r n a t i v e column c o n s t r u c t i o n m a t e r i a l . Unlike g l a s s , fused s i l i c a does not contain m e t a l l i c oxides (Lewis acids) which i f unremoved c o n t r i -bute to r e v e r s i b l e and i r r e v e r s i b l e adsorption i n t e r a c t i o n s with the s o l u t e and subsequent peak t a i l i n g and losses of t r a c e components (Novotny, 1981). In a d d i t i o n to t h e i r i n e r t n e s s , fused s i l i c a columns are very f l e x i b l e , durable and very much l e s s f r a g i l e than those con-s t r u c t e d of glass (Jennings, 1980c; Freeman, 1981a, 1981d). Even more r e c e n t l y , bonded or c r o s s - l i n k e d fused s i l i c a columns have been introduced. These columns also e x h i b i t a high degree of i n e r t -ness as well as e x c e l l e n t thermal s t a b i l i t y (325°-350°C) and very low 24 solvent e x t r a c t a b i l i t y (Grob and Grob, 1981; Freeman, 1981a; Plotczyk, 1982). The immobilized coatings i n these c a p i l l a r y columns o f f e r two important advantages. F i r s t , the dangers p r e v i o u s l y associated with solvent phase s t r i p p i n g are v i r t u a l l y eliminated thus allowing the use of a wider range of solvents ( v i z . , improved solvent strength to more e f f i -c i e n t l y d i s s o l v e the s o l u t e ) . The second advantage i s that these columns can be rinsed with solvents to remove n o n - v o l a t i l e d e p o s i t s , the accumu-l a t i o n of which can reduce column chromatographic e f f i c i e n c y ( q u a l i t a -t i v e , q u a n t i t a t i v e ) , (Grob and Grob, 1981; Pl o t c z y k , 1982). The high e f f i c i e n c y associated with glass open t u b u l a r ( c a p i l -l a r y ) columns has been applied f o r many years to resolve complex mix-t u r e s . A n a l y s i s of environmental p o l l u t a n t s i n a i r and water and use i n the petroleum, food, f l a v o r and fragrance i n d u s t r i e s i s widespread. Only r e c e n t l y has t h i s technology been applied to the a n a l y s i s of b i o l o g i c a l samples f o r metabolic p r o f i l i n g (Jennings, 1980; Freeman, 1981; Novotny, 1981) of c e r t a i n disease s t a t e s , the measurement of human urine and p l a s -ma drug concentrations (Guerret, 1980; VandenHeuvel and Zweig, 1980; Jochemsen and Breimer, 1981) and the determination of drug pharmacokine-t i c s i n man (VandenHeuvel and Zweig, 1980; Jochemsen and Breimer, 1981). The recent i n t r o d u c t i o n of hi g h l y i n e r t fused s i l i c a columns (Freeman, 1980a, 1981d) f a c i l i t a t e s r e l i a b l e and reproducible a n a l y s i s of a v a r i e t y of u n d e r i v a t i z e d ( a c i d i c , b a s i c , n e u t r a l , polar) drugs ( P l o t c z y k , 1982). P r e v i o u s l y , such procedures f o r u n d e r i v a t i z e d substance were only margi-n a l l y successful with the more s u r f a c e - a c t i v e , glass c a p i l l a r y columns. The use of c h i r a l c a p i l l a r y ( g l a s s , fused s i l i c a ) columns to resolve enantiomeric p a i r s and to e s t a b l i s h t h e i r pharmacokinetics has also been 25 r e c e n t l y reported ( S i l b e r et a l _ . , 1982). Open t u b u l a r c a p i l l a r y columns are ch a r a c t e r i z e d by low c a r r i e r gas flow rates (0.5-10 mL.min - 1), reduced sample capacity and f a s t peak p r o f i l e s (Freeman, 1980c). Because of these c h a r a c t e r i s t i c s , v a r i o u s instrument-related parameters require s p e c i a l design and op t i m i -z a t i o n . For example, the ext r a column volume associated with the chroma-tographic i n l e t and low column capacity require special methods of sample i n t r o d u c t i o n . Four sampling modes have evolved: s p l i t , s p l i t l e s s , d i r e c t and on-column. S p l i t and s p l i t l e s s appear to be the most commonly used and are compatible with the instrumentation a v a i l a b l e i n our l a b o r a t o r y so they w i l l be tested during the development of the c a p i l l a r y assay method f o r MCP. In the s p l i t mode of sample i n j e c t i o n an i n l e t s p l i t l i n e r or s p l i t t e r i s used. The sample undergoes f l a s h v a p o r i z a t i o n and the s p l i t -t e r " s p l i t s " the sample i n t o two unequal p o r t i o n s . The smaller p o r t i o n i s introduced onto the column while the l a r g e r i s vented. This reduces the amount of sample entering the column and prevents column overloading. S p l i t r a t i o s ranging from 10:1 t o 500:1 have been r o u t i n e l y used i n c a p i l l a r y gas chromatography (Freeman, 1981c); the s e l e c t i o n of a p a r t i -c u l a r s p l i t r a t i o depends l a r g e l y upon the capacity of the column and the sample component co n c e n t r a t i o n . A v a r i e t y of s p l i t l i n e r c o n f i g u r a t i o n s have been d e s c r i b e d ; these have been discussed and reviewed (Schomburg et a l . , 1977; Jennings, 1975, 1980a). Optimization of the s p l i t t e r f o r a given a n a l y s i s i s required since a p a r t i c u l a r design may provide s u p e r i o r chromatographic r e s u l t s . Some of the more r e a d i l y a v a i l a b l e s p l i t l i n e r s 26 w i l l be examined during the course of t h i s p r o j e c t . Since most of the i n j e c t e d sample i s vented, the s p l i t mode i s often not s u i t a b l e f o r t r a c e l e v e l analyses, however, with the use of s e n s i t i v e and s p e c i f i c detectors (e.g. electron-capture) i t i s p o s s i b l e to extend t h i s range consider-ably. The s p l i t l e s s mode of sample i n j e c t i o n i s more suited to t r a c e l e v e l a n a l y s i s . In t h i s mode the f l a s h vaporized sample reconcentrates (condenses) i n a narrow band ("plug") at the head of the column, the temperature of which i s g e n e r a l l y 20 or more degrees lower than the b o i l -ing point of the solvent f o r i n j e c t i o n . The column temperature i s then increased r a p i d l y causing the solvent to vaporize and the normal chroma-tographic process proceeds. This sampling technique w i l l also be inves-t i g a t e d during the course of the present assay development. The d i r e c t and on-column sampling modes are r e l a t i v e l y new i n j e c -t i o n techniques and requi r e s p e c i a l i n l e t systems l a r g e l y compatible with recent gas chromatographs. This technology i s not yet a v a i l a b l e i n our 1aboratory. Very good d e s c r i p t i o n s of the theory and operational requirements of these four sampling methods ( i . e . , s p l i t , s p l i t l e s s , d i r e c t , on-column) as well as t h e i r advantages and l i m i t a t i o n s have been presented by several authors (Grob and Grob, 1969; Schomburg et al _ . , 1977; Yang et a l . , 1978; Jennings, 1980a; Freeman, 1981c; Schomburg et aj_., 1981). The small c a r r i e r gas flows associated with the use of c a p i l l a r y columns a l s o requires s p e c i a l detector c o n s i d e r a t i o n s ( a d a p t a t i o n s ) . 27 These low flows are i n s u f f i c i e n t to maintain detector c e l l volume and i t i s t h e r e f o r e necessary to add a make-up gas at the entrance of the column i n t o the detector. As optimal flows are required to maximize s e n s i t i v i t y and to minimize band spreading the e f f e c t of various make-up flow rates w i l l be assessed. 28 2. EXPERIMENTAL 2.1. M a t e r i a l s and Supplies 2.1.1. Chemicals The f o l l o w i n g were su p p l i e d by A.H. Robins Canada Inc., Montreal, Quebec : 4-amino-5-chloro-2-methoxy-N-(2-diethyl aminoethyl) benzamide monohydrochloride monohydrate (MCP.HC1.H20) (Lot Nos. 9207 and A105), 4-amino-5-chloro-2-methoxy-N-(diethylaminoethyl) benzamide monohydrochloride i n j e c t i o n , 5mg.mL (Reglan I n j e c t a b l e , -2mL ampule). (Lot no. 80246); diazepam (Lot R-9481) and prazepam by Hoffman La-Roche L t d . , Montreal, Quebec; Maprotiline.HCl (MAR.HC1) (N-methyl-9-10-ethanoanthracene-9(10H) propanamide).lot Al1663096472-0 by Ciba Pharmaceuticals, Mississauga, Ontario. Metoclopramide monohydrochloride -1 R f o r i n j e c t i o n , 5mg.mL (Maxeran I n j e c t a b l e , 2mL ampule; Nordic Pharmaceuticals, Montreal, Quebec), .thiopental sodium f o r i n j e c t i o n R R (Pentothal ; Abbott L a b o r a t o r i e s , Montreal, Quebec), S t e r i s p r a y (Fisons Corporation, Scarborough, O n t a r i o ) , halothane ( F l u o t h a n e R ; Ayerst L a b o r a t o r i e s , Montreal, Quebec) and a m p i c i l l i n f o r i n j e c t i o n P ( P e n b r i t i n , 250mg, 500mg; Ayerst Laboratories) were obtained from Pharmacy, Vancouver General H o s p i t a l , Vancouver, B.C. 2.1.2. Reagents One and f i v e normal sodium hydroxide were prepared from ACS reagent grade Sodium Hydroxide P e l l e t s ( F i s h e r S c i e n t i f i c Co., F a i r Lawn,, N.O., U.S.A.), one normal h y d r o c h l o r i c a c i d from ACS reagent grade H y d r o c h l o r i c . A c i d 37% (American S c i e n t i f i c and 29 Chemical, S e a t t l e , Washington, U.S.A.) and one and four percent ammonium hydroxide from Ammonia S o l u t i o n Strong 27% ( M a l l i n c k r o d t , Inc., St. Lo u i s , M i s s o u r i , U.S.A.). The f o l l o w i n g were obtained from P i e r c e Chemical Co., Rockford, 111., U.S.A.: T r i f l u o r o a c e t i c Anhydride (TFAA), P e n t a f l u o r o p r o p i o n i c Anhydride (PFPA), H e p t a f l u o r o b u t y r i c Anhydride (HFBA), H e p t a f l u o r o b u t y r y l i m i d a z o l e (HFB1), T r i e t h y l amine Sequanal Grade, P y r i d i n e S i l y l a t i o n Grade and Di m e t h y l c h l o r o s i l a n e (DMCS). Pentafluorobenzoyl c h l o r i d e (PFBC) was purchased from Regis Chemical Co., Morton Grove, I I I . , U.S.A.: 2.1.3. Solvents The f o l l o w i n g ( d i s t i l l e d i n g l a s s ) were obtained from Caledon Laboratories Inc., Georgetown, Ont. : Benzene, Toluene, Chloroform, Hexane; Iso-octane from Burdick and Jackson Laboratories Inc., Muskegon, Michigan, U.S.A.; Methanol ACS reagent grade from BDH Chemicals, Toronto, Ontario. 2.2. Equipment A model 5840A Hewlett-Packard r e p o r t i n g gas chromatograph equipped with a Ni Electron Capture. Detector'(ECD), a packed column compatible model 18835B C a p i l l a r y I n l e t System and a model 18850A i n t e g r a t o r t e r m i n a l , Hewlett-Packard Co., Avondale, PA,, D U.S.A.; Vortex-type mixer ( V o r t e x - G e n i e ) , incubation oven P (Isotemp , model 350), F i s h e r S c i e n t i f i c I n d u s t r i e s , S p r i n g f i e l d , Mass., U.S.A.; IEC Model 2K C e n t r i f u g e , Damom/IEC D i v i s i o n , Needham 30 HTS. Mass., U.S.A.; r o t a t i n g - t y p e tube mixer with c l i p s (Labquake R Tube shaker, model 415-110), L a b i n d u s t r i e s , Berke-l e y , C a l i f o r n i a , U.S.A.; F l u i d i z e d dryer ( H i - E F F T m ) , Applied Science Laboratories Inc., State College, PA. U.S.A.; 15 mL Pyrex^ c u l t u r e tubes with T e f l o n ^ - l i n e d screw caps, Canlab, Vancouver, B.C. 2.3 Preparation of Drug Stock S o l u t i o n 2.3.1 Metoclopramide. HC1.H20 Approximately 11.81 mg of MCP.HC1.H20 (equivalent to -10 mg of MCP base) was accurately weighed, t r a n s f e r r e d to a 100 mL volumetric f l a s k and made up to volume with deionized d i s t i l l e d water. A 0.2 mL a l i q u o t of t h i s s o l u t i o n was d i l u t e d with deionized d i s t i l l e d water to volume i n a 100 mL volumetric f l a s k . Ten mL of the l a t t e r s o l u t i o n was f u r t h e r d i l u t e d to volume with deionized d i s t i l l e d water i n a 50 mL v o l u -metric f l a s k to give a f i n a l working stock s o l u t i o n with a concentration of =0.04 yg.mL-1. Volumes of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 mL of t h i s f i n a l stock s o l u t i o n were subsequently used f o r standard ( c a l i b r a -t i o n ) curve pr e p a r a t i o n . 2.4 Preparation of Inte r n a l Standard S o l u t i o n s 2.4.1. Maprotiline.HCl About 11.31 mg of maprotil ine.HCl (MAP.HC1), equivalent to -10 mg of MAP.base, was accurately weighed and t r a n s f e r r e d i n t o a 100 mL volumetric f l a s k . Deionized d i s t i l l e d water was added to volume. A 0.2 mL a l i q u o t of t h i s s o l u t i o n was pipetted i n t o a 50 mL 31 volumetric f l a s k and d i l u t e d to volume with deionized d i s t i l l e d ' water to y i e l d a f i n a l working concentration of - 0.4yg.mL~\ Volumes of 100 or 200yL were used as the i n t e r n a l standard i n the q u a n t i t a t i o n of MCP i n human and sheep plasma employing fused s i l i c a c a p i l l a r y GLC, (2yL i n j e c t i o n s ) . 2.4.2. Diazepam Approximately 2mg was a c c u r a t e l y weighed, t r a n s f e r r e d to a 1L volumetric f l a s k and made up to volume with benzene. An a l i q u o t of t h i s s o l u t i o n (e.g. 100ml) was d i l u t e d f i v e f o l d with benzene to y i e l d a f i n a l concentration of - 0.4yg . m l ~ \ Diazepam served as the i n t e r n a l standard (5yL i n j e c t i o n s ) f o r packed column GLC-ianalysis. 2.4.3. Prazepam About l.Omg of prazepam was a c c u r a t e l y weighed, t r a n s f e r r e d i n t o a 50mL volumetric f l a s k and made up to volume with toluene. A f i n a l c oncentration of - 0.2;u.g .mL~^  was obtained by d i l u t i n g O.lmL of the o r i g i n a l s o l u t i o n to 50mL (volumetric f l a s k ) with toluene. Prazepam was added as a second i n t e r n a l standard during e a r l y fused s i l i c a c a p i l l a r y GLC studies,, (2yL i n j e c t i o n s ) . 2.5. Plasma E x t r a c t i o n Procedure A l l e x t r a c t i o n s were c a r r i e d out i n 15mL Pyrex^,(disposable) R c u l t u r e tubes equipped with Teflon - l i n e d screwcaps. To 0.25 - 0.5mL of plasma (human or sheep) co n t a i n i n g MCP was added 0.5mL of IltT NaOH (pH - 14) and the f i n a l aqueous volume 32 adjusted to 2.0mL with deionized d i s t i l l e d water. In the case of samples to be analyzed by c a p i l l a r y GLC, O.lmL of the i n t e r n a l standard, maprotiline.HCl (0.4yg.mL _ 1) i n water, was a l s o added at t h i s time ( t o t a l aqueous phase volume,2.lmL). Six mL of benzene was added and the mixture rotated f o r 20 min. on a r o t a t i n g rack (Labquake R) to e x t r a c t the MCP and m a p r o t i l i n e . A f t e r c e n t r i f u g a t i o n a t 4000 r.p.m. (2300G) f o r 10 min. 5mL of the organic phase was removed and back-extracted with 2mL of INIiHClffor 20 min. The samples were c e n t r i f u g e d f o r 5 min. and the organic l a y e r a s p i r a t e d and discarded (water vacuum a s p i r a t o r ) . The remaining aqueous l a y e r was washed with two 4mL a l i q u o t s of benzene, a l k a l i n i z e d by adding 0.5mL of 5N NaOH (pH = 14) and then e x t r a c t e d f o r 20 min. with 6mL of benzene. Following c e n t r i f u g a t i o n at 4000 rpm f o r 5 min., 5mL of the organic l a y e r was removed and d r i e d under a gentle stream of nitrogen i n a 40°C water bath. The nitrogen d r i e d residues were then d e r i v a t i z e d and used i n the subsequent GLC-ECD a n a l y s i s . 2.6. Chemistry 2.6.1. D e r i v a t i v e Formation A l l d e r i v a t i z a t i o n r e a c t i o n s described i n the f o l l o w i n g t e x t p p were c a r r i e d out i n 15mL Pyrex c u l t u r e tubes capped with Teflon -l i n e d screw caps. 33 A. Packed Column GLC-ECD ( i ) H e p t a f l u o r o b u t y r i c Anhydride (HFBA) The nitrogen d r i e d residue c o n t a i n i n g MCP (e x t r a c t e d as described i n Sectio n 2.5) was r e c o n s t i t u t e d to a volue of 500yL with 40CyL of diazepam ( i n t e r n a l standard) i n toluene C0.4yg.mL"1) and lOOyL of 0.05M t r i e t h y l a m i n e (.TEA) i n toluene. Twenty yL of HFBA was added, the samples mixed well (vortex) and incubated a t 55°C f o r 30 min. Following i n c u b a t i o n , the samples were allowed to cool to room temperature and the excess reagent (HFBA) was hydrolyzed with 0.5mL of deionized d i s t i l l e d water (vortex f o r 10 s) and 0.5mL of 4% NH^OH (vortex f o r 10 s ) . following c e n t r i f u g a t i o n at 2000 r.p.m. f o r 1 min. the organic l a y e r was immediately t r a n s f e r r e d to a c l e a n , dry 15 mL c u l t u r e tube. Five yL of the d e r i v a t i z e d sample was i n j e c t e d i n t o the GLC-ECD. ( i i ) T r i f l u o r o a c e t i c anhydride (TFAA), P e n t a f l u o r o p r o p i o n i c anhydride CPFPA) The d e r i v a t i z a t i o n procedure with these two reagents was i d e n t i c a l to that employed f o r HFBA above ( s e c t i o n 2.6.1., A ( i ) ) except that the c a t a l y s t , TEA, was not added. The residue was r e c o n s t i t u t e d w i t h 0.5mL benzene c o n t a i n i n g the i n t e r n a l standard diazepam (0.4yg.rnL" 1). I n j e c t i o n s of 5yL of the d e r i v a t i z e d sample were made i n t o the GLC-ECD. 34 ( i i i ) H e p t a f l u o r o b u t y r y l i m i d a z o l e (HFBI) Twenty yL of HFBI was added to the nitrogen d r i e d residue ( e x t r a c t e d as described i n s e c t i o n 2.5), mixed w e l l (vortex) and incubated a t 55°C f o r 30 min. The sample was allowed to reach room temperature, 0.5mL of diazepam i n benzene (0.4yg.mL~^) and 0. 5mL of deionized d i s t i l l e d water were added and the tube contents mixed well (vortex f o r 20 s ) . The organic l a y e r was immediately t r a n s f e r r e d t o , and stored i n , a clean dry 15mL c u l t u r e tube f o l l o w i n g c e n t r i f u g a t i o n a t 2000 r.p.m. f o r 1 min. Five yL i n j e c t i o n s were made i n t o the GLC-ECD. ( i v ) Pentafluorobenzoyl Chloride (PFBC) Two sets of aqueous s o l u t i o n s c o n t a i n i n g 0.04 or 1.0 g.mL"^ of MCP.HC1.H20 (0.5 and l.OmL) were ex t r a c t e d f o r 20 min. with 6.0mL of benzene f o l l o w i n g a l k a l i n i z a t i o n with 0.5mL of IN NaOH ( t o t a l aqueous phase 2.0mL). Samples were c e n t r i f u g e d a t 4000 r.p.m. f o r 5 min., 5.0mL of the benzene l a y e r removed and d r i e d i n a 40°C water bath under a gentle stream of ni t r o g e n . Each set of d r i e d e x t r a c t s was d e r i v a t i z e d by one of the f o l l o w i n g two r e s p e c t i v e procedures: 1. Based on the method of Midha et a l . , 1979. Two yL of 0.1% PFBC i n benzene and 0.5mL of benzene were added, mixed (vortex) and incubated a t 55°C f o r 60 min. The sample was cooled to ambient temperature, 0.5mL of 0.IN NHA0H added, 35 mixed f o r 30 s ( v o r t e x ) and c e n t r i f u g e d a t 2000. rpm f o r 1 min. The organic l a y e r was immediately removed. Samples of l-5yL were i n j e c t e d i n t o the GLC-ECD. 2. Based on the Method of Matin and Rowland, 1972. Five yL of 0.1% PFBC i n benzene and l.OmL of 2.5N NaOH were added and the tube mixed (vortex) f o r 5 min. The d e r i v a t i v e was ext r a c t e d with 0.5mL of benzene by mixing f o r 1 min. ( v o r t e x ) . Following c e n t r i f u g a t i o n at 2000 r.p.m. f o r 1 min. organic l a y e r was immediately removed. I n j e c t i o n s of l-5yL were made i n t o the GLC-ECD. Note: ( i ) Two yL of undi l u t e d PFBC was a l s o s u b s t i t u t e d f o r 0.1% PFBC i n benzene ( i i ) a base concentration of 0.00025N NaOH was a l s o used i n place o f 2..5N NaOH i n some samples to observe the e f f e c t of base s t r e n g t h , i f any, on product recovery. Fused S i l i c a C a p i l l a r y Column GLC-ECD The n i t r o g e n d r i e d e x t r a c t c o n t a i n i n g MCP and the i n t e r n a l standard m a p r o t i l i n e ( e x t r a c t e d as described i n Section 2.5) was r e c o n s t i t u t e d to a volume of 200yL with 150yL of toluene and 50yL of 0.05M TEA i n toluene. Twenty yL of HFBA was added, the sample mixed we l l (vortex) and then incubated at 55°C f o r 36 60 min. The excess reagent was hydrolyzed and n e u t r a l i z e d as described f o r HFBA ( s e c t i o n 2,6.1, A ( i ) ) . The d e r i v a t i z e d organic l a y e r was e i t h e r i n j e c t e d (2 L) i n t o the GLC-ECD t h a t same day or stored a t -20°C f o r up to 4 days u n t i l analyzed. Note: In i n i t i a l s t udies prazepam was included as a second i n t e r n a l standard. In these instances the 200yL of r e c o n s t i t u t i n g s o l v e n t c o n s i s t e d of 150yL of toluene c o n t a i n i n g 0.2pg.mL-1 of prazepam and 50yL of 0.05M TEA i n toluene. 