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A sensitive GLC-ECD method for determination of metoclopramide in biological fluids Tam, Yun Kau 1978

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A SENSITIVE GLC-ECD METHOD FOR DETERMINATION OF METOCLOPRAMIDE IN BIOLOGICAL FLUIDS By YUN KAU^TAM B . S c , U n i v e r s i t y of Minnesota, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the F a c u l t y of Pharmaceutical Science 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 requ i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1978 @ Yun Kau Tarn, 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PHARMACEUTICAL SCINECE The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date FF R 7 1Q7R ABSTRACT Metoclopramide, 4-amino-5*-chloro^2-methoxy-N^(2-diethyl-amino-ethyl) benzamide, a procaine derivative with antiemetic activity, i s currently-used in gastro-intestinal diagnostics and in the treatment of various types of gastro-intestinal disorders. Metoclopramide increases the tone and peristalsis of the stomach and duodenum, distends the duodenal bulb and improves the pyloric activity, thus promoting gastric motility and reducing gastric emptying time. This effect of metoclopramide has led to the modification of the absorption of other drugs when administered concomitant-ly (e.g. digoxin, griseofulvin, the salicylates, and levodopa,) Despite the pharmacological and therapeutic knowledge accumulated in the literature, l i t t l e pharmacokinetic information pertaining to this drug is available. This is due, in part, to the lack of a highly sensitive assay to'quantitate trace amounts of the drug in small volume biological samples. This thesis reports a highly sensitive GLC-ECD assay which is capable of detecting picogram amounts of metoclopramide in the biological fluids in rats. This method involves extraction, derivatization, removal of excess derivatizing agent and quantitation by injecting the derivative solution into a Reporting GLC-ECD. The structure of the derivative was confirmed by using GC/MS electron impact and chemical ionization methods. The optimized conditions are described as follows: Purification - After extraction and drying, the free base was recrystallized three times with, benzene. The crystals were dried at 125°C under vacuum for 2 hrs. This process removed any volatile substances such as benzene. i i i The purity of the free base was confirmed by differential scanning calorimetry. Extraction - Pesticide grade benzene was found to be the best solvent for extraction due to i t s high purity and satisfactory extraction efficiency for the drug after alkalinization of the aqueous layer with 1 ml of 1 N NaOH. The optimum aqueous and organic ratio for extraction was 1 to 3, viz., 2 ml of the aqueous layer was extracted with 6 ml of benzene. Extrac-tion was accomplished by shaking the aqueous and the organic mixture on a wrist-action shaker for 20 minutes. After centrifugation, 5 ml of the organic phase from a sample was pipetted into a separate 15 ml centrifuge tube. One ml of diazepam solution (.750 mcg/ml in benzene) was added to each tube. The content was dried under a gentle stream of nitrogen. The residue was reconstituted with 1 ml of benzene. Derivatization - Twenty y l of heptafluorobutyric anhydride was added to the reconstituted sample. The reaction mixture was incubated at 55°C for 20 minutes. Removal of excess derivatizing agent - The removal of the excess derivatiz-ing agent was achieved by hydrolysis with 0.5 ml of water and neutraliz-ation with 0.5 ml of 4% ammonium hydroxide solution. GLC - One y l of the derivative solution was injected into a GLC-ECD with the following conditions: injection temperature: 250°C oven temperature: 250°C carrier gas(95%/5% argon/methane) flow rate: 40 ml/ min. i v A 1.8 m, 2 mm i . d . g l a s s column packed w i t h 3% OV-17 coated on Chromosorb W (80-100 mesh) was used. GLC/MS - The s t r u c t u r e of the HFB d e r i v a t i v e was e l u c i d a t e d by GLC-electron impact mass spectrometry. The f o l l o w i n g c o n d i t i o n s were used: i n j e c t i o n temperature: 250°C column temperature: 250°C c a r r i e r gas (helium) flow r a t e : 40 ml/min A 1.8 m, 2 mm i . d . g l a s s column packed w i t h 3% 0V-17 coated on Chromosorb W (80-100 mesh) was used. i o n i z a t i o n beam energy: 70 eV e l e c t r o n m u l t i p l i e r v o l t a g e : 2 Kv analyser temperature: 50°C separator oven temperature: 200°C Since the molecular i o n was not r e a d i l y d i s c e r n a b l e from the e l e c t r o n impact mass spectrum, chemical i o n i z a t i o n mass spectrometry was used to i d e n t i f y the molecular i o n . The f o l l o w i n g c o n d i t i o n s were used: probe temperature: 200°C o source temperature: 150 C i o n i z a t i o n v o l t a g e : 70 V i o n i z i n g gas: isobutane samples were introduced by d i r e c t probe method Q u a n t i t a t i v e s t u d i e s - A c a l i b r a t i o n curve was prepared f o r the plasma e x t r a c t s . L i n e a r i t y was observed i n the range studied (91-750 ng/ml). The recovery of drug spiked separately i n t o plasma, whole blood and u r i n e was 85%. This assay i s extremely s e n s i t i v e and s p e c i f i c . The lowest V detectable amount of metoclopramide i s 1 peg. Animal studies - The a p p l i c a b i l i t y of t h i s assay technique was shown by studying the elimination k i n e t i c s of the drug i n the r a t . The time course of the drug elimination a f t e r an i . v . dose can be described by a biexponen-t i a l equation. The h a l f - l i f e calculated was 49.75 - 8 minutes. TABLE OF CONTENTS v i Page ABSTRACT i ± LIST OF TABLES x i LIST OF FIGURES x i l LIST OF ABBREVIATIONS x l v LIST OF SCHEMATICS x V I. INTRODUCTION ! 