2.6.2. E f f e c t of Base Strength (NH^OH) and C a t a l y s t (TEA, p y r i d i n e ) on d e r i v a t i v e s t a b i l i t y (area r a t i o , HFB/MCP/diazepam v a r i a b i l i t y ) . In order to i n v e s t i g a t e p o s s i b l e reasons f o r the d e c l i n e i n area r a t i o s of HFB-MCP/diazepam with time, the f o l l o w i n g two parameters were examined: 1. the e f f e c t of adding a c a t a l y s t to the d e r i v a t i z i n g mixture 2. the e f f e c t of NH^OH concentration on the removal ( n e u t r a l i z a t i o n ) of excess HFBA. 1 To 0.5mL of MCP.HC1.H20 i n deionized d i s t i l l e d water (0.04yg.mL _ 1) was added 0.5mL of IN NaOH and 0.5mL of i n t e r n a l standard, diazepam i n toluene ( t o t a l aqueous phase volume . adjusted to 2.0mL with deionized d i s t i l l e d water). This mixture was e x t r a c t e d f o r 20min..on a r o t a t i n g tube shaker (Labquake R). A f t e r c e n t r i f u g i n g at 4000 r.p.m. (2300G) 37 t h e o r g a n i c phase was p i p e t t e d I n t o a c l e a n , d r y 15mL c u l t u r e t u b e . The samples were d i v i d e d i n t o t h r e e s e p a r a t e groups and e a c h group d e r i v a t i z e d by one o f t h e f o l l o w i n g t h r e e r e s p e c t i v e p r o c e d u r e s : A. Twenty yL o f HFBA was added t o t h e e x t r a c t e d sample, mixed w e l l ( v o r t e x ) and t h e r e a c t i o n m i x t u r e i n c u b a t e d a t 55°C f o r 30 min. Tubes were c o o l e d t o room t e m p e r a t u r e and t h e e x c e s s HFBA was h y d r o l y z e d and n e u t r a l i z e d by a d d i n g 0.5mL o f d e i o n i z e d d i s t i l l e d w a t e r and m i x i n g ( v o r t e x ) f o r 30 s. Samples were c e n t r i f u g e d a t 2000 r.p.m. f o r 1 min, t h e o r g a n i c l a y e r removed and used f o r packed column GLC-ECD a n a l y s i s . ( 5 y l i n j e c t i o n s ) . B. Twenty yL o f HFBA and 100yL o f 0.05M TEA were added t o t h e e x t r a c t e d sample, mixed w e l l and i n c u b a t e d a t 55°C f o r 30 min. The e x c e s s r e a g e n t (HFBA) was removed by e i t h e r : ( i ) h y d r o l y s i s w i t h 0.5mL o f d e i o n i z e d d i s t i l l e d w a t e r , v o r t e x f o r 10 s, and n e u t r a l i z a t i o n w i t h 0.5mL o f 1% NH^OH, v o r t e x f o r 20 s. F o l l o w i n g c e n t r i f u g a t i o n a t 2000 rpm f o r 1 min,the o r g a n i c phase was removed and a n a l y z e d by packed column GLC-ECD (5yL i n j e c t i o n s ) , o r ( i i ) as i n s e c t i o n 2.6.2. B ( i ) e x c e p t 0.5mL o f 4% NH^OH was used i n t h e n e u t r a l i z a t i o n s t e p . Samples were a g a i n a n a l y z e d by packed column GLC-ECD ( 5 y l i n j e c t i o n s ) . 38 C. Procedures used were described i n s e c t i o n 2,6.2. B ( i ) and ( i i ) except 20yL of r e d i s t i l l e d p y r i d i n e was added In place of the TEA to serve as c a t a l y s t . Samples were analyzed by packed column GLC-ECD (5yl i n j e c t i o n s ) . 2.6.3. S i l a n i z a t i o n of Glassware to Test f o r P o s s i b l e MCP Adsorption Losses onto Glass Surfaces A l l glassware (Pyrex c u l t u r e tubes, p i p e t t e s , volumetric f l a s k s , t r a n s f e r v i a l s , e tc.) used i n the p r e p a r a t i o n , e x t r a c t i o n and d e r i v a t i z a t i o n of MCP was washed with chromic a c i d , thoroughly r i n s e d with tap and deionized d i s t i l l e d water, oven d r i e d and then soaked overnight i n a s o l u t i o n of 10% d i m e t h y l c h l o r o s i l a n e (DMCS) i n toluene. The f o l l o w i n g morning the glassware was removed from the DMCS s o l u t i o n , d rained, thoroughly r i n s e d with reagent grade methanol and oven d r i e d . P r i o r to use,culture tubes (15mL) were f u r t h e r r i n s e d with lmL of benzene (vortex) and oven d r i e d . 2.6.4. Optimization of Metoclopramide and M a p r o t i l i n e D e r i v a t i z a t i o n Reaction Time The d e r i v a t i z a t i o n r e a c t i o n time f o r MCP has been p r e v i o u s l y described (Tarn, M.Sc. Thesis 1978) and reported to be instantaneous (Tarn and Axelson, 1978). In the present work, however, maprotiline.HC1, a t e t r a c y c l i c antidepressant compound 39 with a secondary amine s i d e c h a i n , replaced diazepam as the i n t e r n a l standard so the optimal r e a c t i o n time to achieve a constant area r a t i o (HFB-MCP/HFB-HAP) was examined. MCP.HC1.H20, 0.5mL, dO.04yg.mL" 1) w i t h m a p r o t i l i n e . H C l , O.lmL, dO.4yg.mL~ 1) as the i n t e r n a l standard was e x t r a c t e d according to the general e x t r a c t i o n procedure ( s e c t i o n 2.5). The nitrogen d r i e d e x t r a c t was r e c o n s t i t u t e d to 200yL w i t h 50yL o f 0.05M TEA i n toluene and 150yL of toluene. Twenty yL of HFBA was added, the r e a c t i o n mixture mixed w e l l (vortex) and then incubated at 55°C f o r the f o l l o w i n g times: 0, 20, 40, 60, 90, 120 and 180 min. A f t e r c o o l i n g the excess reagent (HFBA) was removed as p r e v i o u s l y described f o r HFBA i n Section 2.6.1. A ( i ) . Two yL i n j e c t i o n s were made onto a 25m x 0.31mm I.D. c r o s s l i n k e d SE-54 fused s i l i c a c a p i l l a r y column. The GLC-ECD co n d i t i o n s employed are l i s t e d i n se c t i o n 2.8.4.2. Standard Curve Preparation f o r Fused S i l i c a C a p i l l a r y Column A n a l y s i s Volumes o f 0.1, 0.2, 0.4, 0.6, 0.8 and l.OmL of MCP.HC1.H20 stock s o l u t i o n d0.04yg.mL~ 1) were p i p e t t e d i n t o separate 15mL Pyrex c u l t u r e tubes c o n t a i n i n g 0.5mL of blank human (or sheep) plasma and O.lmL of i n t e r n a l standard, maprotiline.HCl (0.4yg.mL _ 1). The sampl were a l k a l i n i z e d w i t h 0.5mL of TN NaOH, the t o t a l aqueous phase adjusted to 2.1mL and then e x t r a c t e d as described i n s e c t i o n 2.5. 40 The nitrogen d r i e d extracts, were r e c o n s t i t u t e d , d e r i v a t i z e d and the excess reagent (HFBA) removed as p r e v i o u s l y described i n sec t i o n 2.6.1. B. Two yL of the d e r i v a t i z e d s o l u t i o n was i n j e c t e d i n t o the GLC-ECD equipped with a 25m x 0.31mm I.D. cross!inked SE-54 fused s i l i c a c a p i l l a r y column. Standard curves were constructed by p l o t t i n g the area r a t i o s o f the HFB-derivatives o f MCP/maprotiline against MCP concentrations. 2.8. GLC-ECD 2.8.1. Packed Column Studies During r o u t i n e assessment of an e a r l i e r assay method (Tarn et a l . , 1979) several s t a t i o n a r y phases and s o l i d supports were evaluated i n order to optimize MCP measurement i n human or sheep plasma. P r i o r to packing, columns (1.8m x 2mm I.D. gl a s s ) were washed with chromic a c i d , thoroughly r i n s e d with tap and deionize d d i s t i l l e d water, oven d r i e d and then f u r t h e r r i n s e d with methanol and toluene and again d r i e d . The columns were then s i l a n i z e d with a s o l u t i o n of 10% DMCS i n toluene f o r 30 min. The DMCS s o l u t i o n was drained from the column and i t was r i n s e d with methanol and toluene r e s p e c t i v e l y . A f t e r thoroughly d r y i n g i n a hot a i r oven the column was ready f o r packing. 2.8.1.1. Commercially prepared column packings The f o l l o w i n g commercial and i n - l a b coated packings were t e s t e d : 3% 0V-17 (50% methyl, 50% phenyl s i l i c o n e ) on Chromosorb 41 W HP 10Q-120mesh (Western Chromatographic S u p p l i e s , Annacis I s . , New Westminster, B.C.).,.3% OV-225 (25% cyanopropyl, 25% phenyl methyl s i l i c o n e ) on chromosorb W HP 100-120mesh, 3% OV-225 on Gas Chrom Q 120-140 mesh*, 3% OV-225 on Chromosorb 750 100-120 mesh* (Chromatographic S p e c i a l t i e s , B r o c k v i l l e , O n t a r i o ) . 3% OV-225 on Gas Chrom Q 230-270 mesh ( U l t r a - P a k R ) , 3% Si l a r - 9 C P (90% cyanopropyl phenyl s i l i c o n e ) on Chromosorb W HP 100-120 mesh,3% S i l a r - 9 C P on Gas Chrom Q 120-140 mesh* (Applied Science Laboratories Inc., State C o l l e g e , PA., U.S.A.) 2.8.1.2, In-lab Coated Column Packings 3% SE-30 ( m e t h y l s i l i c o n e ) on Chromosorb W HP 80-100mesh, 3% SP-2300 (36% cyanopropyl s i l i c o n e ) on Chromosorb W HP 100-120 mesh (Supelco Inc., B e l l e f o n t e PA., U.S.A.). 3% OV-225 on Chromosorb W HP 100-120mesh, 3% Si l a r - 5 C P (50% cyanopropyl phenyl s i l i c o n e ) on Chromosorb W HP 100-120 mesh, 3% Si l a r - 9 C P on Chromosorb W HP 80-100 and 100-120 mesh, 3% Si l a r - 9 C P on Chromosorb 750 100-120 mesh*, 3% S i l a r - l O C (.100% 3-cyano-propyl s i l i c o n e ) on Chromosorb W HP 100-120 mesh (Applie d Science Laboratories Inc., State C o l l e g e , PA., U.S.A.). 2.8.1.3. S o l i d Supports Frequently extreme v a r i a b i l i t y (namely a d e c l i n e ) i n the area r a t i o s of HFB-MCP /diazepam was observed from i n j e c t i o n to i n j e c t i o n of the same sample. In order to i n v e s t i g a t e p o s s i b l e on-column adsorption losses of MCP as a source of t h i s v a r i a b i l i t y 42 two d i f f e r e n t s o l i d supports, Gas Chrom Q and Chromosorb 750 were te s t e d . These supports were coated with e i t h e r 0V-225 or S i l a r - 9 C P and are included i n the l i s t i n g s of commercial and i n - l a b packings as i n d i c a t e d by an a s t e r i s k (*) i n s e c t i o n s 2.8.1.1. and 2.8.1.2 r e s p e c t i v e l y . 2.8.2. General Procedure f o r In-Lab S o l i d Support Coating The s o l i d support (Chromosorb W HP 80-100 or 100-210 mesh, Chromosorb 750 100-120 mesh) was coated with a 3% s o l u t i o n of the d e s i r e d s t a t i o n a r y phase (Carbowax 20M, 0V-225, S i l a r - 5 C P , S i l a r - 9 C P , SP-2300, SE-30) d i s s o l v e d i n chloroform. The method b r i e f l y c o n s i s t s of s t r e a k i n g the s o l i d support i n a P e t r i - d i s h with the s o l u t i o n of TM s t a t i o n a r y phase, t r a n s f e r r i n g to a f l u i d i z e d bed dryer (Hi-EFF ) maintained at 60°C, and removing the solvent w i t h a gentle stream of n i t r o g e n . 2.8.3. General Chromatographic c o n d i t i o n s f o r Packed Column Studies Column (oven) temperature 245 or 250°C, I n j e c t i o n temperature, 250°C; Detector (E.C.D.) temperature, 350°C; C a r r i e r gas (Argon-Methane, 95:5) f l o w , 40mL.min.~ 1; A t t e n u a t i o n , 2 7 or 2 8; Chart speed, 0.3 c m . m i n . ; Slope s e n s i t i v i t y , 0.5-1.0. 2.8.4. Fused S i l i c a C a p i l l a r y Column Studies: Assay Development and Optimization of GLC-ECD Parameters . During the course of assay development the f o l l o w i n g three fused s i l i c a c a p i l l a r y columns were used: 12m x 0.2mm I.D. 43 Carbowax 20M, 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d (Carbowax 20M d e a c t i v a t e d ) , supplied by Hewlett-Packard; 25m x 0.31 mm I.D. c r o s s l i n k e d (bonded) SE-54 (5% phenyl methyl s i l i c o n e , U l t r a #2; f i l m thickness 0.15y, s i l o x a n e - d e a c t i v a t e d ) purchased from Hewlett-Packard Co., Avondale, PA., U.S.A. Almost a l l p r e l i m i n a r y work with regard to assay development and o p t i m i z a t i o n of GLC-ECD parameters was c a r r i e d out on the 12m methyl s i l i c o n e f l u i d column. The r e s u l t s from these i n i t i a l s t u d i e s were l a t e r a p p l i e d to and/or confirmed on the 25m cro s s -l i n k e d SE-54 column which was used f o r MCP q u a n t i t a t i o n i n human and sheep plasma. 2.8.4.1. Sample Preparation Samples used i n assay development were e x t r a c t e d and d e r i v a t i z e d by e i t h e r of the f o l l o w i n g two general procedures: A. A sample co n t a i n i n g 0.5mL of MCP.HC1.H20 (lyg.mL - 1 i n deionized d i s t i l l e d water) was ext r a c t e d with s i x mL of benzene f o l l o w i n g a l k a l i n i z a t i o n with 0.5mL of IN NaOH and aqueous phase adjustment to 2.0mL with deionized d i s t i l l e d water. The sample was c e n t r i f u g e d at 4000 r.p.m. f o r 10 min., a 5mL a l i q u o t of the benzene l a y e r removed and d r i e d under a gentle stream of ni t r o g e n i n a 40°C water bath. The residue was r e c o n s t i t u t e d to a volume of 600yL with 500yL of hexane and 100yL of 0.05M TEA i n hexane. The sample was incubated at 55°C f o r 60 min., cooled to room temperature 44 and t h e e x c e s s r e a g e n t (HFBA) h y d r o l y z e d and n e u t r a l i z e d as p r e v i o u s l y d e s c r i b e d i n s e c t i o n 2.6.1. A ( . i ) . B. Samples c o n t a i n i n g v a r i o u s volumes o f MCP.HCl.h^O (0.1 - l.OmL o f 0.04yg.mL _ 1 i n d e i o n i z e d d i s t i l l e d w a t e r ) and MAP.HC1 . (0.4>ig.niL^) as i n t e r n a l s t a n d a r d w i t h and w i t h o u t b l a n k plasma were e x t r a c t e d and d e r i v a t i z e d as d i s c u s s e d i n s e c t i o n s 2.5 and 2.6.1. B r e s p e c t i v e l y . 2.8.4.2. O p t i m i z a t i o n o f GLC-ECD par a m e t e r s U n l e s s o t h e r w i s e i n d i c a t e d t h e f o l l o w i n g g e n e r a l 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 were employed: I n j e c t i o n t e m p e r a t u r e , 220°C; c o l u m n ( o v e n ) t e m p e r a t u r e , 235°C; D e t e c t o r (E.C.D.) t e m p e r a t u r e , 350°C; C a r r i e r gas (hydrogen) f l o w , l.OmL.min" 1; S p l i t v e n t f l o w , 30mL.min _ 1; I n l e t p r e s s u r e 9.5 p . s . i . ; Make-up gas (Argon-Methane, 95:5) f l o w , 60mL.min _ 1; C h a r t s p e e d , 1cm.min - 1; A t t e n u a t i o n , 2 4 ; S l o p e s e n s i t i v i t y , 0.2. The f o l l o w i n g i n s t r u m e n t p a r a m e t e r s were v a r i e d i n d e p e n d e n t l y and t e s t e d f o r o p t i m i z a t i o n o f t h e s e p a r a t i o n and measurement o f MCP: 1. I n j e c t i o n t e m p e r a t u r e : 2 1 0 ° , 2 2 0 9 and 240°C. 2. Column (oven) t e m p e r a t u r e : 2 3 5 ° , 240°and 245°C. 3. C a r r i e r gas: Hydrogen ( H 2 ) , Helium ( H e ) . 4. Column f l o w ( c a r r i e r gas) r a t e : 0.5 and l.OmL.min"^ 45 5. Make-up gas (Argon-Methane, 95:5) f l o w : 20, 30, 40, 50, and eOmL.min"1. 6. I n j e c t i o n mode: S p l i t l e s s , s p l i t . 7. I n j e c t i o n s o l v e n t : I s o - o c t a n e , hexane, benzene, t o l u e n e . 8. S p l i t l i n e r s ( S p l i t t e r s ) : Fused s i l i c a , 2mm I.D., J e n n i n g s t u b e ; ( w i t h and w i t h o u t p a c k i n g as i l l u s t r a t e d i n t h e f o l l o w i n g d i a g r a m s ) : Fused S i l i c a I n s e r t J e n n i n g s Tube h--»3 cm c u t • " t i g h t " s i l a n i z -ed g l a s s wool p l u g cm •* .5 cm s i l a n i z e d g l a s s -* wool p l u g I—-».5 cm GC packing ( 2 % 0V-1, 3% 0V-17) *.5 cm s i l a n i z e d g l a s s -wool p l u g 2.9. Packed Fused S i l i c a C a p i l l a r y Column GLC-ECD A n a l y s i s o f Human and Sheep plasma Packed A s s a y A p p l i c a t i o n t o t h e 2.9.1.Human ( P h a r m a c o k i n e t i c ) Studies:-MCP P l a c e n t a l T r a n s f e r T h i s c o l l a b o r a t i v e s t u d y was c a r r i e d o u t i n t h e Department o f A n a e s t h e s i o l o g y , V a n c o u v e r G e n e r a l H o s p i t a l , V a n c o u v e r , B.C. Normal, h e a l t h y women i n l a b o u r u n d e r g o i n g e l e c t i v e C a e s a r i a n s e c t i o n and r e q u i r i n g MCP as a p r e - a n e s t h e t i c m e d i c a t i o n were used t o s t u d y t h e ( p l a c e n t a l ) t r a n s f e r o f MCP from mother t o f e t u s . A t o t a l o f 23 p a t i e n t s 46 were included i n the study. 2.9.1.1. Experimental P r o t o c o l . P a t i e n t s received intravenous (infused over l-2min.) MCP.HC1 (Maxeran R I n j e c t a b l e ) at the r a t e of 0.15mg.kg -1 or Sodium Chloride f o r I n j e c t i o n on a double-blind b a s i s . P a t i e n t samples were coded and the code maintained u n t i l plasma MCP q u a n t i t a t i o n was completed i n our l a b o r a t o r y . One-point blood samples were drawn at d e l i v e r y from a maternal v e i n (MV) and from a doubly-clamped s e c t i o n of the . u m b i l i c a l cord ( u m b i l i c a l vein (UV), u m b i l i c a l a r t e r y (UA)) immediately a f t e r being severed from the neonate. Blood samples were t r a n s f e r r e d to heparinized Vacutainers (Becton-Dickinson), c e n t r i f u g e d , the plasma removed and stored at -20°C u n t i l analyzed. S t a t i s t i c a l information (age, weight, use of other medications, etc.) and d e t a i l s of MCP dosing and blood sampling times were recorded. 2.9.1.2. Q u a n t i t a t i v e Plasma A n a l y s i s R e p l i c a t e plasma samples (0.25-0.5mL)(MV,UV,UA) were ext r a c t e d and d e r i v a t i z e d as described i n s e c t i o n s 2.5 and 2.6.1.B r e s p e c t i v e l y . M a p r o t i l i n e . H C l (0.4yg.mL - 1) was employed as the i n t e r n a l standard. Two yL i n j e c t i o n s were made i n t o the GLC-ECD equipped with the 47 25m x 0.31mm c r o s s l i n k e d SE-54 fused s i l i c a column. Q u a n t i t a t i v e e s t i m a t i o n of MCP i n unknown plasma samples was determined from area r a t i o s of the HFB-derivatives of MCP/MAP f i t t e d to a standard curve regression l i n e . Standard curve samples were e x t r a c t e d , d e r i v a t i z e d and chromatographed the same day as the t e s t samples. 2.9.2. Sheep Studies This c o l l a b o r a t i v e study of the m a t e r n a l - f e t a l p l a c e n t a l t r a n s f e r of MCP was c a r r i e d out i n the Department of O b s t e t r i c s and Gynaecology, The U n i v e r s i t y of B r i t i s h Columbia, Vancouver, B.C. Experimentation was performed i n an e s t a b l i s h e d sheep model used i n the i n v e s t i g a t i o n of f e t a l oxygen consumption. A l l surgery was performed under a s e p t i c technique and i s only described b r i e f l y h e rein. 2.9.2.1.Animal Care and S u r g i c a l Preparation Ewes were brought i n t o the f a c i l i t y about one week p r i o r to surgery to allow them to become accustomed to the environment. The animals were provided a standard d i e t and water ad l i b i t u m . Surgery was u s u a l l y performed at 110-115 days g e s t a t i o n (term, 145 days). A d m i n i s t r a t i o n of an intravenous (I.V.) bolus of t h i o p e n t a l R -1 sodium (Pentothal , 15mg.kg ) was followed by endotracheal p i n t u b a t i o n and l i g h t anesthesia with halothane (Fluothane , Ayerst L a b o r a t o r i e s , Montreal, Quebec) and oxygen. S i l a s t i c c a t h e t e r s were implanted i n a maternal j u g u l a r vein and femoral a r t e r y . The uterus 48 was exposed by a m i d l i n e abdominal i n c i s i o n . A small u t e r i n e i n c i s i o n was made to expose the f e t a l neck and s i l a s t i c catheters were implanted i n the f e t a l trachea and i n the amniotic c a v i t y . The i n c i s i o n was closed and another made through which the f e t a l hind-limbs and abdomen were e x t e r i o r i z e d . S i l a s t i c catheters were implanted i n a f e t a l femoral a r t e r y , l a t e r a l t a r s a l vein and the common u m b i l i c a l v e i n . The p a r t i a l l y exposed fetus was returned to the uterus. D A l l i n c i s i o n s were sprayed with S t e r i s p r a y , an a n t i b i o t i c mixture, p r i o r to c l o s u r e . The catheters were e x t e r i o r i z e d through a sub-cutaneous tunnel and placed i n a pouch on the ewe's f l a n k . Post-s u r g i c a l a n t i b i o t i c prophylaxis c o n s i s t e d of d a i l y i n j e c t i o n s of P a m p i c i l l i n ( P e n b r i t i n ) to the ewe (500mg I.M. f o r 3 days), and i n t r a - a m n i o t i c a l l y (250mg f o r the duration of the p r e p a r a t i o n ) . Following surgery,ewes' . were kept e i t h e r s i n g l y or i n p a i r s i n pens with f r e e access to food and water. The animals were allowed .to recover f o r at l e a s t 2-3 days p r i o r to experimentation. Catheters were kept patent by f l u s h i n g and f i l l i n g d a i l y with s t e r i l e h e parinized s a l i n e . 2.9.2.2. Experimental Protocol On the day of the experiment, the ewe was t r a n s f e r r e d to a wooden c a r t adjacent to the pens i n f u l l view of companion ewes. This helped to minimize nervousness and anxiety i n the ewe being monitored. 49 MCP.HC1 (Reglan* i n j e c t a b l e ) was administered, on a crossover basis (10, 20, and 40 mg), v i a the maternal j u g u l a r venous catheter over a time i n t e r v a l of 1-2 minutes. Blood samples f o r MCP determination were simultaneously c o l l e c t e d i n p l a s t i c syringes from the maternal and f e t a l femoral a r t e r i a l catheters (1.5 and l.OmL r e s p e c t i v e l y ) at times of -5, 1 , 3 , 5, 10, 15, 20, 30 and 45 min. and 1, 1%, 2, 2% and 3 hr. Samples f o r maternal and f e t a l blood gas a n a l y s i s were c o l l e c t e d from these same catheters at -5, 1, 5, 10 and 30 min. Blood samples were t r a n s f e r r e d to heparinized Vacutainers , c e n t r i f u g e d at 4000 r.p.m. x 15 min. and the plasma removed and stored at -20°C u n t i l analyzed. At the end of the sampling p e r i o d , the t o t a l amount of f e t a l blood c o l l e c t e d was replaced by an equivalent volume of maternal blood. Note: i f the f e t a l femoral a r t e r i a l c atheter was not patent then the l a t e r a l t a r s a l vein was used f o r sampling. For h - 1 hr. before and 1 hr. a f t e r MCP a d m i n i s t r a t i o n , the f o l l o w i n g p h y s i o l o g i c a l parameters were continuously recorded on an 8 - channel polygraph recorder (Beckman R612): maternal a r t e r i a l pressure, f e t a l a r t e r i a l pressure, i n t r a - u t e r i n e pressure, f e t a l i n t r a -t r a c h e a l pressure and maternal and f e t a l heart rates with s t r a i n -gauge transducers and cardiotachmeters r e s p e c t i v e l y . These recordings along with blood gas determinations have y e t to be f u l l y analyzed and t h e r e f o r e w i l l not be reported or discussed i n t h i s t h e s i s . 50 2.9.2.3. Q u a n t i t a t i v e Plasma A n a l y s i s Maternal and f e t a l plasma samples .(0,25-0..5ml) were ex t r a c t e d and d e r i v a t i z e d as described i n se c t i o n s 2.5 and 2.6.1-B r e s p e c t i v e l y . M a p r o t i l i n e . HC1 (0.4yg.mL _ 1) was used as the i n t e r n a l standard. Two yL i n j e c t i o n s were made i n t o the GLC-ECD equipped with the 25m c r o s s l i n k e d SE-54 fused s i l i c a column. Q u a n t i t a t i v e determination of MCP i n t e s t plasma samples was performed using area r a t i o s of the HFB-derivatives of MCP/MAP f i t t e d to a standard curve regression l i n e (area r a t i o MCP/'MAP versus MCP co n c e n t r a t i o n ) . Standard curve samples were e x t r a c t e d , d e r i v a t i z e d and chromatographed the same day as the t e s t samples. 51 3, RESULTS 3.1. Packed Column GLC-ECD 3.1.1. S t a t i o n a r y Phases When attempts were made to apply the method of Tarn et a l . , 1979, to the r o u t i n e a n a l y s i s of MCP i n human or sheep plasma using 3% OV-225 (Chromosorb W HP 100-120 mesh) two i n t e r f e r i n g peaks were observed ( F i g . 1). The presence of these i n t e r f e r i n g substances c o n t r i b u t e d to a s i g n i f i c a n t amount of e r r o r i n MCP q u a n t i t a t i o n , e s p e c i a l l y at low drug l e v e l s ( v i z . , 8-40 ng.mL - 1). I n i t i a l l y , e f f o r t s were made to i d e n t i f y the source of these compounds. So l v e n t s , detergents, n i t r o g e n , and (deionized) d i s t i l l e d water s u p p l i e s were examined. The two i n t e r f e r i n g peaks were thought to be due to components e x t r a c t e d from p l a s t i c s . However, even the use of a g l a s s apparatus d i d not e l i m i n a t e t h i s i n t e r f e r e n c e . Beset with t h i s problem, other l i q u i d ( s t a t i o n a r y ) phases were t e s t e d i n hopes of r e s o l v i n g MCP from these two extraneous substances. A number of s t a t i o n a r y phases ranging from low to high general s e l e c t i v i t y were examined. These are l i s t e d i n Table I along with the summarized f i n d i n g s . Heptafluoro-b u t y r i c anhydride (HFBA) was the d e r i v a t i z i n g agent used i n a l l cases. Representative chromatograms obtained from blank and MCP-spiked human plasma e x t r a c t s on S i l a r - 9 C P are shown i n F i g s . 2a and b. S i l a r - 9 C P , although p r o v i d i n g the d e s i r e d r e s o l u t i o n , e x h i b i t e d broad t a i l i n g peaks and a long a n a l y s i s time. 52 z ID cs A r e p r e s e n t a t i v e e x t r a c t on 3% OV HFB-MCP (R.T. = 5 diazepam (R.T. = are i n d i c a t e d by Column, 3% OV-225 (1.8m 100-120 mesh; I n j e c t i o n (E.C.D.), 350°C; Column Methane, 95:5) flow, 40, chromatogram obtained from a MCP-spiked plasma 225. Five yL i n j e c t i o n , each yL contained .Olmin.) 33.33pg and the i n t e r n a l standard 12.87min.) 0.4ng. I n t e r f e r i n g component peaks the arrows (J,). Chromatographic c o n d i t i o n s : x 2mm'I.D., gl a s s ) on Chromosorb W HP temperature, 250°C; Detector temperature temperature, 245°C; C a r r i e r gas (Argon-OmL.mins Chart speed 0.3cm.min-l. 53 T A B L E 1 S T A T I O N A R Y P H A S E S S t a t i o n a r y P h a s e 1 M c R e y n o l d s C o n s t a n t 2 F i n d i n g s a n d C o m m e n t s 3 5 S E - 3 0 o n C h r o m o s o r b W HP 8 0 - 1 0 0 m e s h I , 5 . 2 1 7 - v e r y b r o a d f l a t p e a k f o r MCP - i n c o m p l e t e r e s o l u t i o n f r o m i n t e r f e r i n g c o m p o u n d s - i n c o m p l e t e r e s o l u t i o n f r o m d i a z e p a m ( i n t e r n a l s t a n d a r d ) 3 5 O V - 1 7 o n C h r o m o s o r b W HP 1 0 0 - 1 2 0 m e s h I , 5 , 8 8 4 - p a t t e r n v e r y s i m i l a r t o O V - 2 2 5 ( F i g . 1 ) - i n c o m p l e t e r e s o l u t i o n f r o m i n t e r f e r i n g c o m p o n e n t s 3 5 O V - 2 2 5 o n C h r o m o s o r b W HP 1 0 0 - 1 2 0 m e s h i , 5 . 1 8 1 3 - i n c o m p l e t e r e s o l u t i o n o f MCP f r o m i n t e r f e r i n g p e a k s ( F i g . 1 ) 3 5 C a r b o w a x 2 0 M o n C h r o m o s o r b W HP 8 0 - 1 0 0 m e s h Z , 5 , 2 3 0 8 - i n c o m p l e t e r e s o l u t i o n o f MCP f r o m i n t e r f e r i n g p e a k s - l o w t e m p e r a t u r e l i m i t ( 2 2 0 ° C ) - MCP t a i l s b a d l y a t t e m p e r a t u r e s < 2 3 0 ° C . 3 5 S P - 2 3 0 0 o n C h r o m o s o r b W HP 1 0 0 - 1 2 0 m e s h r , 5 . = 2 4 0 0 - c h r o m a t o g r a p h i c p a t t e r n v e r y s i m i l a r t o O V - 2 2 5 ( F i g . l ) - i n c r e a s i n g MCP a r e a c o u n t s w i t h s u c c e s s i v e i n j e c t i o n s w h i c h c o u l d n o t be o v e r c o m e b y p r i m i n g 3 5 S i l a r - 5 C P o n C h r o m o s o r b W HP 1 0 0 - 1 2 0 m e s h E , 5 . 2 4 2 8 - i n t e r f e r i n g p e a k o n l e a d i n g e d g e o f MCP 3 5 S i l a r - 9 C P o n C h r o m o s o r b W HP 8 0 - 1 0 0 1 0 0 - 1 2 0 m e s h I , 5 , 3 5 3 6 - r e s o l u t i o n f r o m i n t e r f e r i n g c o m p o n e n t s - MCP p e a k b r o a d a n d o f t e n t a i l i n g - c o n s i d e r a b l e b a t c h t o b a t c h v a r i a t i o n i n r e t e n t i o n t i m e s - s i g n s o f o n - c o l u m n a d s o r p t i o n o f MCP 3 5 S i l a r - l U C o n C h r o m o s o r b W HP 1 0 0 - 1 2 0 m e s h r , 5 , 3 6 8 2 - b r o a d f l a t bump f o r MCP - t h i s r e q u i r e d t h e i n j e c t i o n o f v e r y h i g h c o n c e n t r a t i o n s 1 . P a c k e d i n 1 . 8 m x 2mm 1 . 0 . s i l a n i z e d g l a s s c o l u m n s 2 . C u m u l a t i v e M c R e y n o l d s c o n s t a n t 54 F i g . 2. Representative chromatograms obtained from blank (a.) and MCP-spiked (b.) plasma e x t r a c t s on 3% S i l a r - 9CP. Peaks with r e t e n t i o n , times of 6.66 and 21.04 min. correspond to HFB-MCP (53.33pg.y L""1) and u n d e r i v a t i z e d diazepam ( 0 . 4 n g . y L _ 1 ) , the i n t e r n a l standard. Chromatographic c o n d i t i o n s : as i n F i g . 1, except the column used was 3% S i l a r - 9 C P on Chromosorb W HP 100-120 mesh. Five yL i n j e c t i o n . 55 3.1.2. D e r i v a t i z i n g Agents: HFBA, HFBI, PFPA, TFAA, PFBC Studies employing f l u o r o - a c y l a t i n g agents other than HFBA were a l s o conducted. I t was hoped that by a l t e r i n g the molecular weight and r e l a t i v e p o l a r i t y of the MCP d e r i v a t i v e that r e s o l u t i o n from the i n t e r -f e r i n g components could be obtained on OV-225. The f o l l o w i n g reagents were test e d and chromatographed on 3% OV-225 on Gas Chrom Q 120-140 mesh: he p t a f l u o r o b u t y r y l i m i d a z o l e (HFBI), p e n t a f l u o r o p r o p i o n i c anhydride (PFPA), t r i f l u o r o a c e t i c anhydride (TFAA) and pentafluorobenzoyl c h l o r i d e (PFBC). The HFBI d e r i v a t i v e o f f e r e d no advantages over the anhydride, since r e s o l u t i o n times of the d e r i v a t i v e s were i d e n t i c a l , so the i n t e r f e r i n g peaks were s t i l l a problem. In a d d i t i o n the imidazole by-product r e s u l t e d i n chromatographic dist u r b a n c e s , namely a much l a r g e r solvent f r o n t , such that MCP now e l u t e d on the solvent f r o n t . The pentafluoropropionyl d e r i v a t i v e r e s u l t e d i n a s l i g h t increase i n r e t e n t i o n time (about 45 s) such that the MCP peak was d i r e c t l y c o i n c i d e n t with one of the i n t e r f e r i n g component, peaks. In a d d i t i o n , there was about a 30% decrease i n e l e c t r o n capture response over the HFB-MCP d e r i v a t i v e . T r i f l u o r o a c e t i c anhydride d i d r e s u l t i n a d e r i v a t i v e (TFA-MCP) that was well resolved from i n t e r f e r i n g components (HFB-MCP r e t e n t i o n time, (R.T.) = 4.9 min., TFA-MCP R.T. = 7.4 min). However, there was a s i g n i f i c a n t decrease i n e l e c t r o n - c a p t u r e detector response (about 80%) compared to the HFB d e r i v a t i v e such t h a t q u a n t i t a t i o n of the lower 3/4 of the standard curve ( v i z . , 8-32 ng.mL"^) would not have been p o s s i b l e . Attempts to form a MCP d e r i v a t i v e with PFBC based upon the methods of Midha et a l . , 1979, Matin and Rowland, 1972, and v a r i a t i o n s thereof were g e n e r a l l y 56 unsuccessful. In no instance was a peak f o r MCP.observed ( v e r i f i e d by using d i f f e r e n t MCP co n c e n t r a t i o n s , 40-200 ng.mL -^). A l l chromatograms appeared i d e n t i c a l . Matin and Rowland s t a t e that f r e q u e n t l y the product i s e i t h e r a s o l i d or an o i l . In the case of incubation with 2yL of undiluted PFBC an o i l y f i l m was observed. However, t h e i r recommended method of graviimetrtc a n a l y s i s by e x t r a c t i o n with methylene c h l o r i d e and r e c r y s t a l l i z i n g i n 90% ethanol Was not f e a s i b l e a t 8 - 40 ng.mL"^ drug l e v e l s . HFBA then continued to be the d e r i v a t i z i n g agent of choice and was employed f o r the balance of t h i s p r o j e c t . 3.1.3. S o l i d Supports • While attempting r o u t i n e plasma a n a l y s i s with S i l a r - 9 C P and OV-225 (on Chromosorb W HP 100-120 mesh) considerable v a r i a b i l i t y i n area r a t i o s (HFB-MCP/diazepam) was observed. A 20-25% d e c l i n e i n area r a t i o values f o r d u p l i c a t e or t r i p l i c a t e i n j e c t i o n s of the same sample over a 45-60 minute period was not uncommon. I n i t i a l i n v e s t i g a t i o n s were d i r e c t e d toward the p o s s i b i l i t y of on-column adsorption of MCP. Chromosorb 750 and Gas Chrom Q are reported to be very i n e r t by t h e i r r e s p e c t i v e manufacturers and are recommended f o r biomedical and pharmaceutical a n a l y s i s . Both commercially prepared and i n - l a b coated packings of 3% OV-225 and 3% S i l a r - 9 C P on these supports were t r i e d . The r e s u l t s of these i n v e s t i g a t i o n s are summarized i n Table I I . Neither of these s o l i d supports r e s u l t e d i n any improvement i n area r a t i o ' v a r i a b i l i t y . Chromosorb 750, r e p o r t e d l y the most i n e r t support a v a i l a b l e r e s u l t e d i n severe on-column adsorption of MCP which could not be overcome by column priming. 57 TABLE 11 SOLID SUPPORTS S o l i d Support Findings and Comments Gas Chrom Q.120-140 mesh coated with i ) 3% OV-225 - usual chromatographic patt e r n f o r OV-225 ( F i g . 1) - no improvement i n area r a t i o v a r i a b i l i t y i i ) 3% S i l a r - 9 C P - - poor response f o r MCP; broad, f l a t peak - no improvement i n area r a t i o v a r i a b i l i t y - signs of on-column adsorption (required some priming with MCP p r i o r to use) Chromosorb 750 100-120 mesh coated w i t h : i ) 3% OV-225 required high concentrations (>200ng.mL-l) of MCP to obtain a response priming d i d not improve MCP response very poor MCP peak shape; broad with a long leading edge and severe t a i l i n g . diazepam (the i n t e r n a l standard) now e l u t e s .before MCP. i i ) 3% S i l a r - 9 C P 1 - as i n ( i ) immediately above 1. coated i n - l a b . 58 3.1.4. S i l a n i z a t i o n o f G l a s s w a r e W a l l e and E h r s s o n , 1971, r e p o r t t h a t l o s s e s o f compounds by a d s o r p t i o n t o g l a s s s u r f a c e s can be an i m p o r t a n t p r o b l e m i n t h e q u a n t i -t a t i v e m i c r o a n a l y s i s o f amines. In o r d e r t o examine t h i s as a p o s s i b l e r e a s o n f o r t h e d e c l i n e i n a r e a r a t i o s o v e r time a l l g l a s s w a r e used i n MCP p r e p a r a t i o n , e x t r a c t i o n , d e r i v a t i z a t i o n and s t o r a g e was t r e a t e d w i t h 10% DMCS i n t o l u e n e . In s p i t e o f s u r f a c e p r e - t r e a t m e n t ( s i l a n i z a t i o n ) , extreme v a r i a b i l i t y i n a r e a r a t i o s p e r s i s t e d . 3.1.5. D e r i v a t i v e S t a b i l i t y : E f f e c t o f NH^OH, TEA, P y r i d i n e Having f a i l e d t o r e d u c e a r e a r a t i o v a r i a b i l i t y t h r o u g h the use o f a l t e r n a t e s u p p o r t s and s i l a n i z e d g l a s s w a r e , t h e s t a b i l i t y o f t h e HFB-MCP d e r i v a t i v e i t s e l f was q u e s t i o n e d . I t had been o b s e r v e d t h a t when t h e method o f Chang and Glazko, 1 9 7 2 ( f o r Ketamine. HC1 a n a l y s i s ) , was a p p l i e d t o MCP (namely, the removal o f e x c e s s HFBA w i t h 2mL o f 2N NaOH) noMCP peak c o u l d be o b s e r v e d . In a d d i t i o n , W a l l e and E h r s s o n , 1971, r e p o r t a base c a t a l y z e d (NH^OH, 0.1N NaOH) d e c o m p o s i t i o n o f HFBA phenol e s t e r d e r i v a t i v e s . I t was p o s s i b l e t h e n t h a t a base c a t a l y z e d h y d r o l y s i s o f the h e p t a f l u o r o b u t y r a m i d e l i n k a g e was o c c u r r i n g . I t had a l s o been o b s e r v e d d u r i n g t r i a l t e s t s w i t h TEA t h a t a r e a r a t i o v a r i a b i l i t y d i m i n i s h e d . The a d d i t i o n o f a c a t a l y s t such as TEA d u r i n g d e r i v a t i z a t i o n i s recommended by W a l l e and E h r s s o n , 1970, t o e n s u r e r a p i d and q u a n t i t a t i v e d e r i v a t i z a t i o n o f amines. 59 I t was decided then, to co n c u r r e n t l y evaluate the e f f e c t of adding a c a t a l y s t during d e r i v a t i z a t i o n as w e l l as t o examine the e f f e c t of ammonium hydroxide concentration on excess HFBA n e u t r a l i z a t i o n . Both TEA (lOOyL of 0.05M i n toluene) and p y r i d i n e ( 2 0 y L ) were used as c a t a l y s t s . H y d r o l y s i s and/or n e u t r a l i z a t i o n steps were c a r r i e d out with 0.5mL of deionized d i s t i l l e d water and 0.5mL of 1% or 4% NH^OH r e s p e c t i v e l y . Each sample contained 20ng.mL"1 of MCP.HC1 equivalent to base (extracted and d e r i v a t i z e d as described i n s e c t i o n 2.6.2). Five yL i n j e c t i o n s were made onto a 3% OV-225 (on Gas Chrom Q 120-140 mesh) column. The r e s u l t s of t h i s study are presented i n Table I I I . Area r a t i o v a r i a b i l i t y ( d e c l i n e i n area r a t i o s with time) i s reported as a c o e f f i c i e n t of v a r i a t i o n (C.V.) obtained by i n j e c t i n g each sample on a r o t a t i n g basis over an eleven hour period with a repeat i n j e c t i o n of each 20-24 hours a f t e r the f i r s t . The f i n d i n g s may be summarized as f o l l o w s : ( i ) h y d r o l y s i s and n e u t r a l i z a t i o n with water only r e s u l t e d i n chromatograms w i t h l a r g e s o l v e n t f r o n t s i n d i c a t i n g incomplete removal of h e p t a f l u o r o b u t y r i c a c i d . I n j e c t i o n s of these samples were discontinued due to the r i s k o f column damage. ( i i ) there was a great e r degree of v a r i a b i l i t y i n samples without c a t a l y s t ( e i t h e r TEA or p y r i d i n e ) and most notably i n samples n e u t r a l i z e d w i t h 4% NH^OH (C.V., 23.98%). ( i i i ) there was somewhat more v a r i a b i l i t y i n samples c o n t a i n i n g p y r i d i n e as c a t a l y s t . This i s l i k e l y r e l a t e d to chroma-tographic disturbances (broadening of the solvent f r o n t ) 60 T A B L E 111 E F F E C T OF AMMONIUM HYDROX IDE C O N C E N T R A T I O N AND C A T A L Y S T ON D E R I V A T I V E S T A B I L I T Y S a m p l e T r e a t m e n t A r e a R a t i o 1 C . V . 2 S a m p l e T r e a t m e n t A r e a R a t i o 1 2 c.v/ H N H 4 0 H 3 . 2 0 7 1 ± . 0 2 1 3 1 0 . 9 3 4 ( 2 6 . 2 6 % ) * 4 X N H 4 0 H 5 . 1 9 4 8 i . 0 4 6 7 2 3 . 9 8 ( 4 3 . 1 3 ' . ) 1% N H 4 0 H 6 . 1 0 8 1 ± . 0 0 8 3 7 . 7 2 4% N H 4 0 H ? . 2 8 9 8 t . 0 1 1 4 3 . 9 3 1 0 0 u L . 0 5 M T E A ( 1 9 . 2 4 % ) l O O u L . O S M T E A ( 7 . 9 4 % ) I S N H 4 0 H 3 2 0 u L P y r i d i n e . 0 7 9 1 • . 0 0 7 5 9 . 5 1 ( 2 1 . 1%) 4 % N H 4 0 H 9 2 0 u L P y r i d i n e . 2 2 1 5 ± 0 . 1 1 6 5 . 2 4 ( 1 2 . 7 6 % ) 1 . H F B - M C P d e r i v a t i v e / d i a z e p a m . E x p r e s s e d a s t h e m e a n ( X ) o f 5 o r 6 r e p e a t i n j e c t i o n s i o n e s t a n d a r d d e v i a t i o n ( s ) . 2 . % C o e f f i c i e n t o f v a r i a t i o n , (§- x 1 0 0 ) . x 3 . H y d r o l y z e d w i t h 0 . 5 m L d e i o n i z e d d i s t i l l e d w a t e r , n e u t r a l i z e d w i t h 0 . 5 m L 1% N H 4 0 H . 