1-1. Pharmacodynamic studies ]_ A. Gastrointestinal effects 1 B. Mechanism of action 2 1-2. C l i n i c a l t r i a l s 4 A. Symptomatic r e l i e f 4 B. Radiology 4 C. Gastrointestinal intubation 4 D. Upper gastrointestinal endoscopy 4 E. Gastrointestinal symptoms after vagotomy 5 F. In acid-pepsin disease 5 (i) Gastric ulcer 5 ( i i ) Duodenal ulcer 5 ( i i i ) Reflux esophagitis 5 G. In anaesthesia 5 H. In gastrointestinal disorders 6 I. In migraine 6 J. Side effects 6 1-3. Influence on drug absorption 6 A. Analgesics 7 B. Digoxin 7 (TABLE OF CONTENTS continued . . . 2) I C. A n t i b i o t i c s D. Levodopa E. Ethanol F. Isoniazid 1-4. Metabolism 1-5. Theory of gas l i q u i d chromatography A. I n j e c t i o n port B. Column C. Detectors (i ) Pulsed mode ( i i ) D.C. mode ( i i i ) Comparison of the two modes (iv) Linear mode D. C a r r i e r gas requirement I- 6. Pharmacokinetics I I . EXPERIMENTAL I I - l . Materials II-2. Preparation of metoclopramide II-3. Determination of p u r i t y of metoclopramide monohydrochloride and metoclopramide ( d i f f e r e n t i a l scanning calorimetry). II-4. Preparation of i n t e r n a l standard (diazepam) s o l u t i o n II-5. Preparation of the aqueous s o l u t i o n of metoclopramide monohydrochloride. II-6. Preparation of metoclopramide i n benzene II-7. Preparation of g r i s e o f u l v i n i n benzene II-8. Preliminary GLC-ECD studies A. Choice of d e r i v a t i z i n g agent v i i i (TABLE OF CONTENTS continued . , , 3) Page B. Column s e l e c t i o n 30 I I - 9 . Summary of the p r e l i m i n a r y GLC co n d i t i o n s 30 A. GLC 30 B. D e r i v a t i z i n g procedure 37 C. Removal of excess h e p t a f l u o r o b u t y r i c anhydride. 37 11-10. E f f e c t of h e p t a f l u o r o b u t y r i c anhydride on diazepam — 37 the i n t e r n a l standard. 11-11. Op t i m i z a t i o n of the GLC co n d i t i o n s 38 A. I n j e c t i o n temperature 38 B. Detector temperature 41 C. Oven temperature 41 11-12. Removal of excess d e r i v a t i z i n g agent 41 A. Evaporation method 41 B. ( i ) H y d r o l y s i s w i t h water 48 ( i i ) N e u t r a l i z a t i o n w i t h ammonium hydroxide 48 11-13. Opti m i z a t i o n of d e r i v a t i z a t i o n ( r e a c t i o n time) 48 11-14. S t a b i l i t y of the d e r i v a t i v e 53 11-15. S e l e c t i o n of e x t r a c t i o n solvent 53 11-16. Optimal e x t r a c t i o n of metoclopramide monohydrochloride 58 from aqueous s o l u t i o n s . 11-17. C a l i b r a t i o n curve p r e p a r a t i o n 58 A. Standard curve of metoclopramide 58 B. Standard curves from e x t r a c t i o n 63 11-18. Mass spectrometry 63 A. GLC/MS 63 B. Chemical i o n i z a t i o n 64 i x (TABLE OF CONTENTS continued . . . 4) Page 11-19. Animal s t u d i e s 64 I I I . RESULTS AND DISCUSSION 65 I I I - l . Confirmation of p u r i t y 66 I I I - 2 . O p t i m i z a t i o n of the GLC c o n d i t i o n s 66 A. Column s e l e c t i o n 66 B. Choice of i n t e r n a l standard 71 C. The i n f l u e n c e of h e p t a f l u o r o b u t y r i c anhydride on 73 diazepam, the i n t e r n a l standard. D. I n j e c t i o n temperature 73 E. Oven temperature 76 F. Detector temperature 79 G. C a r r i e r gas flow r a t e 79 I I I - 3 . Reaction time 81 I I I - 4 . Removal of excess h e p t a f l u o r o b u t y r i c anhydride 81 A. ( i ) Optimal aqueous/organic r a t i o i n removing 81 the excess h e p t a f l u o r o b u t y r i c anhydride ( i i ) Aqueous ammonia 84 B. Removal of the excess h e p t a f l u o r o b u t y r i c anhydride 84 by d r y i n g I I I - 5 . S t a b i l i t y of the d e r i v a t i v e 85 I I I - 6 . I d e n t i t y of the d e r i v a t i v e 85 I I I - 7 . E x t r a c t i o n 92 I I I - 8 . GLC of plasma, whole blood and u r i n e samples and the 92 c a l i b r a t i o n curve of the plasma e x t r a c t s . X (TABLE OF CONTENTS continued . , , 5) Page III-9. Recovery 95 111-10. Animal Data 95 I I I - l l . The finalized standard procedure 101 111-12. Conclusions 104 REFERENCES 106 x i LIST OF TABLES TABLE Page Effect of temperature on tl electron capture detector. 6 3 1. Effect of temperature on the sensitivity of the Ni 80 2. Percentage recovery after extraction from biological fluids. 100 LIST OF FIGURES FIGURE 1. The schematic of a typical gas liquid chromatograph, 2. The schematic of an electron capture detector. 3. The effect of pulsating voltage on the electron capture detector. 4. A comparison of the chromatograms obtained from i n -complete reaction between metoclopramide and hepta-fluorobutyric anhydride. 5. Chromatograms obtained from the 0.6 m 2 mm i.d, glass column packed with 10% OV-101 coated on Chromosorb W before and after i t s deterioration. 6. A chromatogram obtained after eluting the derivative and the internal standard through the 1,8 m 2 mm i.d. 3% OV-25 column. 7. A representative chromatogram of the derivative obtained from the 1.8 m 2 mm i.d. 3% OV-17 column. 8. An i l l u s t r a t i o n to show how the response of the metoclopramide heptafluorobutyryl derivative i s affected by injection temperatures. 9. Comparison of the area ratios of the heptafluoro-butyryl derivative of metoclopramide obtained using various detector temperatures, vi z . ( A ) = 350°C, ( • ) = 300°C. 10. The response of the metoclopramide heptafluorobutyryl derivative at different oven temperatures. 11. A comparison between the methods used to remove excess heptafluorobutyric anhydride, a. by hydrolysis with water and neutralization with an aqueous ammonia solution procedure, b. evaporation method. 12. The hydrolysis of excess heptafluorobutyric anhydride by water. 