4 . ( ) , A b s o l u t e I d e c l i n e i n a r e a r a t i o s o v e r t h e 2 0 - 2 4 h o u r p e r i o d o f s t u d y . 5 . A s i s 3 , b u t n e u t r a l i z e d w i t h O.SmL 41 N H 4 0 H 6 . I O O U L 0 . 0 5 M T E A a d d e d a s c a t a l y s t p r i o r t o I n c u b a t i o n w i t h H F B A , h y d r o l y z e d a n d n e u t r a l i z e d a s d e s c r i b e d I n 3 . 7 . A s I n 6 , e x c e p t n e u t r a l i z e d w i t h O.SmL 4 1 N H 4 0 H . 8 . 2 0 J L p y r i d i n e a d d e d a s c a t a l y s t p r i o r t o I n c u b a t i o n w i t h H F B A , h y d r o l y z e d a n d n e u t r a l i z e d a s i n 3 . 9 . A s 1n 8 . b u t n e u t r a l i z e d w i t h O.SmL 4J N H 4 0 H 61 caused by p y r i d i n e . o v e r a l l , t h e a d d i t i o n of TEA and n e u t r a l i z a t i o n of excess HFBA with 4% NH^OH r e s u l t e d i n the l e a s t area r a t i o v a r i a b i l i t y . ( C . V . , 3.92% over the 20-24 hour period of study). A p p l i c a t i o n of these f i n d i n g s to r o u t i n e plasma a n a l y s i s r e s u l t e d i n l i n e a r standard curves and good sample s t a b i l i t y . 62 3.2. Fused S i l i c a C a p i l l a r y Column GLC-ECD 3.2.1. Column S e l e c t i o n During the course of packed column studies three glass c a p i l l a r y columns ( S i l a r - l O C , SP-2300, OV-225) were t e s t e d . In each case a very broad, se v e r e l y t a i l i n g MCP peak was obtained, so f u r t h e r studies with these columns were disc o n t i n u e d . With the a v a i l a b i l i t y of more i n e r t fused s i l i c a columns,renewed e f f o r t s were made to develop a c a p i l l a r y GLC-ECD assay method f o r MCP. The f a i l u r e of 3% S i l a r - 9 C P packed columns to withstand r o u t i n e plasma a n a l y s i s provided f u r t h e r impetus f o r such a development. Of the three fused s i l i c a columns (Carbowax 20M, methyl s i l i c o n e f l u i d , cross l i n k e d SE-54) used during assay development, the ^25rax 0.31mm I.D. c r o s s l i n k e d SE-54 column, was chosen f o r r o u t i n e MCP plasma q u a n t i t a -t i o n because of i t s r e s i s t a n c e to phase s t r i p p i n g by aromatic s o l v e n t s , such as toluene. The 12m x 0.2mm I.D. Carbowax 20{<1 and methyl s i l i c o n e f l u i d columns were used f o r i n i t i a l separation assessments and to optimize various GLC operating parameters. Both provided r e s o l u t i o n of MCP from plasma components but were of l i m i t e d usefulness because of t h e i r s u s c e p t i b i l i t y to phase s t r i p p i n g by aromatic s o l v e n t s . In a d d i t i o n Carbowax 20M has a low operating temperature (220°C) which f u r t h e r r e s t r i c t s i t s use since MCP tends to t a i l badly at temperatures of l e s s than 220°C. 63 3.2.2. Optimization of GLC-ECD Conditions 3.2.2.1.Injection Temperature Figure 3 i l l u s t r a t e s the e f f e c t of i n j e c t i o n temperature on the h e p t a f l u o r o b u t y r y l d e r i v a t i v e s of MCP (HFB-MCP) and MAP (HFB-MAP), the i n t e r n a l standard. The response i s reported as an area r a t i o (HFB-MCP/ HFB-MAP). The samples were ext r a c t e d and d e r i v a t i z e d as discussed i n s e c t i o n 2.5 and 2.6.1.B r e s p e c t i v e l y . Two pL i n j e c t i o n s were made onto the 25m x0.31mm;crosslinked SE-54 column using the chromatographic c o n d i t i o n s l i s t e d i n s e c t i o n 2.8.4.2. An i n j e c t i o n temperature of 220°C r e s u l t e d i n an optimal response and was chosen f o r a l l subsequent s t u d i e s . 3.2.2.2. Column Temperature The e f f e c t of column temperature on the separation of MCP from endogenous plasma components i s shown i n F i g . 4. The sample contained 24ng.mL~1 of MCP.HC1.H20 and 40 ng.mL"1 of MAP. HC1 which was e x t r a c t e d and d e r i v a t i z e d as described i n s e c t i o n s 2.5 and 2.6.1. B r e s p e c t i v e l y . Two pL i n j e c t i o n s were made onto the 25m x 0.31mm I.D. SE-54 column. At 235°C the extraneous peak ( i n d i c a t e d by an arrow) with a r e t e n t i o n time (R.T.) of 3.18'rain,is well resolved from MCP (R.T. = 3.31 min); at 240° ( F i g . 4b) i t begins to merge with MCP (R.T. - 2.87 min.) appearing as a shoulder peak; and at 245°C co-elutes with MCP (R.T. = 2.51 min.). 64 .5n °- A > I CQ U. / / i CD l i . • 2H < < cc .1 < 0 T T 1 200 210 220 230 240 250 INJECTION T E M P E R A T U R E <°C) E f f e c t of i n j e c t i o n port temperature on HFB-MCP and HFB-MAP response. Two yL i n j e c t i o n , each yL contained 277.78pg MAP, and 27.79pg M C P ( _ ) , 83.36pg ( — ) , 138.93pg ( ). n=l, d u p l i c a t e i n j e c t i o n s . 65 1. MCP 2. MAP CM N C\l N Si GI IS F i g . 4. E f f e c t of column temperature on the separation of HFB-MCP from endogenous plasma components, a. 235°C, b.240°C, c. 2450C. Each yL contained MCP 83.36pg, MAP 138.89pg (two iiL i n j e c t i o n s on the 25m SE-54 column). 66 Based on t h e s e o b s e r v a t i o n s a column t e m p e r a t u r e o f 235°C was chosen f o r r o u t i n e plasma a n a l y s i s . 3.2.2.3. C a r r i e r Gas: Helium ( H e ) , Hydrogen ( H 2 ) The e f f e c t s o f h e l i u m and hydrogen c a r r i e r g ases on MCP s e p a r a t i o n and a n a l y s i s t i m e , u s i n g t h e 25m c r o s s l i n k e d SE-54 column, a r e shown i n F i g s . 5a and b r e s p e c t i v e l y . In both i n s t a n c e s MCP (R.T. = 5.01 and 3.11 m i n , r e s p e c t i v e l y ) i s w e l l r e s o l v e d from plasma components and t h e i n t e r n a l standardSsMAP and prazepam,with r e t e n t i o n t i m e s o f 7.72 and 10.04 min f o r He, and 4.46 and 6.20 min. f o r H 2-U s i n g hydrogen, as t h e c a r r i e r g a s , t h e system l o s e s about 27% o f i t s s e p a r a t i o n e f f i c i e n c y (based on t h e t o t a l number o f t h e o r e t i c a l p l a t e s ( n ) , n = 24,180 f o r H 2 and 33,322 f o r He, u s i n g MCP as t h e r e f e r e n c e peak) but t h e a n a l y s i s r e q u i r e s 38% l e s s t i m e . S i n c e s e p a r a t i o n was not a p r o b l e m ( r e s o l u t i o n (R) = 13.5 and 18.3 between MCP and MAP f o r H 2 and He, r e s p e c t i v e l y ) , hydrogen was chosen as the c a r r i e r gas f o r t h e b a l a n c e o f the p r o j e c t . 3.2.2.4. C a r r i e r Gas ( H 2 ) Flow Rate The i n f l u e n c e o f c a r r i e r gas ( H 2 ) , f l o w r a t e on MCP r e s p o n s e was e v a l u a t e d a t 0.5 and l.OmL.min" 1. The r e s u l t s o f t h i s l i m i t e d s t u d y e m p l o y i n g s p l i t r a t i o s o f 30:1 and 60:1 a r e i l l u s t r a t e d i n F i g s . 6a and b. -1 The sample c o n t a i n e d 0,5yg.inL o f MCP.HC1 ,H 20 e x t r a c t e d and d e r i v a t i z e d as d e s c r i b e d i n s e c t i o n 2.8.2.T.A. A c c u r a t e i n j e c t i o n s o f 67 1. MCP 2. MAP 3. Prazepam •X' 6 ro 3. v. E f f e c t s of helium (a.) and hydrogen (b.) on HFB-MCP separation and a n a l y s i s time. The plasma e x t r a c t contained , MCP, 138.93pg.yL~ 1, MAP, 138.89 p g . y L - 1 , and prazepam 0.2ng. L" (2yL i n j e c t i o n ) . Chromotographic c o n d i t i o n s : column, 25m x 0.31mm I.D. c r o s s l i n k e d SE-54; I n j e c t i o n temperature, 220°C; Detector (E.C.D.) temperature, 350OC; Column temperature, 2350C; C a r r i e r gas f l o w , l.OmL.min-l; S p l i t vent flow, 30mL.min - 1 ( s p l i t r a t i o , 30:1); I n l e t pressure, 14.5 p . s . i . (He), 9.5 p . s . i . ( H 2 ) ; Make-up gas (Argon-Methane, 95:5) f l o w , 60mL.min.-l; Septum purge, 1.5mL.min-l; Chart speed 1.0cm.min _l; A t t e n u a t i o n , 2^; Slope s e n s i t i v i t y 0.2. 68 75 n Q. O 5 m 50H UJ 25 Q. 0' 0.5 1.0 0.5 1.0 CARRIER G A S (H 2 ) F L O W R A T E (mL.min. 6. E f f e c t of c a r r i e r gas (H 2) flow r a t e on HFB-MCP response. a. s p l i t vent flow, SOmL.min-1 ( s p l i t r a t i o , 30:1). b. s p l i t vent flow, 60mL.min _ 1 ( s p l i t r a t i o , 60:1). n = 1, d u p l i c a t e i n j e c t i o n s , Chromatographic c o n d i t i o n s : column, 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d ; I n l e t pressure, 19 p . s . i . A l l other GLC-ECD parameters are l i s t e d i n F i g . 5. 69 l y L were made, i n t h e absence o f an i n t e r n a l s t a n d a r d , onto a 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d column. The r e s p o n s e o f MCP t o c a r r i e r gas f l o w i s r e p o r t e d as an a b s o l u t e peak h e i g h t (mm). A f l o w o f 1.0 mL.nrin"^ p r o d u c e d t h e h i g h e s t r e s p o n s e and was t h e r e f o r e chosen f o r s u b s e q u e n t s t u d i e s . 3.2.2.5. Make-up Gas (Argon-Methane, 95:5) Flow Rate An o p t i m a l f l o w o f make-up gas i s r e q u i r e d t o m a i n t a i n c e l l volume and t o e f f e c t i v e l y sweep the e l e c t r o n c a p t u r e d e t e c t o r c e l l d u r i n g c a p i l l a r y column o p e r a t i o n . Flows o f between 20 and 60mL.min~^ a r e u s u a l l y s u f f i c i e n t and t h i s range i s recommended by the i n s t r u m e n t m a n u f a c t u r e r ( H e w l e t t - P a c k a r d C o . ) . F i g u r e 7 r e p r e s e n t s t h e e f f e c t o f make-up gas (Argon-Methane, 95:5) f l o w s o f 30, 40, and 60mL.min"^ on HFB-MCP r e s p o n s e ( a b s o l u t e peak h e i g h t , mm). A c c u r a t e i n j e c t i o n s o f l y L were made onto a 12m xO.2mm I.D. methyl s i l i c o n e f l u i d column i n t h e absence o f an i n t e r n a l s t a n d a r d . The sample was p r e p a r e d as d i s c u s s e d i n s e c t i o n 2.8.2.1.A. A f l o w o f 60mL.min~^ p r o v i d e d optimum s e n s i t i v i t y and was t h e r e f o r e s e l e c t e d f o r a l l s u c c e e d i n g a s s a y s . F i g u r e 8 i l l u s t r a t e s b a s e l i n e ( s i g n a l t o n o i s e ) p l o t s o b t a i n e d w i t h make-up gas f l o w s o f 20, 40 and 60mL.min _ 1 a t a t t e n u a t i o n s o f 2^, 2 2 , 2 4 and 2 6 . A f i o w 0 f 60mL.min~^ p r o v i d e d t h e b e s t b a s e l i n e s u p p o r t i n g t h e f i n d i n g s i n F i g u r e 7. The normal o p e r a t i n g a t t e n u a t i o n 4 f o r MCP plasma a n a l y s i s was 2 . 70 50-i Q. o ~ 30H 20 H 10H 30.0 40.0 50.0 60.0 MAKE-UP G A S F LOW R A T E (mL.min. 1 ) Influence of make-up gas (Argon-Methane, 95:5) flow rate on HFB-MCP response, n = 1, d u p l i c a t e i n j e c t i o n s . Chromatograph c o n d i t i o n s : column, 12m x 0.2mm. I.D. methyl s i l i c o n e f l u i d ; s p l i t vent f l o w , 60mL.min-1; a l l other parameters as reported i n F i g . 5. Hydrogen was used as the c a r r i e r gas. 71 <-CM z r-CE CSi fvj cr z r -K CE CM ca CM «-CM z cr CM Z t-H cr CM «-CM Z I-CE C3 8. Influence of make-up gas (Argon-Methane, 95:5) flow r a t e baseline ( s i g n a l to noise) p l o t s , a. 20mL.min-l, b. 40mL.min~', c. 60mL.min~l. 72 3.2.2.6. S p l i t L i n e r s : Jennings Tube, Fused S i l i c a I n sert A number of s p l i t l i n e r c o n f i g u r a t i o n s e x i s t and have been discussed and evaluated by Schomburg et a l . , 1977, Freeman, 1981c, and Jennings, 1980a. Fused s i l i c a i n s e r t s , and Jennings tubes are r e a d i l y a v a i l a b l e and were used during the course of t h i s p r o j e c t . These two s p l i t l i n e r s are i l l u s t r a t e d i n the diagram i n s e c t i o n 2.8.4.2.(8) and may be used e i t h e r with or without packing ( s i l a n i z e d g l a s s wool, and/or conventional column packing such as 2% 0V-1 or 3% OV-17). The e f f e c t of a 2% 0V-1 packed Jennings tube (see s e c t i o n 2.8.4.2. (8)) on MCP, MAP, and prazepam peak shape (R.T. = 3.34, 4.64 and 6.41 min, r e s p e c t i v e l y ) i s shown i n F i g . 9a. Marked band spreading and t a i l i n g occurred with each of these compounds. This human plasma sample (spiked w i t h MCP and MAP) was extracted and d e r i v a t i z e d as described i n s e c t i o n s 2.5 and 2.6.1. B , r e s p e c t i v e l y . Two yL i n j e c t i o n s were made onto the 25m-x 0'.'31mm i.D. c r o s s l i n k e d SE-54 column. Results of the i n j e c t i o n of t h i s same sample f o l l o w i n g the removal of the 2% OV-1 packing are shown i n Fig.9b. A l l three drugs show a marked improvement i n peak shape. A higher on-column d e l i v e r y a l s o r e s u l t s making p o s s i b l e the q u a n t i t a t i o n of lower drug concentrations ( v i z . , 4-24ng.mL~^). The fused s i l i c a l i n e r (2mm I.D.) was a l s o tested i n both the packed and unpacked c o n f i g u r a t i o n s (see s e c t i o n 2.8.4.2. ( 8 ) ) . When used unpacked a s p l i t vent flow of lOmL.min"^ ( s p l i t r a t i o , 10:1) w i l l y i e l d e s s e n t i a l l y the same r e s u l t s (area r a t i o values) as an unpacked Jennings tube at a 30:1 s p l i t r a t i o . More v a r i a b i l i t y i s , however, 73 Is- co ca ca 1. MCP 2. MAP 3. prazepam * is 9. E f f e c t of a 2% OV-1 packed Jennings s p l i t l i n e r on MCP, MAP and prazepam peak shape, a. with 2% OV-1 packing, b. without packing. V. 74 observed. Use of t h i s s p l i t l i n e r . a t such low s p l i t vent flows has r e s u l t e d i n a l o s s of i n l e t pressure. This could r e s u l t i n an i n t e r r u p t i o n of column flow, and subsequent column damage at operating temperatures. The use of t h i s l i n e r at such low s p l i t r a t i o s i s not recommended as a precautionary measure. Packing the s i l i c a i n s e r t with a " t i g h t " s i l a n i z e d g l a s s wool plug as i l l u s t r a t e d i n s e c t i o n 2.8.4.2.(8) y i e l d s r e s u l t s very s i m i l a r to those obtained with the unpacked Jennings tube at equivalent s p l i t vent flows (30mL.min~^); s p l i t r a t i o , ( 3 0 : 1 ) . This- i s shown i n F i g . 10 where the same samples (from an abbreviated standard curve) were i n j e c t e d (2yL) onto the 25m x 0.31mm .1.D. c r o s s l i n k e d SE-54 column using f i r s t the unpacked Jennings tube and immediately followed by the use of a packed fused s i l i c a l i n e r . There i s very good agreement of the standard curves and t h e i r r e s p e c t i v e s t a t i s t i c a l para-meters ( r = 0.9996, y = 0.0241x + 0.0086 and r = 0.9997, y = 0.0243x + 0.0004). Both s p l i t l i n e r s , the Jennings tube i n the unpacked c o n f i g u r a t i o n and the fused s i l i c a i n s e r t , packed with a " t i g h t " s i l a n i z e d g l a s s wool plug , were used f o r q u a n t i t a t i v e plasma a n a l y s i s during the course of t h i s work. 3.2.2.7. I n j e c t i o n Mode: S p l i t , S p l i t l e s s Of the four i n j e c t i o n modes c u r r e n t l y a v a i l a b l e ( s p l i t , s p l i t l e s s , d i r e c t , on-column) f o r high r e s o l u t i o n gas chromatography, s p l i t and •: s p l i t l e s s were examined during the course of t h i s study. 75 .9-1 MCP C O N C E N T R A T I O N (ng .mL 1 ) F i g . 10. Comparison of standard curve parameters obtained using an unpacked Jennings tube ( ), (y = 0.024 x + 0.0086, r = 0.9996) and a fused s i l i c a i n s e r t packed with a " t i g h t " s i l a n i z e d g l a s s wool plug ( ), (y = 0.0243 x + 0.004, r = 0.9997) (±one standard d e v i a t i o n ) . 76 The s p l i t mode i s the e a s i e s t from the standpoint of ease of instrument conversion, s i m p l i c i t y of the method (good r e s u l t s are r e l a t i v e l y easy to obtain) and q u a n t i t a t i v e r e l i a b i l i t y (Jennings, 1980a). Although not g e n e r a l l y recommended f o r low l e v e l a n a l y s i s , the use of s e n s i t i v e d e t e c t o r s , such as e l e c t r o n - c a p t u r e , make i t p o s s i b l e to extend the a p p l i c a b i l i t y of s p l i t i n j e c t i o n to trac e l e v e l s . A p p l i c a t i o n of t h i s i n j e c t i o n mode to b i o l o g i c a l samples (human and sheep plasma) r e s u l t e d i n c l e a n chromatograms and good r e p r o d u c i b i l i t y over the concentration range (4-40ng.mL _ 1) required f o r MCP q u a n t i t a t i o n . Representative chromatograms employing t h i s technique are shown i n F i g . 5. The s p l i t l e s s mode of sample i n j e c t i o n r e s u l t e d i n very complex chromatograms with p r o t r a c t e d solvent f r o n t s and many e x t r a peaks due to plasma components. Resolution of MCP from endogenous plasma components was not complete. This mode requ i r e s a d d i t i o n a l work to optimize the proper s o l v e n t , i n i t i a l temperature, purge a c t i v a t i o n time, i n j e c t i o n s i z e and sample con c e n t r a t i o n . Based on these observations the s p l i t mode of sample i n j e c t i o n was adopted f o r use i n t h i s p r o j e c t . 3.2.2.8. I n j e c t i o n Solvent During the course of assay development fo u r s o l v e n t s were used: iso-octane, hexane, benzene and toluene. 77 In p r e l i m i n a r y experiments, with the 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d columns, iso-octane and hexane were employed as i n j e c t i o n , s o l v e n t s . The use of aromatic solvents such as benzene and toluene with these columns i s not recommended due to the danger of phase s t r i p p i n g and subsequent l o s s of column e f f i c i e n c y . Iso-octane e x h i b i t e d very poor s o l u b i l i t y and e x t r a c t a b i l i t y c h a r a c t e r i s t i c s towards MCP. The base (MCP) was very i n s o l u b l e i n t h i s solvent and e x t r a c t i o n of the hydrochloride s a l t from aqueous s o l u t i o n s y i e l d e d extremely v a r i a b l e r e s u l t s . Hexane was t r i e d as an a l t e r n a t i v e although e x h i b i t i n g somewhat b e t t e r s o l u b i l i t y and e x t r a c t a b i l i t y c h a r a c t e r i s t i c s , i t too was found to be a poor solvent choice f o r MCP (Tarn, M.Sc. Thesis, 1978). Hexane, however, was used with high MCP concentrations(0..5yg.mL~ 1) i n e a r l y developmental work u n t i l c r o s s l i n k e d columns became a v a i l a b l e . With the a v a i l a b i l i t y of a c r o s s l i n k e d (or bonded) fused s i l i c a c a p i l l a r y column(25m x 0.31mm I.D;, SE-54) aromatic solvents such as benzene and toluene could be used without the p r o b a b i l i t y of phase s t r i p p i n g . Both of these solvents e x h i b i t good MCP s o l u b i l i t y and e x t r a c t a b i l i t y c h a r a c t e r i s t i c s and allowed development of the assay such that MCP q u a n t i t a t i o n i n the d e s i r e d range of 4-40ng.mL~1 could be c a r r i e d - o u t . . Of the two, toluene was u l t i m a t e l y chosen as the d e r i v a t i z i n g and i n j e c t i o n solvent because of i t s higher b o i l i n g p o i n t . The use of higher b o i l i n g p o i n t solvents i s recommended by Schomburg et a l . , 1981, to minimize sample d i s c r i m i n a t i o n i n the s p l i t l i n e r . 78 3.2.2.9. S e l e c t i o n of Int e r n a l Standard Three drug compounds (diazepam, prazepam, m a p r o t i l i n e ) were used as i n t e r n a l standards during development of the c a p i l l a r y assay method. Diazepam had been i n use since development of the GLC-ECD MCP assay method (Tarn, M.Sc. Thesis, 1978) so i t was tested on the c a p i l l a r y columns as w e l l . Resolution of diazepam from MCP (R = 1.07, using H 2 as c a r r i e r gas) on the 12m. x. 0.2mm methyl s i l i c o n e f l u i d columns was not complete. However, with the 25m x 0.31mm c r o s s l i n k e d SE^54 column, r e s o l u t i o n of these compounds was no longer a problem (R=5.79, using H 2 as c a r r i e r gas). Since the assay was to be a p p l i e d to h o s p i t a l i z e d p a t i e n t plasma samples the p o s s i b i l i t y of therapy with diazepam e x i s t e d . This drug was, therefore, not a good i n t e r n a l standard choice. Prazepam, another benzodiazepine t r a n q u i l i z e r , was s u b s t i t u t e d f o r diazepam. I t i s very w e l l r e s o l v e d from MCP as i l l u s t r a t e d i n the chromatogram of Fig.5b (MCP, R.T. = 3.11 min.; prazepam, R.T. = 6.20. min.). This drug i s not y e t a v a i l a b l e f o r therapeutic use i n Canada so i t was chosen i n p r e l i m i n a r y s t u d i e s as a s u i t a b l e i n t e r n a l standard. M a p r o t i l i n e (MAP) was u l t i m a t e l y s e l e c t e d as the i n t e r n a l standard i n the f i n a l assay method. This drug, a secondary amine, i s a v a i l a b l e as the hydrochloride s a l t and therefore, undergoes the same e x t r a c t i o n and HFBA d e r i v a t i z a t i o n procedures as MCP. Resolution from MCP i s e x c e l l e n t as shown i n the chromatogram of Fig.5b (MCP, . „ . 79 R.T. = 3.11 min, -MAP, R.T. = 4.46 min.). The use of MAP as i n t e r n a l standard r e s u l t e d i n consider a b l y b e t t e r standard curve c o r r e l a t i o n s than those obtained w i t h prazepam. The l a t t e r , although used as a secondary i n t e r n a l standard, f o r a short period of time;, was discontinued i n q u a n t i t a t i v e plasma s t u d i e s . The use of MAP al s o provided the a d d i t i o n a l b e n e f i t of a shor t e r a n a l y s i s time. 3.2.2.10. Optimization of HFBA D e r i v a t i z a t i o n Time f o r MCP and MAP. Figure 11 dep i c t s the r e a c t i o n k i n e t i c s (at 55°C) f o r the HFBA d e r i v a t i z a t i o n of MCP and MAP i n the presence of the c a t a l y s t t r i e t h y l a m i n e (0.05M). The samples, co n t a i n i n g 20ng of MCP.HCl.H2O and 40ng of MAP.HC1, were e x t r a c t e d and d e r i v a t i z e d according t o t h e procedure i n s e c t i o n 2.6.4. The d e r i v a t i z a t i o n r e a c t i o n was observed to be ra p i d and was r e l a t i v e l y constant throughout the period of study. An incubation time of s i x t y minutes was chosen f o r a l l f o l l o w i n g s t u d i e s . The f i n a l plasma e x t r a c t i o n and d e r i v a t i z a t i o n procedures used i n the a p p l i c a t i o n of the GLC-ECD fused s i l i c a c a p i l l a r y assay method f o r MCP q u a n t i t a t i o n are shown i n Scheme I. 3.3. A p p l i c a t i o n of the Fused S i l i c a C a p i l l a r y GLC-ECD Assay: Q u a n t i t a t i v e Plasma (Human, Sheep) MCP A n a l y s i s 3.3.1. Human Pl a c e n t a l Transfer Studies: Data C o l l e c t i o n The data c o l l e c t i o n sheet i l l u s t r a t e d i n F i g . 12 was used to record p a t i e n t (maternal, i n f a n t ) s t a t i s t i c a l i n f o r m a t i o n , (weights, age, use of other medications, etc.) as well as MCP dosing and blood sampling d e t a i l s . 80 . 9 H CL < CQ Ii. i .6 CL O 5 .5. CQ LL 1 .4' < 2 cc < 04 — i — i — i — j — i — i — i — i — i 0 20 40 60 80 100 120 140 160 180 TIME (min.) ;Fi.g. 11. Optimization of HFBA d e r i v a t i z a t i o n r e a c t i o n time at 55°C f o r MCP and MAP i n the presence of 0.05M TEA. Two yL i n j e c t i o n s were made onto the 25m SE-54 column. Each yL of sample contained 69.79pg of MCP and 138.89pg of MAP. n = 3, d u p l i c a t e i n j e c t i o n s (j/ one standard deviation)-. 81 SCHEME I E x t r a c t i o n a n d D e r i v a t i z a t i o n P r o c e d u r e f o r F u s e d S i l i c a C a p i l l a r y G L C - E C D A s s a y P l a s m a ( h u m a n / s h e e p ) , 0 . 2 5 - 0 . 5 m L M a p r o t i l i n e - H C l ( 0 . 4 y g . m L - l ) , O . l m L I N NaOH 0 . 5 m L D e i o n i z e d d i s t i l l e d H 2 0 q . s . 2 . 1 m L B e n z e n e 6 . 0 m L S h a k e f o r 2 0 m i n . , C e n t r i f u g e a t 4 0 0 0 r . p . m . x 5 m i n . , P l a c e 1n i c e b a t h = 5 m i n . . C e n t r i f u g e a t 4 0 0 0 r . p . m . f o r 5 m o r e m i n . D i s c a r d a q u e o u s l a y e r ' 5mL b e n z e n e l a y e r - A d d 2mL I N HCL C e n t r i f u g e a t 2 0 g O r . p . m . +-x 1 m i n . i I m m e d i a t e l y t r a n s f e r o r g a n i c l a y e r t o c l e a n , d r y 1 5 n t c u l t u r e t u b e J- 0 S t o r e a t - 2 0 U C u p t o 4 d a y s 2 uL i n t o G L C - A d d O . SmL 4 1 N H . O H V o r t e x a t s p e e d 1 0 f o r 1 0 s A d d 0 . 5 m L d e i o n i z e d d i s t i l l e d H 2 0 , V o r t e x a t s p e e d 1 0 f o r 1 0 s S h a k e f o r 2 0 m i n . , C e n t r i f u g e a t 4 0 0 C r . p . m . x 5 m i n . Wash a q u e o u s l a y e r w i t h 2 x 4mL b e n z e n e I A d d O .SmL 5N NaOH a n d 6 . 0 m L b e n z e n e 1 S h a k e f o r 2 0 m i n . , C e n t r i f u g e a t 4 0 0 0 r . p . m . x 5 m i n . 5 m l b e n z e n e l a y e r I E v a p o r a t e w i t h N , a t 4 0 ° C ( H 2 0 b a t h ) . •1 R e c o n s t i t u t e r e s i d u e w i t h : 1 5 0 p L t o l u e n e 5 0 U L 0 . 0 5 M T E A A d d 2 0 u L HFBA - V o r t e x I I n c u b a t e a t 5 5 ° C f o r 6 0 m i n . I ' C o o l t o r o o m t e m p e r a t u r e A s p i r a t e a n d d i s c a r d b e n z e n e l a y e r D i s c a r d a q u e o u s l a y e r 82 METOCLOPRAMIDE DATA SHEET Patient/Sample Code No. VITAL STATISTICS METOCLOPRAMIDE (MCP) DATA MOTHER: DOSE Age Route of Administration: o r a l Weight I.V. Smoker Nonsnoker Time of Administration Other Orugt Clocktloe of Maternal Sample Ciocktlme of Umbilical Sample INFANT: (venous, a r t e r i a l ) Hale Female Weight et b i r t h Apgar Score H.R. B.P. Signs of adverse drug e f f e c t Gestational Age ADDITIONAL COMMENTS: F i g . 1 2 . MCP Data C o l l e c t i o n Sheet 83 Information on maternal drug use, smoking s t a t u s and p e r t i n e n t comments are presented i n Table IV. A l l p a t i e n t s received a standard general anaesthetic regimen of t h i o p e n t a l 250mg, halothane 0.5%, d-tubocurare 3mg, and s u c c i n y l c h o l i n e lOOmg except as i n d i c a t e d by a change of dose. P a t i e n t 5 S was given a s p i n a l ( e p i d u r a l anaesthetic (Marcaine ) p r i o r to general anaesthesia. The use of other medications and comments f e l t to have a bearing on i n f a n t response such as, prolonged labour, are a l s o included i n the t a b l e . Only one p a t i e n t (19 S) showed any signs of an adverse drug r e a c t i o n to MCP. Infant s t a t i s t i c a l d e t a i l s and r e l e v a n t comments are presented i n Table V. A n a l y s i s of the accumulated data on each of the f o l l o w i n g parameters: g e s t a t i o n a l age, b i r t h weight, Apgar scores, heart r a t e , blood pressure, maternal age and weight, dose of MCP and a d m i n i s t r a t i o n times was completed using the Students' t - t e s t subprogram of the S t a t i s t i c a l Package f o r the S o c i a l Sciences v e r s i o n 9 (SPSS:9) (Nie, et a l . , 1982). S i g n i f i c a n c e was determined at the .05 l e v e l . No s i g n i f i c a n t d i f f e r e n c e e x i s t e d between the t r e a t e d and untreated groups i n g e s t a t i o n a l age, sex d i s t r i b u t i o n , b i r t h weight or c a r d i o v a s c u l a r parameters (HR, BP) as i n d i c a t e d i n Table V. S t a t i s t i c a l l y , the Apgar scores at 1 minute were s i g n i f i c a n t l y b e t t e r i n the t r e a t e d group, however, t h i s f i n d i n g was l i k e l y biased since there was one baby (3S*) i n the untreated group who had a low Apgar score because of unexpected problems at d e l i v e r y . When t h i s baby's score i s deleted there i s no apparent d i f f e r e n c e between the groups. Babies 4 S, 15 S and 35 S were premature. They were t r a n s f e r r e d to the i n t e n s i v e care nursery (ICN) and were not included i n the s t a t i s t i c a l a n a l y s i s . A l l i n f a n t s (except 4 S, 15 S, 35 S) underwent a v a r i e t y of 84 TABLE IV Maternal Data Pat ien t Code Smoker Other Drugs^ - MCP TREATED -Comments 2S 5S 9S 12S 34S 4S 19S 22S 29S No No No No No Yes (4 pack/day) Yes Yes (1 pack/day) Yes (1 pack/day) - t h iopenta l 500 mg - v i t . B 6 -Natabec -Seconal (at 2235) -Marcaine .5S(20mL) - N a . c i t r a t e preop. - s u c c i n y l c h o l i n e 200 mg - f a i l u r e to progress, prolonged labour -maternal a g i t a t i o n lOmin. p o s t - i n j e c t i o n , passed in 2-3 min. CONTROLS -IS 7S 8S 10S 16S 26S 28S 30S 32S 33S 35S 3S 15S 42S No No No No No No No No NO No Yes Yes(3-4 /day) Yes (1 pack/day) - t h i o p e n t a l 373mg -Na. c i t r a t e -halothane 0.5-0.75% -d - tubocurare 15mg - th iopenta l 275mg - s u c c i n y l c h o l i n e 140mg -v i tamins - th iopenta l 325mg - s u c c i n y l c h o l i n e 140mg -Na. c i t r a t e - th iopenta l 300 mg - th iopenta l 300 mg -ethanol 12/wk. -repeat C/Sc - repeat C /S" - d e l i v e r y 12min.after induct ion -very prolonged d e l i v e r y - d e l i v e r y 15min.after induct ion -prolonged labour - d e l i v e r y 12min. a f t e r induct ion - repeat C / S c - a s p i r a t i o n of g a s t r i c contents A l l pa t ients rece ived : th iopenta l 250mg, halothane 0.5%, d-tubocurare 3mg, s u c c i n y l c h o l i n e lOOmg, except where ind ica ted by change of dose and/or add i t i ona l agents. 2. C /S , Caesarian sect ion 85 Table V Infant Data Patient Code Gestational Sex Birth Weight Apgar1 H.R. B.P. Adverse Drug Age (M.F) (9) Score (systolic) Effects/Comments - MCP TREATED -2S 40 M 3800 8. 9 148 55 5S 40 M 3060 8. 9 122 65 - difficult delivery 9S 40 cried at once F 4060 9. 10 140 58 12S 39 F 3540 9, 9 160 56 - very alert 34 S 39 F 4760 9, 10 160 68 4S*t 37 F 2200 9. 9 122 65 - baby not followed 39 transferred to ICN2 19S* F 2690 9, 9 150 58 - difficult Intubation Apgars likely 7,8 at 22S* 40 the most M 3070 8. 9 148 56 29S* 39 F 3670 9. 9 150 50 -X 39.5 3581 8.6, 9.3 147 58 S.D. ±0.5 ±655 ±0.5,±0.5 ±12 ±8 - PLACEBO IS 40 F 3340 9. 9 150 55 7S 38 F 3910 8. 9 145 54 8S 38 F 3240 8. 9 152 68 10S 38 F 2960 8, 10 145 62 16S 39 F 3380 8. 9 160 46 _ 26S 40 3050 7. 9 140 58 _ 28S 30S 40 f 3220 8. 9 176 52 - exceptional ly alert 40 f 3858 8. 9 150 58 32S 40 M 4680 8. 9 148 60 33S 38 M 3040 8. 9 160 56 35S + 37 M 2310 8, 8 144 61 - respiratory problems (mucus) - transferred 3S* 40 to ICN2 F 3900 3, 9** 169 74 - very flat and blue at birth; slow recusita-15S*t 37 F 3520 5. 8 162 74 tion - baby aspirated fluid at delivery, trans-ferred to ICN2 42S* 40 (tachypnea) M 2840 9, 10 140 50 X 39.3 3451.5 7.7.9.2 153 58 S.D. ±0.1 ±154 ±1.6,±0.4 ±11 ±8 1. Apgar scores determined at 1 and 5 minutes post delivery, ( , ). • Infants born to mothers who smoke. 2. ICN, Intensive Care Nursery ** Unanticipated problems at delivery likely contributed to low 1 min Apgar score. + Not Included 1n statistical analysis (Students' t-Test, (SPSS-9)) since babies were lost to follow-up. 86 n e u r o l o g i c a l f u n c t i o n a l t e s t s at 2, 4, 6 and 24 hours p o s t - d e l i v e r y to observe any adverse e f f e c t s MCP may have had as a r e s u l t of p l a c e n t a l t r a n s f e r . The r e s u l t s of these assessment studies w i l l be reported i n a f u t u r e p u b l i c a t i o n . (Bylsma-Howell et a l . , 1982) 3.3.2. Human P l a c e n t a l Transfer : Plasma MCP Determination Plasma (MV, UV, UA) MCP a n a l y s i s was c a r r i e d out using the developed GLC-ECD fused s i l i c a c a p i l l a r y column assay. The 25m x 0.31mm I.D. c r o s s l i n k e d SE-54 column was used f o r a l l MCP q u a n t i t a t i o n . A l l plasmas (blank, MCP-spiked, t e s t ) were e x t r a c t e d and d e r i v a t i z e d as described i n s e c t i o n s 2.5 and 2.6.1. B, r e s p e c t i v e l y . A r e p r e s e n t a t i v e standard curve used f o r MCP plasma q u a n t i -t a t i o n i s shown i n F i g . 13. The l i n e of best f i t through the data p o i n t s obtained from l i n e a r r e g r e s s i o n a n a l y s i s was described by: Y = 0.0254 x - 0.0023 with r = 0.9993 Chromatographic response was found to be l i n e a r over the concentration range studied ( v i z . , 4 - 40ng.mL _ 1). This represents ~ 0.9 - 9 pg at the e l e c t r o n - c a p t u r e detector employing a 30:1 s p l i t r a t i o . C o e f f i c i e n t s of v a r i a t i o n ( C V . ) f o r each data p o i n t (MCP concen-t r a t i o n ) are reported i n F i g . 13. The mean C V . f o r t h i s standard curve was 4.91%. A more extensive determination of assay r e p r o d u c i -b i l i t y i s reported i n Table VI f o r two extreme points i n the standard curve ( v i z . , 8.02 and 40.2ng.mL"1 of MCP.HC1.H20 eq u i v a l e n t to MCP base). In g e n e r a l , a range of c o e f f i c i e n t of v a r i a t i o n from 2-10% was observed f o r a l l standard curves. 87 1 . 2 n CL < i CD CL O Sample^ A.R . 2 C V . 3 4.02 0.1055+0.0114 10.85 8.04 0.1893+0.0159 8.41 16.08 0.4263+0.0119 2.80 24.12 0.5939+0.0235 3.95 32.16 0.8094+0.0178 2.20 40.20 1.0256+0.0127 1.24 T 30 35 40 MCP CONCENTRATION (ng.mL1) F i g . 13. A r e p r e s e n t a t i v e standard curve of human plasma e x t r a c t s obtained by p l o t t i n g the area r a t i o of HFB-MCP/HFB-MAP versus MCP c o n c e n t r a t i o n , n = 2, d u p l i c a t e i n j e c t i o n s _, (2LIL). L i n e a r i t y was observed between 4.02 - 40.20 ng.mL" (13.96 - 139.68 pg.pL' 1). (MAP c o n c e n t r a t i o n , 40 ng.mL"1, cj.pL- 1" 138.89 pg ) . An unpacked Jennings tube was used. 1. MCP c o n c e n t r a t i o n , ng.mL - 1 2. Mean area r a t i o , HFB-MCP/HFB-MAP ± one standard d e v i a t i o n 3. C o e f f i c i e n t of v a r i a t i o n , %. 88 TABLE VI\ C o e f f i c i e n t of V a r i a t i o n Study Sample 8.02 (6) 40.20 (5) 4 0.3416±0.0159 4.65 1.215210.0815 6.71 1. MCP.HCl.r^O concentration (equivalent to MCP base), ng.mL"1 2. Mean area r a t i o (HFB-MCP/HFB-MAP)± one standard d e v i a t i o n . 3. C o e f f i c i e n t of v a r i a t i o n , %. 4. ( ), number of samples e x t r a c t e d ; t r i p l i c a t e i n j e c t i o n s . A fused s i l i c a i n s e r t packed with a " t i g h t " s i l a n i z e d g l a s s wool plug was used i n t h i s study. 89 The r e s u l t s of the plasma analyses, (pne-poi.nt; MV, UV, UA) are presented i n Table V I I . Determinations were made on two d i f f e r e n t occasions.(i ,e. r e p l i c a t e plasma e x t r a c t i o n s and d e r i v a t i z a t i o n s ) except i n the case of some UA samples (2.S, 5 S, 9 S, 19 S) where there were i n s u f f i c i e n t plasma. Nine out of the twenty-three p a t i e n t s i n v o l v e d i n the double b l i n d study were administered MCP, the remaining fourteen received Sodium Ch l o r i d e f o r I n j e c t i o n as evidenced by the lack of a chromatographic peak f o r MCP. The r e s u l t s of the plasma determinations were i n agreement with the coded f i n d i n g s when the code was broken f o l l o w i n g completion of the plasma analyses. This study demonstrates that MCP does cross the placenta r e s u l t i n g i n an average f e t a l : m a t e r n a l r a t i o of 0.60 ( f 0.17), (Table V I I ) . In a l l cases MV plasma MCP concentrations were higher than those i n e i t h e r the UV or UA. U m b i l i c a l venous MCP concentrations were higher than UA except i n the case of p a t i e n t 29 S. This apparent discrepancy may be due to a n a l y t i c a l e r r o r or to i n c o r r e c t venipuncture during u m b i l i c a l cord blood sampling. No s i g n i f i c a n t d i f f e r e n c e s between smokers'(4 S, 19 S, 22 S, 29 S) and nonsmokers' plasma MCP l e v e l s (MV, UV, UA) were noted, however, a much l a r g e r sample population would be required to i n t e r p r e t the e f f e c t of smoking. Attempts to p l o t plasma versus time p r o f i l e s f o r paired maternal and i n f a n t ( u m b i l i c a l ) samples were i n c o n c l u s i v e . No s i g n i f i c a n t c o r r e l a t i o n was found between MV and UV, MV and UA metoclopramide concentrations or between these i n d i v i d u a l concentrations and time. A c o r r e l a t i o n of r = 0.9014 was obtained between UV and UA metoclopramide concentrations. The l i n e of best f i t ( l i n e a r r e g r e s s i o n a n a l y s i s ) was 90 T a b l e V I I . P l a s m a A n a l y s i s D a t a P a t i e n t M a t e r n a l S a m p l e E l a p s e d T i m e ( m 1 n ) P l a s m a MCP c o n c e n t r a t i o n s C o d e A g e Wt I . V . D o s e M V 1 U M B 2 ( n g . m L " 1 ) 3 F / M 5 ( y r s ) ( k g ) ( m g / k g ) MV UV UA - MCP T R E A T E D -2 S 2 8 6 8 0 . 1 5 3 6 2 9 6 1 . 1 6 ± 0 . 2 2 1 8 . 8 2 * 0 . 1 3 1 8 . 2 8 ? 0 . 3 1 5S 24 7 0 0 . 1 0 34 4 2 5 7 . 2 2 t l . 9 7 3 8 . 6 4 * 2 . 4 3 3 8 . 0 6 * 0 . 6 8 9 S 2 3 8 0 0 . 1 5 21 3 0 6 3 . 0 U 3 . 1 2 3 8 . 7 5 * 3 . 2 7 3 2 . 7 8 4 0 . 6 2 12S 2 7 84 0 . 1 4 5 0 5 3 4 9 . 0 6 ± 3 . 5 1 4 5 . 5 7 ± 3 . 6 4 4 3 . 0 2 t l . 0 5 0 . 9 3 3 4 S 33 7 5 0 . 1 5 30 34 7 4 . 3 4 ± 2 . 5 0 3 7 . 0 5 * 0 . 6 2 3 4 . 8 0 * 1 . 9 5 0 . 5 0 4 S * + 2 2 6 2 0 . 1 4 1 8 2 3 7 1 . 2 4 * 0 . 2 3 4 9 . 5 0 ± 0 . 7 9 4 3 . 8 9 j l . 7 7 0 . 6 9 1 9 S * 20 6 8 0 . 1 5 31 3 0 8 5 . 0 3 ± 2 . 0 5 4 8 . 5 7 * 0 . 7 8 4 8 . 1 4 * 0 . 5 7 2 2 S * 2 3 6 3 0 . 1 4 1 5 1 9 5 7 . 7 2 * 2 . 0 2 3 5 . 7 1 * 0 . 5 3 2 4 . 7 9 ± 1 . 1 0 0 . 6 2 2 9 S * 31 9 0 0 . 1 5 32 3 6 4 2 . 1 9 * 0 . 4 9 2 5 . 8 7 ± 0 . 1 5 3 0 . 5 3 ± 2 . 3 1 0 . 6 1 X 2 6 . 1 3 7 4 . 7 5 0 . 1 4 3 1 . 1 3 3 4 . 1 3 6 1 . 2 2 3 6 . 1 2 3 3 . 8 0 0 . 6 0 S . D . • 4 . 4 2 • 9 . 2 4 • 0 . 0 2 ±10 .37 ±10 .08 ± 1 3 . 5 3 ± 9 . 7 3 • 9 . 5 9 ±0 .17 - P L A C E B O -I S 31 6 0 0 . 1 5 34 3 7 _ _ _ 7S 2 9 8 2 0 . 1 2 2 7 3 5 _ _ 8 S 3 3 64 0 . 1 4 24 2 8 _ 10S 2 7 5 5 0 . 1 0 12 2 0 _ _ _ 16S 2 8 7 5 0 . 1 5 4 5 5 6 _ _ 2 6 S 31 8 6 0 . 1 4 2 8 3 6 _ 2 8 S 3 0 7 0 0 . 1 4 21 2 2 _ _ _ 3 0 S 3 7 9 0 0 . 1 5 3 0 31 _ _ 3 2 S 34 8 6 0 . 1 4 2 5 3 6 _ 3 3 S 3 0 6 2 0 . 1 5 4 0 4 6 _ _ _ 3 5 S + 31 64 0 . 1 4 31 3 2 _ _ 3 S * 2 2 6 2 0 . 1 5 51 5 9 _ _ _ 1 5 S * t 2 3 9 8 0 . 1 5 4 5 4 9 _ _ 4 2 S * + 2 7 6 3 0 . 1 4 2 6 3 0 - -X 2 9 . 9 2 7 1 . 2 5 0 . 1 4 3 0 . 2 5 3 6 . 3 3 S . D . ± 3 . 8 5 ±12 .06 ± 0 . 0 1 ±10 .77 ±12 .09 1. M C P a d m i n i s t r a t i o n t i m e - MV s a m p l e t i m e 2 . U m b i l i c a l s a m p l e s ( U V , U A ) ; M C P a d m i n i s t r a t i o n t i m e - d e l i v e r y t i m e 3 . M e a n (X) o f t w o s e p a r a t e d e t e r m i n a t i o n s ± o n e s t a n d a r d d e v i a t i o n ( S . D . ) 4 . S i n g l e d e t e r m i n a t i o n 5 . F e t a l - m a t e r n a l r a t i o , U V MV + N o t i n c l u d e d 1 n s t a t i s t i c a l a n a l y s e s s i n c e b a b i e s w e r e l o s t t o f o l l o w - u p * , S m o k e r s 91 described by: y = 0.9540 x + 4.2991 3.3.3. Sheep P l a c e n t a l Transfer Study : Plasma MCP Determination Chromatograms obtained from blank and MCP-spiked sheep plasma e x t r a c t s were e s s e n t i a l l y i d e n t i c a l to those obtained with human plasma (see F i g s . 14a and b). Again, no i n t e r f e r e n c e was observed from endogenous plasma components f o r e i t h e r MCP or MAP. A s i n g l e plasma study i s included here only to show a p p l i c a b i l i t y of the developed c a p i l l a r y assay to the q u a n t i t a t i o n of MCP i n sheep plasma. The e l i m i n a t i o n k i n e t i c s of MCP i n the ewe (53) and the c h r o n i c a l l y c a t h e t e r i z e d fetus ( i n utero) are shown i n F i g . 