13. Neutralization of the heptafluorobutyric acid with various concentrations of ammonium hydroxide (% v/v), The kinetics of the derivatizing reaction with respect to time. The s t a b i l i t y of the heptafluorobutyryl derivative with time. The extractability of metoclopramide with methylene chloride, benzene, chloroform and hexane. The extent of extraction of a fixed amount of meto-clopramide (3 meg) in a fixed volume (2 ml) of the alkalinized solution (pH=13) by various volumes of benzene (aqueous:organic 1:1-5). A differential scanning calorimetric spectrum of metoclopramide monohydrochloride. The differential scanning calorimetric spectra of the free base before and after drying at 125 C under vacuum. Chromatograms to show the difference obtained before and after incubation of diazepam with 20 u 1 of hepta-fluorobutyryl anhydride at 55 C for 20 minutes. Representative chromatograms showing the change of retention time of the sample components in relation to the column temperature change. A chromatogram to show the incomplete removal of heptafluorobutyric anhydride from the sample. Mass spectra of "the heptafluorobutyryl derivative, a. electron impact, b. chemical ionization. Sample chromatograms from the extracts of biological specimens. A calibration curve of the plasma extracts. A calibration curve of metoclopramide. A representative semi-log plot of the plasma profile of metoclopramide in a rat after an i.v. dose (10 mg/kg). x i v ABBREVIATIONS ADP adenosine diphosphate ATP adenosine triphosphate C.I. chemical i o n i z a t i o n CNS cent r a l nervous system CTZ chemoreceptor t r i g g e r zone D.C. d i r e c t current D.S.C. d i f f e r e n t i a l scanning calorimetry (or e r ) . ECD electron capture detector E.I. electron impact F.I. D. flame i o n i z a t i o n detector GLC gas l i q u i d chromatography HFB heptafluorobutyryl HFBA heptafluorobutyric anhydride HPLC high performance l i q u i d chromatography HVA homovanillic acid i . d . i n t e r n a l diameter i.m. intramuscular i . v. intravenous MCP metoclopramide MCP.HC1 metoclopramide monohydrochloride MS mass spectrometry (or e r ) . TCD thermal conductivity detector TLC thi n layer chromatography LIST OF SCHEMATICS A schematic of the metabolites of metoclopramide recovered from rabbit urine. The postulated fragmentation pattern of the derivative of metoclopramide (GLC-MS electron impact). The postulated derivatization reaction between met-oclopramide and heptafluorobutyric anhydride. x v i To Theresa Acknowledgement The author would like to thank Dr. J.E. Axelson for his supervision and his provision of a friendly atmosphere which made this work a smooth and enjoyable one. The author i s particularly indebted to Dr. F. Abbott of Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, B.C. for his constructive advice and his help in gas liquid chromatography and mass spectrometry. Special thanks would be directed to Dr. D. Hasman of Hewlett Packard Ltd., Dr. W. Godolphin of Vancouver General Hospital for their help and assistance in the mass-spectrometry analysis. The author i s grateful to Dr. F. Jamali of University of Tehran and Mr. R. Venkataramanan for their orientation to and assistance in the laboratory. Thanks to Miss C.J. Chung for her help in the cannulation. This work was supported by U.B.C. grant #32-9400, B.C. Heart grant #65-0566 and M.R.C. grant #MA-5358. 1 I. INTRODUCTION Metoclopramide (MCP), 4-amino-5-chloro-2 methoxy-N-(2-diethyl-amino-ethyl) benzamide, was synthesized by Thominet in 1953. The pharmacological properties of this new drug were f i r s t evaluated in the early sixties. Unlike i t s analog, procainamide, MCP has no significant cardiac effects. However, i t has been observed that MCP was a very potent anti-emetic agent and that i t has profound effect on the gastro-intestinal tract (1-12). /C 2 H 5 -NH-CH2CH2-N^ C 2 H 5 3 Metoclopramide I - l . Pharmacodynamic Studies A. Gastro-intestinal effects - Extensive studies in man (1-11), dog(13,14),guinea pig and rat(15,16) have established the effect of MCP on the gastrointestinal tract. MCP increases the lower esophageal sphincter pressure and the force of p e r i s t a l i c contractions (17-19) without any effect on relaxation of swallowing in man. MCP increases the p e r i s t a l i c strength of the gastric antrum, relaxes the pyloric canal and duodenal cap and increases the synchronization of the gastric antral and duodenal motility (6) in humans. Therefore, the gastric emptying time is reduced (4). Effects resembling those in the esophagus and stomach after MCP have been observed in the small intestine (5,6). The transit time through the duodenum and jejunum is reduced in man as a result of the 2 drug induced increased motility (4,8), No marked effect on colonic motor activity i n vivo is observed (20, 21). B. Mechanism of action - MCP has been postulated to have both peripheral and CHS actions. It has been shown that MCP inhibits emesis in animals to locally acting emetics such as copper sulfate and to centrally acting drugs such as hydergine and apomorphine (11). However, the actual pathways are yet uncertain. Esophagus and gastric contractions induced by MCP are blocked by anticholinergic drugs such as atropine and potentiated by cholinergic drugs such as carbachol and methacholine (14,22). MCP has no anticholinesterase activities (1) and i t s actions are unaffected by the ganglion blocking agents such as chlorisondamine. Eisner (1) has shown that MCP has no action on the isolated human smooth muscles from the body or the antrum of the stomach. However, MCP sensitizes the preparations to acetylcholine. It is postulated that MCP acts via the intramural cholinergic neurons responsible for modifying gastric motility but not gastric secretion(1). A recent study showed that MCP partially or significantly reduces the relaxation effect of adenosine 5-triphosphate (ATP), adenosine diphosphate (ADP) and adenosine but potentiates the effect of noradren-aline on the atropine pretreated taenia c o l i , rabbit ileum and rat duo-denum(23). The inhibiting effect of ATP, ADP and adenosine on the peri-stalsis of guinea-pig ileum was decreased, the effect of adrenaline was potentiated and the effect of theophylline ethylenediamine was not af-fected by MCP. This specific action of MCP was postulated to be due to its sensitive blockade, at the post-synaptic sites, of the effect of the inhibitory purinergic transmitter (ATP and related nucleotides)released 3 during peristalsis C 2 3 ) . Such action of MCP in