15. The ewe received a lOmg I.V. i n f u s i o n of MCP (Reglan Injectable*^) v i a a maternal j u g u l a r v e i n over 3 minutes. Paired (simultaneous) blood samples were withdrawn from the ewe (femoral a r t e r y ) and f e t u s ( l a t e r a l t a r s a l vein) a t 1, 5, 15, 30, 60, 90, 120, 150 and 180 minutes. Plasmas were ext r a c t e d and d e r i v a t i z e d as d e t a i l e d i n s e c t i o n s 2.5 and 2.6.1. B, r e s p e c t i v e l y . The 25M c r o s s l i n k e d SE-54 column was used f o r MCP q u a n t i t a t i o n . The l i m i t of the assay ( v i z . , 4ng.mL~^) was. reached at 120 and 150 minutes f o r the ewe and the f e t u s , r e s p e c t i v e l y . Maternal plasma MCP e l i m i n a t i o n was observed to f o l l o w a biexponential decay described by the f o l l o w i n g general equation: 92 I S 1. MCP 2. MAP oi r--ei C'J oa c\i ro io 14, Representative chromatograms obtained from blank (a.) and MCP-spiked (b.) plasma e x t r a c t s on the 25m 5E-54 fused s i l i c a column. The spiked sample contained MCP, 139.58pg.yL - 1 and MAP, 138.89pg.viL~' (2yL i n j e c t i o n ) . Chromatographic co n d i t i o n s as reported i n F i g . 5 employing hydrogen as the c a r r i e r gas. 93 200, 20 40 60 80 100 120 140 160 TIME (min.) 15. A semi-log p l o t of the plasma p r o f i l e of MCP i n a pregnant ewe f o l l o w i n g a lOmg I.V. dose. (•—•) maternal plasma l e v e l s , (*—A) f e t a l plasma l e v e l s . The terminal e l i m i n a t i o n h a l f - l i v e s i n the ewe and fetus were c a l c u l a t e d to be 40 and 54 minutes r e s p e c t i v e l y . 94 C p = A e " a t + Be" where Cp i s the plasma concentration at time t . A and B are the i n t e r c e p t s of the a and g phases, r e s p e c t i v e l y . The parameters a and 3 represent the d i s t r i b u t i o n and terminal e l i m i n a t i o n r a t e constants, r e s p e c t i v e l y . The h a l f - l i f e of the a phase ( t ^ ) was c a l c u l a t e d to be 6 minutes i n d i c a t i n g r a p i d d i s t r i b u t i o n of MCP f o l l o w i n g the I.V. i n j e c t i o n . A terminal e l i m i n a t i o n h a l f - l i f e ( t r J of 40 minutes was -i> P .calculated f o r the e phase. Fetal plasma l e v e l s exceeded maternal MCP concentrations at 90 minutes and then followed a s i m i l a r e l i m i n a t i o n p a t t e r n . An e l i m i n a t i o n h a l f - l i f e (t, ) of 54 minutes was c a l c u l a t e d f o r the f e t u s . 95 4. DISCUSSION 4.1 Packed Column GLC-ECD 4.1.1 S t a t i o n a r y Phase S e l e c t i o n Numerous attempts to p o s i t i v e l y i d e n t i f y the source of the two i n t e r f e r i n g component peaks on 3% OV-225 ( F i g . 1) were unsuccessful. These i n t e r f e r i n g peaks are f e l t to be due to compounds extracted from p l a s t i c s since they could be r e a d i l y reproduced by e x t r a c t i n g p l a s t i c s and water ( d e i o n i z e d , d i s t i l l e d ) from a v a r i e t y of sources. However, even i f a t o t a l glass set-up was used f o r MCP e x t r a c t i o n and d e r i v a t i z a -t i o n t h i s i n t e f e r e n c e could not be e l i m i n a t e d ; the p o s s i b i l i t y e x i s t s that e i t h e r solvent and/or gases used i n the a n a l y t i c a l procedure may have contributed i n part to the observed i n t e r f e r i n g peaks. I t was not p o s s i b l e to continue use of OV-225 f o r MCP plasma a n a l y s i s at l e a s t at the desired concentration range of 4-40 ng.mL*1. A l a r g e number of s t a t i o n a r y phases with widely varying general s e l e c t i v i t i e s were i n v e s t i -gated i n attempts to accentuate p o l a r i t y d i f f e r e n c e s between MCP and the two i n t e r f e r i n g shoulder peak compounds (Table I ) . Of a l l the l i q u i d phases tested only 3% S i l a r - 9 C P provided the desired r e s o l u t i o n ( F i g . 2 ) . There were however, problems associated with the use of S i l a r - 9 C P and u l t i m a t e l y i t did not prove to be very useful f o r routine plasma (human, sheep) MCP a n a l y s i s . The f o l l o w i n g were some of the l i m i t a t i o n s a s s o c i a -ted with i t s use: 96 i ) i t took at l e a s t seven days to f u l l y c o n d i t i o n and respond i n a reproducible manner, i i ) i t e x h i b i t e d considerable batch to batch (commercial, i n - l a b ) v a r i a b i l i t y both i n peak c h a r a c t e r i s t i c s ( d i f f e r e n c e s i n peak height, width, degree of t a i l i n g ) and i n r e l a t i v e r e t e n t i o n times. This was most l i k e l y the r e s u l t of on-column adsorption, i i i ) i t had a very short l i f e span, l a s t i n g only about two weeks with r o u t i n e use. This was most probably due to column overload with n o n v o l a t i l e plasma components, a r a p i d rate of bleed g e n e r a l l y a s s o c i a t e d with h i g h l y p o l a r phases,as well as the s u s c e p t i b i l i t y of the l a t t e r to oxygen. In a d d i t i o n , the operating temperature (245° or 250°C) required to obtain reasonable MCP peak shape and r e t e n t i o n times was r e l a t i v e l y c l o s e to the operating maximum (275°C) which would a l s o tend to speed d e t e r i o r a t i o n , i v ) a n a l y s i s times were long (often 20-30 minutes) which i s not g e n e r a l l y d e s i r a b l e - f o r a r o u t i n e assay method. 4.1.2. D e r i v a t i z i n g Agent S e l e c t i o n Of a l l the f l u o r o a c y l a t i n g reagents t r i e d , HFBA remained the d e r i v a t i z i n g agent of choice. Reaction with TFAA d i d r e s u l t i n a d e r i v a t i v e (TFA-MCP) t h a t was w e l l resolved from the i n t e r f e r i n g component peaks on 3% OV-225 but the large decrease i n electron-capture response (- 80%) over the HFB-derivative made q u a n t i t a t i o n below 97 32ng.mL~l impossible. 4.1:3. I n v e s t i g a t i o n s of (HFB-MCP/diazepam, HFB-MAP) Area Ratio V a r i a b i l i t y During attempts t o use e i t h e r 3% OV-225 or 3% S i l a r - 9 C P f o r routi n e plasma MCP a n a l y s i s , considerable v a r i a b i l i t y i n the area r a t i o s of HFBrMCP/diazepam was observed w i t h i n and among samples. This v a r i a b i l i t y was f e l t to be due to e i t h e r on-column adsorption of MCP, adsorption losses onto glass s u r f a c e s , to i n s t a b i l i t y of the HFB-MCP d e r i v a t i v e i t s e l f or to combinations thereof. Each of these aspects wis examined. Two d i f f e r e n t s o l i d supports (Gas Chrom Q, Chromosorb 750) were test e d f o r on-column adsorption of MCP i n a d d i t i o n to the Chromosorb W HP normally used. Neither r e s u l t e d i n any improvement i n area r a t i o v a r i a b i l i t y (Table I I ) so Chromosorb W HP was r e t a i n e d . Chromosorb 750, rep o r t e d l y the most i n e r t support a v a i l a b l e , i n f a c t e x h i b i t e d severe adsorption problems which could not be overcome by priming the column with high MCP concentrations. The adsorption of p o l a r compounds, such as MCP, onto glass surfaces has been reported to r e s u l t i n s i g n i f i c a n t losses e s p e c i a l l y at low drug concentrations (Walle and Ehrsson, 1971). This then, could a l s o account f o r the considerable sample to sample area r a t i o v a r i a b i l i t y observed. Treatment of a l l glassware with 10% DMCS i n toluene to block p o t e n t i a l adsorption s i t e s , however, d i d not reso l v e t h i s problem. 98 Having f a i l e d to improve w i t h i n and between sample area r a t i o v a r i a b i l i t y through the use of a l t e r n a t i v e s o l i d supports and surface pre-treatment of glassware,the s t a b i l i t y of the HFB-MCP d e r i v a t i v e i t s e l f was examined. I t had been observed that when base concentrations stronger ( v i z . , 2N NaOH) than the 4% NH^OH normally used to n e u t r a l i z e the excess reagent (HFBA) were employed,no product was obtained (absence of a chromatographic MCP peak). The e f f e c t of NH^OH concentration (0.5-10%) on product recovery (HFB-MCP) had been studied during development of the o r i g i n a l MCP assay (Tarn, M.Sc. Th e s i s , 1978), with no s i g n i f i c a n t d i f f e r e n c e s i n area r a t i o s being reported. However, the MCP concentrations i n v e s t i g a t e d were some 10-25 times higher than those c u r r e n t l y being assayed. I t i s p l a u s i b l e then, that any base c a t a l y z e d h y d r o l y s i s (or degradation) of the HFB-MCP butyramide l i n k a g e t h a t may be o c c u r r i n g would be much more r e a d i l y observable and c o n t r i b u t e to a l a r g e r degree (percentage) of area r a t i o v a r i a b i l i t y at present MCP concentrations. I t can be observed from Table I I I t h a t n e u t r a l i z a t i o n w i t h 4% NH^OH.as opposed to l % , d i d r e s u l t i n approximately a two f o l d greater d e c l i n e i n area r a t i o s over the 20-24 hour period of study. I t had a l s o been observed t h a t i f the d e r i v a t i z e d organic l a y e r was allowed to remain i n contact with the n e u t r a l i z i n g aqueous NH^OH phase f o r periods of - 30 minutes or more,that the chromatographic response (area r a t i o , HFB-MCP/HFB-MAP) was reduced 80-90% over that of an 99 a l i q u o t of the same sample which had been immediately removed and stored i n a separate tube. Further, at concentrations < 16ng.mL~^ the peak f o r MCP was absent. Reports of i n s t a b i l i t y of HFBA-derivatives have a l s o appeared i n the l i t e r a t u r e . Leppard and Reid, 1978, s t a t e t h a t MCP may be unstable at a l k a l i n e pH (aqueous ammonia; s p e c i f i c g r a v i t y , 0.9). Ehrsson, et a l . , 1971, report the r a p i d degradation of h e p t a f l u o r o -b u t y r y l phenol e s t e r d e r i v a t i v e s i f allowed to remain standing on aqueous s o l u t i o n s with a pH of > 6.0. I n s t a b i l i t y of pentafluorobenzoyl d e r i v a t i v e s of ephedrine and 3, 4-dimethoxy amphetamine,when l e f t i n contact with 0.1N NH^OH f o r greater than 20 minutes*has a l s o been reported (Midha, et a l . , 1979). However, even r a p i d removal ( w i t h i n 2-5 minutes) of the organic phase from the aqueous NH40H n e u t r a l i z i n g l a y e r d i d not resolve the problem of d e c l i n i n g area r a t i o s with time, i n d i c a t i n g perhaps t h a t the postulated h y d r o l y s i s was s t i l l o c c u r r i n g w i t h i n the organic (benzene, toluene) l a y e r . The a d d i t i o n of c a t a l y s t s such as TEA (Walle and Ehrsson, 1970) or p y r i d i n e , during HFBA d e r i v a t i z a t i o n of primary and secondary amines as w e l l as a l c o h o l s and phenols i s g e n e r a l l y recommended. I t was found i n the c u r r e n t s t u d i e s t h a t the a d d i t i o n of e i t h e r 0.05M TEA or p y r i d i n e during d e r i v a t i z a t i o n r e s u l t e d i n a marked improvement i n area r a t i o r e p r o d u c i b i l i t y over the 20-24 hour period of study (Table I I I ) . V a r i a b i l i t y was l e s s with TEA than with p y r i d i n e (Table I I I ) most probably because p y r i d i n e causes some chromatographic disturbances e s p e c i a l l y at high e l e c t r o n - c a p t u r e s e n s i t i v i t i e s . O v e r a l l , the use of 0.05M TEA 100 during dertvatizati.on and n e u t r a l i z a t i o n with 4% NH40H r e s u l t e d i n the s m a l l e s t d e c l i n e i n area r a t i o s (Table III) and has subsequently been adopted with e x c e l l e n t r e s u l t s . I t i s p o s t u l a t e d that an a c i d c a t a l y z e d breakdown of the HFB-MCP butyramide bond may have been o c c u r r i n g ( w i t h i n the organic l a y e r ) as a r e s u l t of incomplete removal and/or n e u t r a l i z a t i o n of the HFB-acid formed during the h y d r o l y s i s of excess HFBA with water. Triethylamine then, through i t s a b i l i t y to accept the proton from the primary amino group on MCP,may prevent the formation of HFB-acid (or co n s i d e r a b l y reduce i t s concentration) such that i t remains i n the i o n i z e d form and thus p a r t i t i o n s more completely i n t o the aqueous NH^OH l a y e r during the h y d r o l y s i s - n e u t r a l i z a t i o n step. 4.2. Fused S i l i c a C a p i l l a r y Column GLC-ECD 4.2.1. Column S e l e c t i o n Two 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d columns were used f o r assay development and o p t i m i z a t i o n of chromatographic c o n d i t i o n s . The high e f f i c i e n c y of these c a p i l l a r y columns provided r e s o l u t i o n of MCP from i n t e r f e r i n g components s o l v i n g a serious problem.with the 3% OV-225 packed columns ( F i g . 1). Hexane and iso-octane were employed as i n j e c t i o n solvents i n these q u a l i t a t i v e studies to minimize the danger of phase s t r i p p i n g . One of the columns was i r r e v e r s i b l y damaged by the use of the aromatic solvents benzene and toluene. This p a r t i c u l a r column d e t e r i o r a t e d q u i t e r a p i d l y (over a period of about 3 weeks) as evidenced by increased MCP peak t a i l i n g and subsequent l o s s of response. 101 E a r l y i n 1982 c r o s s l i n k e d or bonded phases ( m e t h y l s i l i c o n e ; m e t h y l p h e n y l s i l i c o n e ) , coated onto s i l o x a n e deactivated fused s i l i c a became commercially a v a i l a b l e (Hewlett-Packard Co., J.and W. S c i e n t i f i c ) . These c a p i l l a r y columns e x h i b i t a high degree of i n e r t n e s s , e x c e l l e n t thermal s t a b i l i t y (325°-350°C) and low solvent e x t r a c t a b i l i t y (Freeman, 1981a; P l o t c z y k , 1982). This new t e c h n o l o g i c a l advance v i r t u a l l y e l i m i n a t e s phase s t r i p p i n g by aromatic solvents such as benzene and toluene which are required f o r good MCP s o l u b i l i t y . The a v a i l a b i l i t y of a c r o s s l i n k e d SE-54 column allowed development of the assay f o r r o u t i n e q u a n t i t a t i v e measurement of plasma MCP i n the d e s i r e d concentration range of 4-40ng.ml_"\ Resolution of MCP from endogenous plasma components i s good ( F i g s . 5 and 14) but requires a b a c k - e x t r a c t i o n technique as described i n s e c t i o n 2.5. A s i n g l e a l k a l i n e plasma e x t r a c t i o n d i d not remove endogenous components and i n t e r f e r e n c e with MCP was observed. The SE-54 column has been found to be very s t a b l e and t o y i e l d r e p r o d u c i b l e r e s u l t s . Minimal t a i l i n g has been observed with MCP on t h i s column as compared to a l l previous columns used f o r the separation and q u a n t i t a t i o n of t h i s drug. Routine r e p e t i t i v e i n j e c t i o n s of plasma e x t r a c t s are p o s s i b l e every 5-6 minutes without b a s e l i n e disturbances from l a t e e l u t i n g plasma components. The column has been very h e a v i l y used and s t i l l e x h i b i t s e x c e l l e n t chromatographic p r o p e r t i e s . The three glass c a p i l l a r y columns ( S i l a r - l O C , SP-2300, OV-225) te s t e d during packed column studies a l l e x h i b i t e d broad, severely t a i l i n g peaks f o r both MCP and diazepam, the i n t e r n a l standard. This was most l i k e l y due to inadequate surface d e a c t i v a t i o n ( f a i l u r e to remove Lewis acids and f u l l y d e a c t i v a t e , surface s i l a n o l groups) which 102 can r e s u l t i n surface adsorption and subsequent t a i l i n g . Fused s i l i c a columns have the advantage of being f r e e of m e t a l l i c oxides (Lewis a c i d s ) and r e q u i r e d e a c t i v a t i o n of only surface s i l a n o l f u n c t i o n s thus they tend to be considerably more i n e r t (Freeman, 1981d). The band spreading and t a i l i n g noted with the glass columns may a l s o have been due to the 3% OV-17 packing employed i n the Jennings s p l i t l i n e r used i n these t r i a l s . The p o s s i b l e reason f o r t h i s w i l l be discussed i n a l a t e r s e c t i o n . 4.2.2. Optimization of GLC-ECD Conditions 4.2.2.1. I n j e c t i o n Temperature: S p l i t Mode of Sample I n j e c t i o n The peak shape and response of the sample are, i n p a r t , dependent upon the temperature of the i n j e c t i o n port. The temperature of the s p l i t l i n e r should approach the b o i l i n g point of the higher-b o i l i n g components i n the sample i n order to achieve adequate sample v a p o r i z a t i o n and to avoid d i s c r i m i n a t i o n (Jennings, 1980a). Too high an i n j e c t i o n port temperature may r e s u l t i n sample decomposition with a subsequent l o s s i n s e n s i t i v i t y . D i s c r i m i n a t i o n against low b o i l i n g - p o i n t components i n the sample may a l s o occur at e x c e s s i v e l y high i n j e c t i o n temperatures, such t h a t these compounds vaporize much more q u i c k l y and are p r e f e r e n t i a l l y vented p r i o r to sample s p l i t t i n g and t r a n s f e r onto the column (Schomburg, et a l . , 1978). I f the i n j e c t i o n port temperature i s too low some band broadening w i l l occur as the sample w i l l vaporize more slowly and enter the column over a longer period of time. This has been observed but i s minimal compared to the e f f e c t s seen on packed columns. Schomburg, e t , a l . , 1977, 103 emphasize t h a t with s p l i t sampling, the i n f l u e n c e of the time of sample i n t r o d u c t i o n and t r a n s f e r i n t o the column on f i n a l band width i s n e g l i g i b l e because of the high c a r r i e r gas flow rates between the c a r r i e r gas i n l e t and the s p l i t t i n g region. D i s c r i m i n a t i o n against high b o i l i n g - p o i n t s o l u t e s i n the sample may a l s o occur i f the i n j e c t i o n temperature i s too low. These compounds w i l l be incompletely vaporized at temperatures below t h e i r b o i l i n g points so the amount introduced to the column i s reduced. While using the s p l i t mode of sampling then, i t i s necessary to s e l e c t an i n j e c t i o n temperature which w i l l minimize d i s c r i m i n a t i o n e f f e c t s that may otherwise occur with e i t h e r temperature extreme. In g e n e r a l , the i n j e c t i o n port temperature i s higher than that of the column. In t h i s study, however, an i n j e c t i o n temperature of 220°C was chosen while the column was maintained at 235°C. No s i g n i f i c a n t d i f f e r e n c e s i n area r a t i o s were seen by varying the i n j e c t i o n temperature between 210° and 240°C ( F i g . 3). Area r a t i o s (HFB-MCP/HFB-MAP) were somewhat higher at 220°C so t h i s temperature was s e l e c t e d f o r plasma MCP q u a n t i t a t i o n . The higher thermal c o n d u c t i v i t y of hydrogen, the c a r r i e r gas, may have provided more complete v a p o r i z a t i o n at temperatures lower than can be used f o r other gases. For example, i n t e s t s with helium (which i s more dense than hydrogen) an i n j e c t i o n temperature of 250°C r e s u l t e d i n optimal area r a t i o s (the column temperature was 235°C). 104 4.2.2.2. S e l e c t i o n of Column Temperature Column temperature can be a major f a c t o r a f f e c t i n g the e f f i c i e n c y of an assay as i t has a d i r e c t e f f e c t on peak shape and a n a l y s i s time. Increasing the column temperature, f o r example, r e s u l t s i n a reduced a n a l y s i s time but a l s o decreases r e s o l u t i o n . The thermal s t a b i l i t y of the column i s a l s o an important c o n s i d e r a t i o n . I t i s necessary then, to weigh a l l of these f a c t o r s ( r e s o l u t i o n , column thermal s t a b i l i t y , a n a l y s i s time) and to choose,if p o s s i b l e , a temperature which optimizes each of them. Column temperatures of 235°, 240° and 245°C were examined. Although higher temperatures were d e s i r a b l e from the standpoint of reduced MCP t a i l i n g and a sh o r t e r a n a l y s i s time, a column temperature of 235°C was chosen i n order to obtain r e s o l u t i o n of MCP from an extraneous peak which was present i n some samples ( F i g . 