antagonizing the action of the in t r i n s i c inhibitory mechanism may well be complementary to the documented muscarinic sensitizing action reported by others(14,24-25). MCP i s reported to raise the threshold of activity in the chemo-receptor trigger zone (CTZ) and decreases the sensitivity of visceral nerve which transmits afferent impulses from the gastrointestinal tract to the emetic center in the lateral reticular formation. Thus, MCP prevents vomiting induced by central emetics (11,26). Cannon (27) has indicated that drugs which stimulate the CTZ are dopamine-like. The action of MCP in the CTZ is believed to be due to i t s dopamine antagon-i s t i c effect. More evidence has been accumulated recently to support this hypothesis. Peringer et a l . (28) have observed that after injecting MCP intraperitoneally, MCP has no effect on the dopamine concentrations on the whole brain, but i t increases the homovanillic acidj(HVA)(a dopamine metabolite), concentration both in the corpus striatum and in the mesolimbic area. It has been postulated that MCP blocks dopamine receptors (28,29) and as a result MCP causes an increase in f i r i n g of the dopaminergic neurons. Hence, the turnover of dopamine is increased. MCP has been shown to stimulate prolactin secretion both in animals(30,31) as well as in humans(32-34). This effect i s abolished by CB154, a dopaminergic stimulant (31). Behavioral studies (35) indicate that MCP resembles pimozide in antagonizing the effect of apomorphine or amphetamine in producing turning behavior in mice with unilateral lesions of the nigrostriatal pathway. It also inhibits the apomorphine induced stereotopy and the reversal of reserpine-induced suppression of locomotor activity (36). It appears that MCP is a relatively potent antagonist of s t r i a t a l dopamine receptors. 4 1-2. C l i n i c a l t r i a l s . As a result of the wide c l i n i c a l implications of i t s properties, MCP has been tested in various conditions of upper gastro-intestinal distress and disease since the 1960's. A. Symptomatic re l i e f - MCP is very effective in relieving post-operative nausea and vomiting (37,38). In a c l i n i c a l study of 1,500 patients, MCP has been shown to be as effective as phenothiazine but with less side effects (39). However, pre-operative nausea and vomiting i s not significantly relieved by MCP (40). This is probably due to i t s CNS as well as peripheral effect-B. Radiology - Barium meal examination is usually a time consuming procedure, especially for those patients who have pyloric obstruction which makes the visualization of the duodenum more d i f f i c u l t . MCP, with i t s properties in accelerating gastric emptying by increasing peri-s t a l t i c activities and relaxing the pyloric canal, reduces the radiolog-i c a l examination time significantly (41-43). Furthermore, this agent is particularly useful in small-bowel examination (4). C. Gastrointestinal intubation - MCP when given i.m. (44) or i.v. (45) shortens the time required to introduce a biopsy capsule or aspirating catheter through the pylorus into proximal jejunum. This is due to the effect of MCP to relax the pyloric canal. D. Upper gastrointestinal endoscopy - Emergency endoscopy for upper gastrointestinal hemorrhage is benefited by giving MCP i.v.. This stimulates the passage of blood into the small bowel, cleaning the f i e l d for improved inspection (46) by the action of increasing the strength of peristalsis of the gastrointestinal tract. 5 E. Gastroinetstiiial symptoms after vagotomy - Post-vagotomy symptoms like postprandial vomiting, belching, epigastric distress, and diarrhea are alleviated by MCP in patients 2 to 3 years after vagotomy (47) simply due to the acceleration of the gastric emptying after MCP. F. In acid-pepsiri disease -(i) gastric ulcer - Impaired gastric emptying time has been implicated in the pathogenesis of benign gastric ulceration, therefore, an agent which enhances gastric emptying may be bene-f i c i a l . A c l i n i c a l study (48) showed that MCP is almost equiva-lent to carbenoxolone in treating a single, chronic, lesser-curve gastric ulcer with fewer side effects. However, the results of controlled t r i a l s are not conclusive (48). Further well designed studies are awaited to prove the efficacy of MCP in gastric ulcer treatment. ( i i ) duodenal ulcer - MCP is found to be highly effective in preventing relapse of duodenal ulceration, but no beneficial effects are shown with patients who have acute exacerbation of duodenal ulcer (49). MCP is postulated to exert i t s effect by reducing gastric emptying time, thus, i t reduces the acid content next to the site of hemorrhage. ( i i i ) Reflux esophagitis - Despite the action of MCP on the lower esophagus (50), i t does not improve the condition of patients with severe symptomatic reflux esophagitis (51). G. In anaesthesia - MCP significantly increases gastric emptying in pregnant women during labor thereby reducing the incidence of vomiting and aspiration during emergency anaesthesia resulting in a reduction in the death rate caused by Mendelson's syndrome (52). 6 Aspiration of stomach contents into the bronchial tree is a major cause of mortality and morbidity in emergency anaesthesia. MCP i s a most effective agent to induce gastric emptying in a short time (53). H. In gastrointestinal disorders - MCP is found to be quite effective in dilating the pylorus of those patients with pyloric stenosis. This enables the patients to avoid emergency operations and a better radiological evaluation and c l i n i c a l preparation is obtained before surgery. MCP i s s t i l l not the ultimate treatment of pyloric stenosis (54). I. In migraine - Some beneficial effects on migraine after MCP treatment are observed (55). However, the mechanism of action of MCP in migraine is not known. It has been suggested that MCP enhances the absorption of analgesics (56) with i t s potent anti-emetic properties(37). MCP reduces the incidence of nausea and vomiting caused by the analgesics used in migraine (55). J. Side effects - In a literature survey (57), a total of 1,023 patients showed an incidence of 11% of side effects. The most common adverse effects are drowsiness and lassitude (4%), bowel disturbances (1.2%), others (4,3%), and dizziness or faintness (0.8%). Also f a c i a l dyskinesia (58) and tetanus-like motor disorder resembling phenothia-zine induced "pseudo-tetanus" (59) have been documented. 1-3. Influence on drug absorption - Most drugs are absorbed from the gastro-intestinal tract as the unionized form by passive diffusion. The forma-tion of the unionized species i s pH dependent. It was previously assumed that acidic drugs w i l l be absorbed faster in the stomach where 7 the pH in the environment is low (60). However, Nimmo et a l . (56) showed that the absorption of the low molecular weight and easily diffus-ible compounds such as ethanol and weakly acidic drugs such as aspirin, warfarin and pentobarbital are in fact slower from the stomach. The site where maximal absorption occurs i s the upper small intestine due to the high relative surface area of the small intestine as compared to the stomach. With this in mind, i t can be postulated that the time required for a drug to be delivered into the small intestine may affect the rate and/or extent of i t s absorption. It has been shown by Heading et a l . that the absorption of paracetamol was dependent on the rate of gastric emptying. MCP, an agent which modifies gastric motility, may influence the absorption of those drugs which are administered concomit-antly. Specifically, MCP may affect the peak plasma level attained and the area under the curve of concentration vs time curve (61). A. Analgesics - The effects of MCP for migraine headache are s t i l l uncertain. However, i t has been observed that the absorption of aspirin is delayed during a migraine attack (62). MCP, when administered i.m. immediately before the ingestion of aspirin, increases the rate of absorb tion of the latter (62). Therefore, i t is postulated that MCP decreases the gastric emptying time during a migraine attack. This fa c i l i t a t e s the absorption of the analgesic. Similar observations have been obtained by Heading et: a l . (56) in the study of paracetamol in man. B. Digoxin - MCP decreased the steady state blood level of digoxin (from 0.72 to 0.46 ng/ml) when administered in multiple doses (tablets) to man. Although there is no direct evidence showing that this i s a result of increased gastric motility, the digoxin blood level is raised by propantheline which has the opposite effect of MCP (63). 8 C. Antibiotics - It has been observed by Jamali and Axelson (61) that MCP enhances the absorption of griseofulvin given in solution while depressing the absorption of griseofulvin given as a suspension dosage form. It was postulated that the time required to deliver the drug to the small intestine w i l l affect the rate and extent of absorption. MCP accelerates the absorption of tetracycline and pivampicillin in normal subjects as well as convalescent patients (64). The time to achieve maximum plasma level was significantly reduced. It was suggested that i t was due to the effects of MCP on the gastrointestinal tract. D. Levodopa -- The absorption of L-dopa is variable (65). MCP effectively increases the gastric motility resulting in an increase in the rate and extent (3-fold increase) of levodopa absorption when tested in Parkinsonian patients (65). E. Ethanol - MCP given orally or i.v. before the ingestion of a standard dose of ethanol increases the absorption of ethanol (6) in man. This i s due to the rate of gastric emptying effect of MCP. F. Isoniazid - MCP has no apparent effect on the absorption of isoniazid in tuberculosis patients (67). 1-4. Metabolism - The major metabolic pathway of MCP is conjugation (68). 4 4 Arita (68) reported that N -glucuronide and N -sulfonate are the major metabolites. Cumulative 24 hour urine collection resulted in the recovery of approximately 85% of the 24 hour urinary excretion products as metabolite. De-ethylation i s the major metabolic reaction in rabbits. An in vitro study (69) showed that MCP had eight metabolites. Later, Cowan (70) was able to identify four additional metabolites (scheme I). These metabolites exist in very minute quantities (<1% of a dose). 9 SCHEME I: A schematic of the metabolites of metoclopramide (MCP) recovered from rabbit urine. The major metabolic pathway of MCP i s the formation of conjugates, namely, the glucuronide and sulfonate. The major metabolic re a c t i o n i s de-ethylation. Other metabolites are shown i n the scheme: I. N-(diethylaminoethyl)-4-amino-5 chloro-2-methoxy-benzamide (MCP). I I . N-(ethylaminoethyl)-4-amino-5-chloro-2-methoxy-benzamide. I I I . N-(aminoethyl)-4-amino-5-chloro-2-methoxybenzamide. IV. N-(2'-hydroxyethyl)-4-amino-5-chloro-2-methoxy benzamide. V. N-(diethylaminoethyl)-4-amino-5-chloro-2-hydroxy-benzamide. VI. N-(ethylaminoethyl)-4-amino-5-chloro-2-hydroxy-benzamide. VII. MCP-N 4-sulfonate. 4 VIII. MCP-N -glucuronide. IX. N 4-Acetyl MCP. 10 11 1-5. Theory of gas liquid chromatography (GLC) For this thesis, a gas liquid chromatograph (GLC) equipped with an electron capture detector (ECD) was used to analyze biological samples. It i s appropriate to discuss the theory of GLC-ECD in this section. Chromatography is a technique used to simultaneously separate and quantitate a substance from a mixture. In 1941, Martin and Synge (71) suggested that instead of using a liquid mobile phase, gas could be used. The f i r s t apparatus was described by James and Martin in 1952(72). The f i r s t commercial gas liquid chromatograph was available in 1955. A GLC comprises three major sections (Fig. 1), namely 1) the injection port; 2) column and 3) detector. Samples are introduced into the injection port, vaporized and carried by the carrier gas through the column where separation between substances