4 ). This temperature s t i l l r e s u l t s i n a very short a n a l y s i s time {- 6 minutes) which i s d e s i r a b l e ' i n a r o u t i n e assay method. 4.2.2.3. S e l e c t i o n of C a r r i e r Gas and Flow Rate The most e f f i c i e n t c a r r i e r gas and i t s optimal flow rate (^ 0p t) are g e n e r a l l y determined from Van Deemter p l o t s of height equivalents to a t h e o r e t i c a l p l a t e (h) versus the average l i n e a r c a r r i e r gas v e l o c i t y (vi). The smaller the value of h, the more e f f i c i e n t the column at that p a r t i c u l a r flow r a t e . 105 Height e q u i v a l e n t , to a t h e o r e t i c a l p l a t e (h) values were c a l c u l a t e d (h = L/n; where L i s the column length i n mm and n the t o t a l number of p l a t e s ) f o r both He and H 2 and found to be 0.75mm and 1.03mm r e s p e c t i v e l y . These c a l c u l a t i o n s i n d i c a t e t h a t He i s approximately 25% more e f f i c i e n t than H 2 i n t h i s p a r t i c u l a r a n a l y s i s (MCP was used as the reference peak i n the c a l c u l a t i o n s ) . Even though H 2 was l e s s e f f i c i e n t when considered on t h i s basis or on the basis of the t o t a l number of pl a t e s (n) (n = 24,180 and 33,322 f o r H 2 and H , r e s p e c t i v e l y ) i t was chosen as the c a r r i e r gas f o r the assay, s i n c e r e s o l u t i o n of MCP from the i n t e r n a l standard, MAP, and plasma components was not a problem ( F i g . 5). The advantage of using H 2 i s that i t r e s u l t s i n a considerably s h o r t e r a n a l y s i s time (- 38%) ; such a l o s s i n e f f i c i e n c y was deemed acceptable. I f however, the e f f i c i e n c y of these two c a r r i e r gases i s viewed on the basis of the number of p l a t e s generated per second ( n / t ^ ; where n i s the t o t a l number of p l a t e s and t ^ the r e t e n t i o n time of MCP i n se c o n d s ) , i t was found that H 2 was about 15% more e f f i c i e n t than He (H 2: 130 p l a t e s . s - 1 ; He: 110 p l a t e s . s - 1 ) . Freeman, 1981b, st a t e s t h a t "since a n a l y s i s time as well as r e s o l u t i o n enter i n t o the determination of column performance, the p l a t e s / second concept may be the best way of c h a r a c t e r i z i n g system performance." Jennings, 1980b, a l s o s t a t e s t h a t "viewed i n t h i s l i g h t ' ( i . e . p l a t e s / second)', hydrogen i s the c a r r i e r gas of choice." One of the major advantages of using H 2 i s that as the c a r r i e r gas v e l o c i t y (£•) i s increased above i t s y n n t » the increase i n 106 h (or l o s s of e f f i c i e n c y ) per u n i t increases i n y i s f a r l e s s than f o r more dense c a r r i e r gases ( v i z . , N2 and He) i . e . H2 e x h i b i t s a f l a t t e r Van Deemter curve (Jennings, 1980b; Freeman, 1981b). A f l a t t e r Van Deemter curve means th a t l e s s e f f i c i e n c y i s s a c r i f i c e d as the c a r r i e r gas v e l o c i t y i s increased (and a n a l y s i s times are shortened); H2 emerges as the best with He a good second. Also l e s s dense gases, such as H2, can be operated at higher v e l o c i t i e s because l e s s time i s required to e s t a b l i s h e q u i l i b r i u m (of the s o l u t e molecules between the molecules i n the l i q u i d phase and those i n the gas phase) at each t h e o r e t i c a l p l a t e (Jennings, 1980b). The small l o s s i n e f f i c i e n c y ( r e s o l u t i o n ) a s s o c i a t e d with a higher v e l o c i t y then, i s .usually outweighed by the advantages of a s h o r t e r a n a l y s i s time. Flow rates of 0.5 - lOmL.min."^ are t y p i c a l l y used f o r c a p i l l a r y columns (Freeman, 1981c), t h i s v e l o c i t y i s considerably lower than those g e n e r a l l y used f o r packed columns (20 - 60mL.min~^). In t h i s study flows of 0.5 and l.OmL.min"^ were t e s t e d ( F i g s . 6a, b). A column flow of l.OmL.min ^ produced the highest response and was t h e r e f o r e chosen f o r q u a n t i t a t i v e plasma MCP a n a l y s i s . This t r a n s l a t e s to a y. of 63 cm.s-^ and 40 cm.s"^ f o r H2 and H e , r e s p e c t i v e l y (y = L/Tm; where y i s the average l i n e a r v e l o c i t y i n cm.s~\ L the column length i n cm (SE-54 column = 2500 cm), and Tm the r e t e n t i o n time of an unretained peak i n seconds). The l e a d i n g edge of the solvent f r o n t was used f o r Tm e s t i m a t i o n . . I d e a l l y more column flow rates should have been t e s t e d and a Van Deemter p l o t (h versus y) constructed. From t h i s p l o t the optimal flow ( f o r H ? the c a r r i e r gas) f o r MCP a n a l y s i s on the 107 c r o s s l i n k e d SE-54 column could have been determined. In a d d i t i o n , a p l o t of p l a t e s . s - 1 versus the corresponding average l i n e a r v e l o c i t i e s (y) determined at each column flow ( I . e . , 0.5, l.OmL.min - 1, etc.) could have been made. U t i l i z i n g both of these p l o t s then, i t may have been p o s s i b l e to s e l e c t an even higher c a r r i e r gas flow r a t e to shorten the a n a l y s i s time f u r t h e r but with minimal l o s s i n e f f i c i e n c y ( r e s o l u t i o n ) . 4.2.2.4. S e l e c t i o n of Make-Up Gas Flow Rate The small c a r r i e r gas flows associated with the use of c a p i l l a r y columns ( v i z . , 0.5 - lOmL.min - 1) are not s u f f i c i e n t to adequately maintain c e l l volume or to e f f e c t i v e l y sweep the e l e c t r o n capture detector c e l l (ECD). I t i s t h e r e f o r e , necessary to add a make-up (or scavenger) gas to the c a r r i e r gas flow at the entrance of the column i n t o the ECD. The ECD i s a concentration-dependent detector so i t s s e n s i t i v i t y i s i n v e r s e l y p r o p o r t i o n a l to flow. A low make-up flow would,therefore, appear to be b e t t e r as i t would provide higher s e n s i t i v i t y . However, i t may be i n s u f f i c i e n t to e f f e c t i v e l y sweep the c e l l r e s u l t i n g i n a mixing of s o l u t e peaks. High make-up flows on the other hand, reduce the concentration of s o l u t e i n the detector per. u n i t time r e s u l t i n g i n . a drop i n s e n s i t i v i t y . I t i s a l s o necessary that the end of the c a p i l l a r y column be located i n an area of high gas v e l o c i t y to minimize band broadening as a r e s u l t of e x t r a column volumes. 108 When using a ECD then, both, optimal s e n s i t i v i t y and minimal band width cannot be obtained a t the same make-up flow. Depending upon the requirements of the assay, the make-up gas flow r a t e should be se l e c t e d to give the best d e s i r e d r e s u l t s (Freeman, 1981c). Make-up flow rates of 20, 30, 40, 50 and 60mL.min~^ were teste d ( F i g s . 7 and 8). A flow r a t e of 60mL.min~^ provided the highest s e n s i t i v i t y (and the lowest S/N r a t i o ) with minimal band spreading and . was ther e f o r e chosen f o r the q u a n t i t a t i o n of MCP i n human and sheep plasma. This make-up gas flow r a t e f a l l s w i t h i n the 20-60mL.min~1 range recommended by the instrument manufacturer (Hewlett-Packard Co., Avondale, PA.). 109 4.2.2.5. S p l i t L i n e r s : Jennings Tube, Fused S i l i c a I n s e r t The low c a r r i e r gas flow r a t e s a s s o c i a t e d w i t h high r e s o l u t i o n c a p i l l a r y columns req u i r e s the use of s p e c i a l l y designed gas chromatographic i n l e t systems. I n l e t s p l i t l i n e r s or s p l i t t e r s are one of the s i m p l e s t and most popular means of reducing e x t r a column volumes as s o c i a t e d with the i n j e c t i o n port and f u n c t i o n to avoid the d e p o s i t i o n of broad bands on the column. A number of s p l i t t e r c o n f i g u r a t i o n s e x i s t and have been discussed and reviewed (Jennings, 1975, 1980a; Schomburg, et a l . , 1977). Glass or fused s i l i c a are the recommended c o n s t r u c t i o n m a t e r i a l s as exposure of the sample to metal surfaces may r e s u l t i n c a t a l y t i c rearrangement or degradation of s e n s i t i v e samples. Regardless of c o n f i g u r a t i o n (Freeman, 1981c; Jennings, 1980a) a l l s p l i t t e r s are designed t o : i ) r e s t r i c t the amount of sample en t e r i n g the column (to prevent overload) by s p l i t t i n g the i n l e t flow stream i n t o two unequal p o r t i o n s , the smaller of which i s d i r e c t e d onto the column and the l a r g e r vented v i a the s p l i t flow vent, i i ) permit r a p i d f l u s h i n g of the i n j e c t i o n chamber so that the sample on the column i s followed by pure c a r r i e r gas. This i s l i k e l y the s p l i t t e r ' s most important f u n c t i o n since f a i l u r e to do so would cause e x p o n e n t i a l l y d i l u t e d sample to enter the column r e s u l t i n g i n severe band broadening (Jennings, 1980a). n o i i i ) provide e f f i c i e n t heat t r a n s f e r to the i n j e c t e d sample so t h a t i t i s r a p i d l y and completely vaporized. Incomplete v a p o r i z a t i o n can r e s u l t i n d r o p l e t or aerosol formation which can have a d e l e t e r i o u s e f f e c t on the l i n e a r i t y and r e p r o d u c i b i l i t y of the s p l i t t e r , i v ) provide thorough mixing (homogenization) of the vaporized sample so that the p o r t i o n e n t e r i n g the column i s r e p r e s e n t a t i v e of the t o t a l sample, v) prevent n o n v o l a t i l e m a t e r i a l s such as plasma components from e n t e r i n g and contaminating the column. This i s most often accomplished by use of a small s i l a n i z e d g l a s s - or quartz-wool plug and/or a small amount of column packing (e.g. 2% OV-1, 37o OV-17) i n the s p l i t l i n e r . A term f r e q u e n t l y encountered i n d i s c u s s i o n s on i n l e t s p l i t t e r s i s l i n e a r i t y . This r e f e r s to the degree of accuracy achieved as the s p l i t t e r d i r e c t s only a p o r t i o n of the i n j e c t e d sample onto the column (Jennings, 1980a). S p l i t t e r s with poor l i n e a r i t y demonstrate d i s c r i m i n a t i o n and may p r e f e r e n t i a l l y d i s c a r d or emphasize one component i n the i n j e c t e d sample over another. For example, based on i n l e t temperature h i g h - b o i l i n g components may be r e t a i n e d while l o w - b o i l i n g components are vented or v i c e versa. The nature of the s p l i t ( i . e . d i s c r i m i n a t i o n ) may a l s o be i n f l u e n c e d by d i f f e r e n c e s i n molecular weights, component c o n c e n t r a t i o n s , p o l a r i t y , the s p l i t r a t i o , volume i n j e c t e d and i n l e t pressure (Freeman, 1981c). These are r e f e r r e d to as i n l e t r e l a t e d parameters and can be minimized by choosing the proper i n i n l e t ( s p l i t t e r ) c o n f i g u r a t i o n and o p t i m i z i n g i t f o r a given a n a l y s i s (Freeman, 1981c). Jennings, 1980a, s t a t e s that " i g n o r i n g sample composition, then, the two f a c t o r s that e x e r c i s e the greatest e f f e c t on s p l i t t e r l i n e a r i t y are the s p l i t r a t i o (or flow v e l o c i t y through the s p l i t t e r ) and the temperature." In t h i s p r o j e c t the e f f e c t of i n j e c t i o n temperature (210°, 220°, and 240°C) r e s u l t e d i n no marked d i f f e r e n c e s i n HFB-MCP/ HFB-MAP area r a t i o s ( F i g . 3). A temperature of 220°C, however, d i d y i e l d a somewhat higher response and was t h e r e f o r e chosen f o r subsequent s t u d i e s . The somewhat lower area r a t i o s obtained at the other two i n j e c t i o n temperatures ( v i z . , 210° and 240°C) may i n d i c a t e p r e f e r e n t i a l venting of MCP and r e t e n t i o n of the i n t e r n a l standard, MAP. The e f f e c t of s p l i t r a t i o on the l i n e a r i t y of the s p l i t l i n e r s used could not be f u l l y evaluated. Operation at s p l i t r a t i o s ( i n l e t flow/column flow) greater than 30:1 was not p o s s i b l e s i n c e a 30:1 s p l i t was required to achieve MCP q u a n t i t a t i o n i n the d e s i r e d 4 - 40ng.mL~1 standard curve c o n c e n t r a t i o n range. S p l i t r a t i o s below 30:1 were test e d ( v i z . , 10:1, 20:1) but r e s u l t e d i n c o n s i d e r a b l y more v a r i a t i o n i n area r a t i o s . More band broadening and MCP peak t a i l i n g were a l s o observed;this may have been due to lower l i n e a r c a r r i e r gas v e l o c i t i e s through the s p l i t t e r r e s u l t i n g i n d e p o s i t i o n of the sample onto the column over a longer period of time as w e l l as to an increased on-column load ( e s p e c i a l l y at a 10:1 s p l i t ) . A 10:1 s p l i t a l s o r e s u l t e d i n a l o s s of i n l e t pressure on one occasion (fused s i l i c a l i n e r ) and an i n t e r r u p t i o n of column flow ; t h i s could r e s u l t i n column damage at 112 operating temperatures. Based on these observations, then, a s p l i t r a t i o of 30:1 was s e l e c t e d f o r a l l subsequent MCP analyses. S p l i t l i n e r s may be used i n e i t h e r the packed or unpacked c o n f i g u r a t i o n (see diagram, s e c t i o n 2.8.4.2. ( 8 ) ) . The packing may includ e such m a t e r i a l s as glass beads, conventional chromatographic column packing (e.g. 2% OV-1, 3% OV-17) or s i l a n i z e d g l a s s - or quartz - wool (Schomburg, et a l . , 1977). The use of the s p l i t l i n e r with or without packing w i l l depend l a r g e l y on the nature of the sample and should be matched to i t s a n a l y t i c a l requirements. For example, with high b o i l i n g point compounds, such as s t e r o i d s (Freeman, 1981c), i t may be necessary to use a packed s p l i t l i n e r . The packing provides a d d i t i o n a l heat c a p a c i t y to the l i n e r and thereby minimizes s p l i t d i s c r i m i n a t i o n by ensuring complete sample v a p o r i z a t i o n . With v o l a t i l e compounds, however, a packed l i n e r can r e s u l t i n a broadening of sample component peaks due to m u l t i p a t h d i f f u s i o n through the packing m a t e r i a l (Freeman, 1981c; Jennings, 1980a). This phenomena was observed i n the present p r o j e c t while using a Jennings l i n e r packed with 2% OV-1 and r e s u l t e d i n severe band spreading of MCP, MAP, and prazepam as i l l u s t r a t e d i n F i g . 9a. Removal of the 2% OV-1 packing and replacement with a small s i l a n i z e d g l a s s wool plug to prevent n o n v o l a t i l e components from contaminating the column r e s u l t e d i n a marked improvement i n the peak shape of a l l three compounds ( F i g . 9b). (Note: Some band broadening and/or peak t a i l i n g may r e s u l t from i n t e r a c t i o n (adsorption) of the sample with a c t i v e s i t e s w i t h i n the packing material (Schomburg, et . a l . , 1977). Based 113 on these observations the Jennings s p l i t l i n e r was used i n the unpacked c o n f i g u r a t i o n (see s e c t i o n .2,8.4.2.(8)) f o r the balance of the p r o j e c t . The fused s i l i c a i n s e r t was a l s o t e s t e d i n the packed and unpacked c o n f i g u r a t i o n s (see s e c t i o n .2.8.4.2. ( 8 ) ) . When used without packing q u a n t i t a t i o n of the lower points on the standard curve ( v i z . , 4, 8, 16ng.mL _ 1) was not p o s s i b l e a t s p l i t r a t i o s above 10:1. Considerable area r a t i o (HFB-MCP/HFB-MAP) v a r i a t i o n was al s o observed at a 10:1 s p l i t . Packing the s i l i c a i n s e r t with a " t i g h t " s i l a n i z e d glass wool plug permitted operation a t a 30:1 s p l i t and provided r e p r o d u c i b l e q u a n t i t a t i o n of the complete MCP standard curve range (4-40ng.mL _ 1). The u n r e s t r i c t e d c o n f i g u r a t i o n of the fused s i l i c a l i n e r (compared to the Jennings) and i t s small o v e r a l l i n s i d e diameter (2mm) q u i t e l i k e l y r e s u l t s i n high l i n e a r c a r r i e r gas v e l o c i t i e s through the c a p i l l a r y i n l e t . Because of t h i s , i t i s very p o s s i b l e t h a t a large part o f the i n j e c t e d sample was vented p r i o r to complete v a p o r i z a t i o n . The a d d i t i o n of a " t i g h t " s i l a n i z e d g l a s s wool plug provides added heat c a p a c i t y to the l i n e r ensuring more complete v a p o r i z a t i o n o f the sample components and b e t t e r homogenization Schomburg, et a l . , 1977) thereby reducing the " d i s c r i m i n a t i o n " ( i .e., i n a b i l i t y to q u a n t i t a t e below 24ng.mL~1 MCP l e v e l s ) observed i n i t s absence. The fused s i l i c a l i n e r was used i n the packed c o n f i g u r a t i o n during the balance of t h i s p r o j e c t . 114 Both s p l i t l i n e r s , the Jennings tube i n the unpacked c o n f i g u r a t i o n and the fused s i l i c a i n s e r t packed with a " t i g h t " s i l a n i z e d g l a s s wool plug were found to be l i n e a r (using a 30:1 s p l i t r a t i o ) over the concen-t r a t i o n range ( F i g . 13) stu d i e d (4-40ng.mL"^) and could be used i n t e r -changeably w i t h very s i m i l a r r e s u l t s ( F i g . 10). These two s p l i t l i n e r c o n f i g u r a t i o n s are r e a d i l y a v a i l a b l e (Hewlett-Packard Co., Avondale, PA.) and have been recommended over a v a r i e t y of others (Schomburg, et a l . , 1977). Both can be r e a d i l y removed from the gas chromatograph p f o r easy c l e a n i n g and repacking. The Pyrex Jennings tube required surface d e a c t i v a t i o n with 10% DMCS i n toluene p r i o r to use otherwise severe MCP peak t a i l i n g was observed. The fused s i l i c a l i n e r d i d not g e n e r a l l y r e q u i r e t h i s surface pre-treatment ( s i l a n i z a t i o n ) . 4.2.2.6. I n j e c t i o n Mode: S p l i t , S p l i t l e s s The s p l i t l e s s and s p l i t modes of c a p i l l a r y column sample i n j e c t i o n were test e d during the course of t h i s p r o j e c t . The s p l i t l e s s mode r e s u l t e d i n very complex chromatograms with m u l t i p l e peaks and p r o t r a c t e d solvent f r o n t s . Metoclopramide was not resolved from endogenous plasma components. Because of the g e n e r a l l y u n s a t i s f a c t o r y p r e l i m i n a r y r e s u l t s obtained, t h i s sampling technique was not examined f u r t h e r . This sampling mode, with i t s more s t r i n g e n t o p e r a t i o n a l requirements, has been thoroughly reviewed by a number of workers i n the high r e s o l u t i o n gas chromatography f i e l d (Freeman, 1981c, 1981e; Jennings, 1980a; Schomburg, et a l . , 1977). 115 The s p l i t mode of sample i n j e c t i o n by comparision provided very c l e a n , uncomplicated chromatograms ( F i g s . 5 , 14) with MCP being well resolved from plasma components. Of the two methods t e s t e d i t was the e a s i e s t to use from the standpoint of o p e r a t i o n a l c o n s i d e r a t i o n s and good r e s u l t s were r e l a t i v e l y easy to o b t a i n . Proper s e l e c t i o n of s p l i t l i n e r c o n f i g u r a t i o n s (see s e c t i o n 4.2.2.5) coupled with a s e n s i t i v e e l e c t r o n capture detector provided reproducible q u a n t i t a t i v e r e l i a b i l i t y over the d e s i r e d 4-40ng.mL~1 plasma MCP concentration range (see c o e f f i c i e n t s of v a r i a t i o n i n Table VI and F i g . 