occur. The eluted sample components are detected by a sensitive and specific detector and the response is reported by a recorder as peaks. A. Injection port - Samples are introduced to the column for subsequent separation after being preheated and vaporized in the inject-ion port. For a particular analysis, the injection temperature should be high enough to rapidly vaporize the sample components. There should also be an adequate linear velocity of the carrier gas flow to rapidly sweep the vaporized sample onto the column where separation takes place. Furthermore, the injection port should be constructed of or lined with a material which is inert to the sample components to prevent destructive catalysis or sample adsorption and loss. B. Column - The column is the "heart" of the GLC wherein separation occurs. Conditions such as column temperature, detector temperature, injection temperature and carrier gas flow rate are 12 FIGURE 1: The schematic of a t y p i c a l gas l i q u i d chrbmatograph. Samples are introduced into the i n j e c t i o n port using a syringe. The samples are vaporized and swept into the column where separation occurs. The sample com-ponents t r a v e l down the column with d i f f e r e n t veloc-i t i e s . This i s due to d i f f e r e n t p a r t i t i o n properties of the sample components. Aft e r e l u t i o n , each compo-nent i s detected by the detector with the response being recorded as i n d i v i d u a l peaks. PRESSURE REGULATOR FLOW CONTROL! VALVE AND METER T TEMP CONTROL INJECTION] _poai COLUMN DETECTOR TEMP ICONTROLl TEMP CONTROL! ELECTROMETER RECORDER 14 adjusted in such a way to achieve maximum, separation and optimal quantitation. A column consists of three components: the container (i t i s either made of metals or glass), the inert solid support (usually diatomaceous earth which has been calcined, then screened using standard wire mesh screen to provide a narrow range of particle size (60-80 mesh)), and the liquid stationary phase. The container and the solid support have to be inert to the sample components. To achieve this goal, the solid support may be acid washed in order to remove trace amounts of metal and the container i s silanized before the column is packed. Chromato-graphic columns may be either polar or non-polar. Depending on what kind of sample is being analysed, one has to choose the right stationary phase. RohrSchneider constants (73) are always used as a guide. Besides the polarity of a stationary phase, factors like column length and percentage loading of the liquid phase onto the solid support may affect the efficiency of a column. Normally, a loading of 3-10% of the liquid phase and a column length from 1 to 4 m are used. The mechanism of separation can be visualized as a series of partitions where the sample goes into solution in the liquid phase and is subsequently revaporized. The a f f i n i t y of the sample for the liquid phase determines the length of time the individual sample component w i l l remain in solution. Those compounds with least a f f i n i t y wi£l elute f i r s t while the opposite i s true for those high af f i n i t y components. The various components in the sample are then separated in discrete bands and travel at different velocities down the column. C. Detectors - After the sample components are eluted in separate bands, elution into the detector occurs where detection i s 15 r e a l i z e d . A number of detectors are a v a i l a b l e f o r q u a n t i t a t i o n based on d i f f e r e n t theory of o p e r a t i o n . The most commonly used d e t e c t o r s are thermal c o n d u c t i v i t y detector(TGD), Flame i o n i z a t i o n detector(FID) and e l e c t r o n capture detector (ECD). Of these, the ECD i s the most s p e c i f i c and s e n s i t i v e . ECD i n p a r t i c u l a r i s a v a i l a b l e to those substances which are h i g h l y e l e c t r o n e g a t i v e such as halogenated s p e c i e s , carbonyl compounds, et c . I t s minimum detectable amount i s at the p i c o -gram l e v e l and o c c a i o n a l l y femtogram l e v e l r e p r e s e n t i n g a d e t e c t i o n l i m i t roughly 1000 x more s e n s i t i v e than FID and TCD. F i g . 2 shows a schematic of the detector used i n our study. fs - emission from the r a d i o a c t i v e source bombards the c a r r i e r gas (such as an argon.methane mixture) and produces secondary e l e c t r o n s and p o s i t i v e i o ns. A f l u x of e l e c t r o n s i s created when a p o t e n t i a l i s a p p l i e d across the detector c e l l . T his produces a constant standing -9 current of about 10 amp. Compounds capable of c a p t u r i n g e l e c t r o n s when el u t e d i n t o the detector form e i t h e r negative ions or r a d i c a l s . These ions are much heavier than the e l e c t r o n s . The negative ions formed may combine w i t h the p o s i t i v e ions to form a n e u t r a l species. This produces a decrease i n e l e c t r o n flow through the d e t e c t o r . Through m u l t i - m a g n i f i c a t i o n , the response i s recorded as a d e f l e c t i o n . The v o l t a g e a p p l i e d across the c e l l can e i t h e r be i n D.C, pulsed or l i n e a r mode. ( i ) Pulsed mode - Using the pulsed sampling technique, the e l e c t r o n c o n c e n t r a t i o n i n the c e l l i s not constant but v a r i e s i n a saw-tooth f a s h i o n . With the a p p l i c a t i o n of a p u l s e , the e l e c t r o n c o n c e n t r a t i o n drops to zero ( F i g . 3 ) . This represents the t o t a l c o l l e c t i o n of the e l e c t r o n s i n the c e l l . In the i n t e r v a l from A to B, the c o n c e n t r a t i o n of e l e c t r o n s b u i l d s up due to £ - p a r t i c l e 16 FIGURE 2: The schematic of an e l e c t r o n capture d e t e c t o r . 63 g emission i s generated from the N i source. The c a r r i e r gas that enters the detector c e l l i s bom-barded by the g emissions to form secondary e l e c t r o n s and p o s i t i v e ions. A f l u x of these e l e c t r o n s i s created by a p p l y i n g a p o t e n t i a l across the detector c e l l ( i . e . the cathode and the anode). Hence a standing current i s formed (10~^ amp). The poten-t i a l a p p l i e d can e i t h e r be D.C. , pulsed or l i n e a r mode. 1 7 GAS OUTLET _t_ O O O O o o o o q a a B B B B B B B B B B 0 B a e a a a CATHODE o o o o oKo o b o o o o 63 Ni SOURCE a STREAM. SPLITTERj o o oooo o o.o o oooo oooo t a a a a a a a a a a a a a a B a a a a a a a ANODE GAS INLET INSULATOR <—-METAL JACKET 18 FIGURE 3: The e f f e c t of p u l s a t i n g v o l t a g e on the e l e c t r o n capture d e t e c t o r . D. Pulse d u r a t i o n : 0.75 psec. X. Pulse i n t e r v a l : 5-150 ysec. 19 T i m e (microsecond) 20 emission from the radioactive source. The magnitude of B C i s dependent on the pulse interval 0 0, i.e. as X decreases, the detector sensitivity decreases. ( i i ) D.C. Mode - A continuous potential i s applied across the detector. Therefore, both electrons and migrated ions are collected in the electrode. This i s a composite rather than a pure electron current. ( i i i ) Comparison of the two modes - In the D.C. mode detector, electrons are not in thermal equilibrium with the carrier gas and are less for electron capture because the electron in the atmosphere is comparatively dilute. Also, the electron concentra-tion in the detector space is comparatively less. With the pulsed mode, the pulse interval (0.5% of the time) allows thermal equilibrium between the electron and the carrier gas molecules to be achieved. During the passage of an electron - capturing component through the c e l l under some circumstances, the substance w i l l plate out or be adsorbed onto one of the metallic surfaces of the c e l l , (probably onto the f o i l i t s e l f ) , and thus create a potential which may enhance or oppose that applied across the c e l l . This phenomenon i s called contact potential. This i s shown on a chromatogram as an excursion below the baseline or a long t a i l on a sample peak which makes quantitation d i f f i c u l t and inaccurate. It i s very obvious that the contact potential w i l l affect the low D.C. voltage more than the high pulsed mode. For example, the contact potnetial i s 5 volts. The D.C. voltage would be 25 x 5 or twenty percent variation, whereas, in the pulsed mode, the 21 voltage would be 50 i 5 volts which is less than a 10% variation. Since the response of a D.C, mode detector is highly dependent on the voltage, a 25% variation can lead to significant variations. (iv) Linear Mode - Pulsed mode detectors have specific advant-ages over D.C. detectors: a) the response of a D.C. mode detector changes sensitively with a change in potential across the detector c e l l . This may render a huge fluctuation in response when contact potential i s produced, b) a pulsed mode detector i s more sensitive than a D.C. mode detector. However, i t can be shown that both detection methods are inherently non-linear over a range of sample concentration. The linear mode, an outgrowth of the pulsed mode, is designed to respond in a linear fashion over a range of sample concentration. This is achieved by using solid-state electronics to operate the c e l l i n the pulsed mode so that a constant current i s produced. A basic requirement is that the pulse frequency be increased when the sample enters the c e l l and captures electrons so that the collection rate (which is the c e l l current) is maintain-ed. Under these operating conditions i t can be shown that the pulse frequency, which is adjusted automatically by the electronics, is linearly related to sample concentration over a very wide range. The linear electron capture c i r c u i t also produces a voltage signal proportional to the pulse frequency which i s fed to a standard recorder. Due to the superiority of the linear mode detector over the previous two discussed, a Hewlett Packard model 5833A GLC 63 equipped with a Ni pulsed closed loop constant current ECD was used for quantitation of MCP in the biological fluids. 22 D. Carrier gas requirement - An argon/methane mixture (95%/5%) is preferred because the high, electron mobility attainable complements the pulse system. 1-6. Pharmacokinetics - Despite the significant number of papers published on MCP, very l i t t l e information pertaining to i t s pharmacokinetics is presently available. The general complaint is the lack of a highly sensitive assay. Tunon (74), in a study using rats, showed that MCP followed bi-exponential elimination after an i.v. dose (10 mg/kg). The h a l f - l i f e of the drug reported was extremely short (13 minutes). A spectrophotometry technique was employed to analyse the blood samples taken from the same rat. In samples after 16 minutes, MCP could not be detected due to the lack of sensitivity of the method. The h a l f - l i f e calculated may be erroneous. Bakke et a l . (75) observed that MCP followed a f i r s t order elimination ( t ^ ^ ~ 0^ minutes) in rats. The h a l f - l i f e of MCP after oral administration was longer but the mechanism is s t i l l not known. By comparing the area under the plasma concentration versus time curve (AUC), i t appeared that the availability of the drug administered orally was only about a tenth of an equivalent i.v. dose. With no evidence of incomplete absorption, first-pass metabolism was postulated. These results, however, have to be confirmed by using a more sensitive method because each datum obtained by Bakke was by sacrificing individual rats, and the standard deviation (probably due to the inter-individual variations) observed was very high. The TLC-photodensitometry method was capable of detecting 0.25 mcg/ml of MCP provided 1 ml of plasma was used. That i s to say that the capability of this assay is limited to detect lower nanogram levels of MCP. Continuous sampling in small animals such as rats i s prohibited because