13). 4.2.2.7 S e l e c t i o n of I n j e c t i o n Solvent Four i n j e c t i o n solvents were used during the course of t h i s p r o j e c t : iso-octane, hexane, benzene and toluene. Iso-octane and hexane were found to be g e n e r a l l y u n s a t i s f a c t o r y . Both e x h i b i t e d poor s o l u b i l i t y and e x t r a c t a b i l i t y properties.towards MCP and as such y i e l d e d extremely v a r i a b l e and u n r e l i a b l e chromatographic r e s u l t s ( e s p e c i a l l y i s o - o c t a n e ) . Hexane, however, was used with high MCP concentrations (0.5'u.g.mL"'') f o r q u a l i t a t i v e c a p i l l a r y assay development employing two 12m x 0.2mm I.D. methyl s i l i c o n e f l u i d columns. The purchase of a c r o s s l i n k e d fused s i l i c a c a p i l l a r y column (SE-54) permitted the use of aromatic sol vents such as benzene and toluene with minimal r i s k of phase s t r i p p i n g . Both benzene and toluene e x h i b i t good MCP s o l u b i l i t y and e x t r a c t i b i l i t y c h a r a c t e r i s t i c s and allowed development of the assay f o r plasma MCP q u a n t i t a t i o n , i n the d e s i r e d concentration range of 4-40ng.mL~1, Benzene had been s u c c e s s f u l l y 116 used as the e x t r a c t i n g and d e r i v a t i z i n g solvent i n the l a b since development of the MCP packed column GLC-ECD assay method i n 1977 (Tarn, M.Sc. Th e s i s , 1978; Tarn and Axelson, 1978; Tarn, et.al.» 1979). Schomburg, et a l . , 1978, 1981, however, recommend the use of higher b o i l i n g point (b.p.) solvents to minimize d i s c r i m i n a t i o n e f f e c t s i n the s p l i t l i n e r based on d i f f e r e n c e s i n sample component v o l a t i l i t i e s and molecular weights. On t h i s b a s i s . t h e n , toluene which has a somewhat higher b o i l i n g p o i n t (110°C) than benzene (b.p. = 80°C) was chosen as the d e r i v a t i z i n g and i n j e c t i n g solvent f o r MCP a n a l y s i s by fused s i l i c a c a p i l l a r y GLC-ECD. 4.2.2.8. S e l e c t i o n of Inte r n a l Standard Diazepam, prazepam and m a p r o t i l i n e (MAP) were t e s t e d as i n t e r n a l standards during the development of the fused s i l i c a c a p i l l a r y assay method. Diazepam, although w e l l resolved from MCP (R = 5.79 on the SE-54 column), was not chosen as i t was p o s s i b l e t h a t p a t i e n t s involved i n the MCP p l a c e n t a l t r a n s f e r study may a l s o have received t h i s drug. Prazepam was s e l e c t e d as a s u i t a b l e a l t e r n a t i v e since i t i s not yet a v a i l a b l e f o r th e r a p e u t i c use i n Canada. This agent was a l s o w e l l resolved from MCP ( F i g . 5b) but d i d not y i e l d standard curves as good as those obtained with MAP. I t was included as a secondary i n t e r n a l standard during o p t i m i z a t i o n of the assay method. M a p r o t i l i n e (I) was s e l e c t e d as the f i n a l i n t e r n a l standard f o r plasma MCP q u a n t i t a t i o n . This drug (a t e t r a c y c l i c antidepressant) although s t r u c t u r a l l y unrelated to MCP ( I I ) f u l f i l l s some of the I 117 d e s i r e d p r o p e r t i e s o f an i d e a l i n t e r n a l s t a n d a r d : i ) l i k e MCP i t i s a v a i l a b l e as the h y d r o c h l o r i d e s a l t and t h e r e f o r e undergoes t he same e x t r a c t i o n s t e p s (Scheme I) i i ) i t p o s s e s s e s a se c o n d a r y amine f u n c t i o n which l i k e t h e p r i m a r y amine o f MCP undergoes d e r i v a t i z a t i o n w i t h HFBA i i i ) i t s e l e c t r o n c a p t u r e r e s p o n s e i s s i m i l a r t o t h a t o f MCP r e s u l t i n g i n an a r e a r a t i o o f = 1.0 ( F i g . 13) a t e q u i v a l e n t c o n c e n t r a t i o n s (l.OmL o f MCP.HCL.H2O, = 0.04yg.mL _ 1 e q u i v a l e n t t o base (= 139 p g . y L " 1 ) ; 0.1 mL o f MAP.HCL,= O.Ayg.mL" 1 e q u i v a l e n t t o base (= 1 3 8 p g . y L - 1 ) i v ) f t e x h i b i t s no a p p a r e n t c h e m i c a l i n t e r a c t i o n s w i t h MCP and l i k e t h e l a t t e r has good s t a b i l i t y f o l l o w i n g d e r i v a t i z a t i o n (> 4 days a t - 2 0 ° C ) . The use o f MAP as i n t e r n a l s t a n d a r d r e s u l t e d i n good s t a n d a r d c u r v e c o r r e l a t i o n s ( F i g . 1 3 ) and a s h o r t a n a l y s i s time (= 6.0 m i n u t e s ) Maprotiline (MAP) Metoclopramide (MCP) CD ( I D 4.2.2.9. O p t i m i z a t i o n o f HFBA D e r i v a t i z a t i o n R e a c t i o n Time f o r MCP and MAP The d e r i v a t i z a t i o n o f MCP and MAP w i t h HFBA a t 55°C i n t h e 118 presence of the c a t a l y s t t r i e t h y l a m i n e (0.05M) was found to be r a p i d and r e l a t i v e l y constant. No s i g n i f i c a n t d i f f e r e n c e s i n response (area r a t i o , HFB-MCP/HCB-MAP) were observed between 20 and 180 minutes ( F i g . 11). A r e a c t i o n time of 60 minutes was chosen f o r a l l subsequent MCP quan-t i t a t i o n s t u d i e s to ensure complete d e r i v a t i z a t i o n . Borga and G a r l e , 1972, a l s o r e p o r t a 60 minute r e a c t i o n time (at 63°C) f o r the d e r i v a t i z a t i o n of MAP with HFBA. They used hexane with N, N' - dimethylformamide as the d e r i v a t i z i n g s o l v e n t ; p y r i d i n e was added as the c a t a l y s t . Geiger, et a 1 ., 1975 report an optimal incubation time of 90 minutes, however, no s i g n i f i c a n t d i f f e r e n c e s i n t h e i r area r a t i o data ( m a p r o t i l i n e / n o r t r i p t y l i n e ) are apparent from 60-180 minutes. The r e a c t i o n with HFBA was c a r r i e d mt at 70°C i n a c e t o n i t r i l e - n - h e p t a n e without a c a t a l y s t . Tarn (M-Sc. Th e s i s , 1978) studied the r e a c t i o n k i n e t i c s of MCP i n benzene (at 55°C) i n the absence of a c a t a l y s t . No s i g n i f i c a n t d i f f e r e n c e s i n area r a t i o s (HFB-MCP/diazepam) were reported over the 0-60 minute period of i n c u b a t i o n . A 20 minute r e a c t i o n time was chosen f o r subsequent s t u d i e s . Gas chromatographic mass spectrometric (GC-MS) a n a l y s i s to determine the i d e n t i t y of the HFB-derivatives formed with MCP and MAP was not conducted during the course of t h i s p r o j e c t . Mono-substitution of the primary amine f u n c t i o n a l group on MCP has been reported by Tarn and Axelson, 1978, f o l l o w i n g e l e c t r o n impact and chemical i o n i z a t i o n GC-MS. Ross-Lee, e t ; a ! . , r e p o r t monoheptafluorobutyryl d e r i v a t i v e s f o r 119 both MCP and MAP f o l l o w i n g chemical i o n i z a t i o n GC-MS. They used HFBI rather than HFBA i n t h e i r MCP assay method. 4.3 Human Placental T r a n s f e r Study Metoclopramide with i t s low molecular weight and high l i p i d s o l u -b i l i t y has been postulated to be t r a n s f e r r e d across the placenta subse-quent to maternal a d m i n i s t r a t i o n (Smith and S a l t e r , 1980; Schulze-Del-r i e u , 1981), however, there are no reports on t h i s t o p i c i n the l i t e r a -t u r e . This double-blind study c o n c l u s i v e l y demonstrates that MCP does undergo maternal-fetal exchange (Table V I I ) . With the exception of pat i e n t 22S, UA metoclopramide l e v e l s were somewhat lower than those i n the u m b i l i c a l v e i n (UV) (Table V I I ) . This arterio-venous d i f f e r e n c e most probably i n d i c a t e s d i l u t i o n i n the f e t a l c i r c u l a t i o n volume, some uptake by f e t a l t i s s u e s ( d i s t r i b u t i o n , binding) and, perhaps, f i r s t - p a s s e f f e c t i n the f e t a l l i v e r . Since f e t a l concentrations ( u m b i l i c a l cord: UV, UA) i n baby 12S were c l o s e to maternal MCP l e v e l s (Table V I I ; F/M concentration ratio=0.93) i t would be valuable i n future studies to obtain one or two i n f a n t blood samples f o l l o w i n g d e l i v e r y to see i f neonatal l e v e l s exceed maternal concentrations at a l a t e r time i n t e r v a l . This has been observed f o r procainamide, a c l o s e s t r u c t u r a l analogue of MCP, at 8 hours a f t e r b i r t h (Lima et , 1978). A l e s s i n v a s i v e procedure would i n v o l v e cumu-l a t i v e u r i n e c o l l e c t i o n from the neonate f o r a s u i t a b l e time period a f t e r b i r t h (e.g. 48 hours). This type of study would provide considerably more information about the pe r s i s t e n c e of MCP i n the neonate. 120 Although one-point plasma drug determinations at b i r t h are the usual method of placental t r a n s f e r study i n humans they provide only l i m i t e d pharmacokinetic information (Krauer and Krauer, 1977; Levy, 1981; Waddell and Marlowe, 1981). One must be c a r e f u l , t h e r e f o r e , not to draw any conclusions about the extent of drug t r a n s f e r between mother and fetus since e q u i l i b r i u m between the two may not yet have been reached at the time of cord sampling ( i . e . d e l i v e r y ) . The number of p a t i e n t s (9) r e c e i v i n g MCP i n t h i s study was i n s u f f i c i e n t to enable c o n s t r u c t i o n of composite maternal and f e t a l con-c e n t r a t i o n - t i m e p r o f i l e s . I t was not p o s s i b l e i n t h i s instance then, to f i t the data to any of the k i n e t i c s i mulations proposed by Levy and Hayton, 1973, or Waddell and Marlowe, 1981. No adverse e f f e c t s were noted i n any of the i n f a n t s r e c e i v i n g MCP as a r e s u l t of placental t r a n s f e r . The Apgar scores between the treated and untreated groups were not s i g n i f i c a n t l y d i f f e r e n t (Table V). This observation i s i n agreement with previous Apgar assessments i n s t u d i e s examining MCP's antiemetic and g a s t r i c emptying e f f e c t s during labour and d e l i v e r y (McGarry, 1971; Howard and Sharp, 1973). Cardiovascular para-meters (H.R., B.P.) were a l l w i t h i n the normal range, with no s i g n i f i c a n t d i f f e r e n c e s between the two groups (Table V). Although the B.P. appear low (-20 p o i n t s ) t h i s was f e l t to be the r e s u l t of a measurement a r t i f a c t associated with the use of the Arteriosonde. A l l babies included i n the s t a t i s t i c a l e v a luation (Table V) were observed to be pink and well per-fused at b i r t h with good peripheral pulses. The i n f a n t s ' charts were als o c a r e f u l l y reviewed f o r records of abnormal movements, r e s t l e s s n e s s , undue drowsiness or d i f f i c u l t y i n f e e d i n g ; none of the i n f a n t s i n e i t h e r 121 group experienced any i d e n t i f i a b l e problems. One mother (19S) experienced t r a n s i e n t dysphoric r e s t l e s s n e s s 10-15 minutes p o s t - i n j e c t i o n , however, t h i s p a t i e n t ' s baby showed no s t a t i s t i c a l d i f f e r e n c e i n e i t h e r Apgar scores or c a r d i o v a s c u l a r para-meters. Maternal g a s t r i c volumes were also measured i n the present study. The group r e c e i v i n g MCP had s i g n i f i c a n t l y lower volumes confirming the f i n d i n g s of an e a r l i e r study (Howard and Sharp, 1973). Since Apgar t e s t -ing may be i n s e n s i t i v e to s u b t l e drug e f f e c t s , the i n f a n t s were subjected t o a more extensive set of neurological f u n c t i o n a l t e s t s , again no s i g n i -f i c a n t d i f f e r e n c e s were observed between the two groups. The d e t a i l s of these two studies (maternal g a s t r i c volume, neonatal neurological evalua-t i o n ) w i l l be reported i n a f u t u r e communication (Bylsma-Howell et a l . , 1982). 1 2 2 4.4 Sheep P l a c e n t a l Transfer Study Since e t h i c a l c o n s i d e r a t i o n s preclude i n utero f e t a l blood samp-l i n g i n humans, a more extensive study of MCP placental t r a n s f e r i n c h r o n i c a l l y c a t h e t e r i z e d pregnant sheep has been i n i t i a t e d . P r e l i m i n a r y f i n d i n g s i n d i c a t e that the t r a n s f e r of MCP across the placenta i s rapid with measurable concentrations i n the fetus at 1 minute f o l l o w i n g the 10 mg i . v . i n j e c t i o n to the ewe. Plasma concentrations peaked i n the fetus at 20 minutes and were observed to exceed maternal l e v e l s at about 90 minutes ( F i g . 15). E l i m i n a t i o n h a l f - l i v e s of 40 and 54 minutes were c a l c u l a t e d f o r the ewe and f e t u s , r e s p e c t i v e l y . The pattern of f e t a l drug l e v e l s exceeding maternal corresponds to one of the computer simulated concentration-time p r o f i l e s reported by Levy and Hayton, 1973, and Waddell and Marlowe, 1981. In t h i s instance the mother and fetus are each represented as a s i n g l e compartment, with the f e t a l compartment having the pharmacokinetic c h a r a c t e r i s t i c s of being deep (Levy and Hayton, 1973). Under these circumstances the drug enters the fetus r e l a t i v e l y s l o w l y , but because i t i s cleared more r a p i d l y by the mother, f e t a l concentrations e v e n t u a l l y exceed maternal l e v e l s . Accumulation i n the fetus may also occur as a r e s u l t of i o n - t r a p -ping due to the m a t e r n a l - f e t a l blood pH gradient (-7.4 and =7.3, respec-t i v e l y ) . Based on the pka of MCP (9.3) there could be a net t r a n s f e r to the f e t u s , i n utero. Both of these explanations have been proposed f o r procainamide (pka = 9.4) a s t r u c t u r a l analogue of MCP, f o l l o w i n g the observation that 123 neonatal concentrations exceeded those i n cord and maternal blood 8 hours a f t e r d e l i v e r y (Lima et a l _ . , 1978). I t should be noted that t h i s type of curve ( F i g . 15) could also r e s u l t from extensive binding to f e t a l plasma. Differences i n the a f f i -n i t i e s of maternal and f e t a l plasma p r o t e i n s have been reported f o r a number of drugs (e.g. l o c a l a n a e s t h e t i c s , s a l i c y l a t e s , phenobarbital) i n humans (Reynolds, 1979), however, the s i g n i f i c a n c e of t h i s f o r MCP i n the present study i s not known. A v a i l a b l e evidence suggests that MCP binding i s minimal i n man (13-22%) and rat (<20%) but nothing i s known of MCP bind i n g to sheep plasma. In summary, t h i s p r e l i m i n a r y study i n d i c a t e s that MCP i s r a p i d l y transported across the ovine placenta to the fetus f o l l o w i n g i . v . mater-nal a d m i n i s t r a t i o n . Confirmation of the maternal and f e t a l concentra-tio n - t i r n e p r o f i l e s observed i n t h i s s i n g l e study ( F i g . 15), however, re q u i r e s completion of MCP assays. A comparison of MCP pharmacokinetics i n gravid and non-gravid ewes i s also underway (4-way cross-over i n 6 non-gravid ewes: 10, 20, 40, 80 mg MCP i . v . ; 3-way cross-over i n 7 g r a v i d ewes: 10, 20, 40 mg MCP i . v . ) . The plasma samples have been c o l l e c t e d and are c u r r e n t l y under a n a l y s i s . 124 SUMMARY AND CONCLUSIONS A modified GLC-ECD assay f o r MCP i n human and sheep plasma was developed. Components i n t e r f e r i n g with MCP estimation could not be resolved using a p r e v i o u s l y developed packed column GLC-ECD method (Tarn et a l _ . , 1979); t h i s problem was overcome through the use of a 25 m x 0.31 mm c r o s s - l i n k e d fused s i l i c a c a p i l l a r y column. Changes to the previous a n a l y t i c a l method inv o l v e d : i . s u b s t i t u t i o n of MAP f o r diazepam as the i n t e r n a l standard. Unlike the l a t t e r , MAP undergoes the same e x t r a c t i o n and d e r i v a t i z a t i o n procedures as MCP. i i . a d d i t i o n of the c a t a l y s t 0.05 M TEA during d e r i v a t i z a t i o n , which provided d e r i v a t i v e (HFB-MCP) s t a b i l i t y at 4-40 ng.mL"-'- MCP c o n c e n t r a t i o n s , i i i . a r e - e v a l u a t i o n of the a c y l a t i o n reaction-time f o r MCP with HFBA employing MAP as the i n t e r n a l standard and TEA as a c a t a l y s t ; 60 minutes at 55°C was found to be optimal, i v . use of a fused s i l i c a c a p i l l a r y column f o r plasma MCP q u a n t i -t a t i o n employing the s p l i t mode of sample i n j e c t i o n . In summary, the developed c r o s s - l i n k e d fused s i l i c a c a p i l l a r y GLC-ECD method: i . i s l i n e a r over the concentration range studied ( v i z . , 4-40 ng.mL - 1). i i . i s r e l i a b l e , r e p r o d u c i b l e and provides improved s e n s i t i v i t y over the previous packed column method ( v i z . , 4-40 ng of MCP base.mL - 1 of plasma f o l l o w i n g the e x t r a c t i o n of 0.25-0.5 mL of plasma (human, sheep) compared to 7-93 ng of MCP.mL-1 of plasma f o l l o w i n g the e x t r a c t i o n of 0.5 mL of human plasma). 125 i i i . provides a short a n a l y s i s time (-6 minutes), a desired property f o r a ro u t i n e a n a l y t i c a l method. i v . has been applied to pat i e n t samples i n the c l i n i c a l s e t t i n g without i n t e r f e r e n c e from the other drugs used during general anaesthesia i n t h i s study ( v i z . , t h i o p e n t a l , halothane, d-tubocurare, s u c c i n y l c h o l i n e and the l o c a l anaesthetic Marcaine R ( b u p i v i c a i n e ) i n one pat i e n t (5S) undergoing a spina l block p r i o r to general anaesthesia), v. has been used to demonstrate placental t r a n s f e r of MCP, both i n humans and i n sheep, which has not been reported to date (Smith and S a l t e r , 1980; Schulze-Del r i e u , 1981). v i . has shown a p p l i c a b i l i t y to the study of MCP pharmacokinetics i n a placental t r a n s f e r study i n sheep. Pl a c e n t a l t r a n s f e r of MCP was observed i n both man and sheep f o l l o w i n g i . v . maternal a d m i n i s t r a t i o n . MCP q u a n t i t a t i o n i n maternal and f e t a l plasma was c a r r i e d out using the developed c a p i l l a r y assay. Twenty-three p a t i e n t s undergoing general anaesthesia f o r Caesar-ian s e c t i o n f o r healthy term pregnancies were included i n the human study. The p a t i e n t s received e i t h e r i . v . MCP (0.15 mg.kg-1) or an equivalent volume of normal s a l i n e on a double b l i n d b a s i s . One-point blood samples were c o l l e c t e d at d e l i v e r y from a maternal vein (MV) and from the u m b i l i c a l cord (UV, UA). The f i n d i n g s may be summarized as f o l l o w s : i . the average of fetal:maternal MCP concentration r a t i o was 0.60 ± 0.17. 126 i i . i n no instance did f e t a l ( u m b i l i c a l cord:UV, UA) MCP concen-t r a t i o n s exceed maternal over the period of study (elapsed times ranged from 15-50 minutes), i i i . no adverse e f f e c t s were noted i n the 9 i n f a n t s r e c e i v i n g MCP as a r e s u l t of placental t r a n s f e r ; t h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n Apgar scores or c a r d i o v a s c u l a r parameters (H.R., B.P.) between the treated and placebo groups. A s i n g l e c h r o n i c a l l y c a t h e t e r i z e d ewe was used to observe the pl a c e n t a l t r a n s f e r of MCP to the f e t u s , i n utero. The ewe received a 10 mg i . v . i n j e c t i o n of MCP v i a a j u g u l a r venous cat h e t e r . 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