compara-tively large volumes of plasma samples (1 ml) are required. A color-imetric technique was used by Arita (76) to study the metabolites of MCP. The sensitivity of this method i s only comparable to the TLC-photodensitometry technique but not superior. A GLC-FID assay (77) was employed by forensic researchers to isolate and identify MCP qualitatively. The sensitivity of this method was not reported. In a recent report, a HPLC assay of MCP has been reported (78). The lowest detectable concentration of this assay i s about 5 ng/ml provided 5 ml of plasma i s being analysed. Again, this assay is not sensitive enough to be used to analyse small volume samples. In view of the available technology to analyse MCP, the development of a highly sensitive assay was of prime importance to f a c i l i t a t e the study of the pharmacokinetics of this drug. The purpose of this thesis i s to report an extremely sensitive GLC-ECD assay for MCP capable of detecting small amounts (peg) of this drug in small volumes (0.1 ml) of biological samples. II EXPERIMENTAL 25 II EXPERIMENTAL 1 2 3 I I - l . Materials - I N NaOH solution , benzene , methylene chloride , chloroform 4, hexane5, NH^ OH^ , heptafluorobutyric anhydride 7 (HFBA) 8 9 10 metoclopramide-HCI (MCP-HCI) , diazepam , and griseofulvin . II-2. Preparation of metoclopramide - About 30 mg of MCP*HCI was dissolved in 1 ml of 1 N NaOH. The solution containing the free base formed was extracted repeatedly with 10 ml of benzene unt i l the white precipitate in the aqueous layer disappeared. After removing the organic layer into a 50 ml Erlenmeyer flask, the content was dried under a gentle stream of nitrogen at 60°C. The residue was recrystallized three times with benzene and dried at 125°C for two hours under vacuum"'""'' (20 m i l l i -torrs of vacuum). II-3. Determination of Purity of MCP-HCI and MCP (Differential Scanning Calorimetry (DSC)) - A Perkin-Elmer DSC, Model DSC-lB, equipped for effluent gas analysis was employed to confirm the purity of MCP'HCI and MCP. A l l samples were placed in metallic pans and crimped. The rate of "*" Mallinckrodt Chemical Works, St. Louis, Montana. 2-5 D i s t i l l e d in Glass, Caledon, Georgetown, Ontario. 6 ' Allied Chemical Canada Ltd., Claire, Quebec, reagent A.C.S. Code 1293. 7 Pierce Chemical Co., Rockford, I l l i n o i s . 8 Merck and Dohme Research Lab., West Point, Pa. (Cat. no.: L-593, 856-01F06) Hoffman - La Roche Limited, Montreal (Lot no.: R-6685). 1 0 Supplied by Dr. Milo Gibaldi, S.U.N.Y. at Buffalo, Buffalo, N.Y. ^ Vac Torr S35 vacuum pump, Precision Scientific Subsidiary of Corporation, Chicago, 111. (Cat. no.: 10011). 26 temperature increase was 10 uC/min f o r each determination. P r e p a r a t i o n of i n t e r n a l standard (diazepam) s o l u t i o n - An average weight of ten mg of diazepam was weighed a c c u r a t e l y and t r a n s f e r r e d i n t o a 50 ml volu m e t r i c f l a s k . The i n t e r n a l standard s o l u t i o n was prepared by r e c o n s t i t u t i o n w i t h benzene to 50 ml. A h a l f ml of t h i s s o l u t i o n was p i p e t t e d i n t o a 50 ml vo l u m e t r i c f l a s k . The d i l u t i o n was accomplished by d i l u t i n g to volume w i t h benzene(2 mcg/ml). I I - 5 . P r e p a r a t i o n of the aqueous s o l u t i o n of MCP-HC1 - About 5-10 mg of MCP-HC1 was weighed a c c u r a t e l y i n t o a 50 ml vo l u m e t r i c f l a s k and added to volume w i t h d i s t i l l e d water. One ml of the MCP-HC1 s o l u t i o n was d i l u t e d to volume w i t h water i n a 50 ml volu m e t r i c f l a s k to y i e l d an average c o n c e n t r a t i o n of 1 - 2 mcg/ml. This d i l u t e d s o l u t i o n was used f o r subsequent e x t r a c t i o n s . I I - 6 . P r e p a r a t i o n of metoclopramide i n benzene - Two to f i v e mg of MCP was weighed a c c u r a t e l y and d i s s o l v e d i n t o 25 ml of benzene. One ml of t h i s s o l u t i o n was t r a n s f e r r e d i n t o a 100 ml volu m e t r i c f l a s k . The content was made to volume w i t h benzene. This d i l u t e MCP s o l u t i o n was used f o r subsequent a n a l y s i s . I I - 7 . P r e p a r a t i o n of g r i s e o f u l v i n i n benzene - Fi v e to ten mg of g r i s e o f u l v i n was weighed a c c u r a t e l y and d i s s o l v e d i n 50 ml of benzene. One ml of t h i s s o l u t i o n was t r a n s f e r r e d i n t o a 100 ml volu m e t r i c f l a s k . The contents were d i l u t e d to volume with benzene. 27 II-8. Preliminary GLC-ECD studies A. Choice of derivatizing agent - Heptafluorobutyric imidazole and pentafluorobenzoyl chloride have been reported to form highly electron withdrawing derivatives with drugs possessing hydroxy1 and/or amino groups (79,80). These agents have been tested in our laboratory. It has been found that a l l but HFBA gave chromatographic problems, viz. numerous interfering peaks and the detector had low response to the derivative. Hence, HFBA was chosen for reaction with MCP. One ml of MCP solution (500 mg/ml in benzene) was pipetted into ten 15-ml centrifuge tubes. To the centrifuge tubes, 10 y 1 of HFBA was added. The reactants were incubated at 55°C for 0, 5, 10, 15, 20, 25, 20, 40, 50 and 60 minutes respectively. The excess KFBA was removed by hydrolysis with 0.5 ml of water and neutralization with 0.5 ml of 1% 12 NH^ OH. One y l of the derivative formed was injected into a GLC-ECD The following conditions were used: injection temperature, 300°C; oven temperature, 280°C; detector temperature, 350°C; carrier gas (95%/5% argon/methane) flow rate, 40 ml/min and a 0.6 m 10% OV-101 coated on Chromosorb-W (80-100 mesh), 2mm i.d. glass column was used. The derivative peak was observed with minimal interfering peaks (Fig.4b). Samples incubated for less than 15 minutes resulted in severe t a i l i n g indicating incomplete reaction (Fig.4a). However, i f 20 y l instead of 10 y1 of HFBA was added for derivatization, the reaction seemed to go to completion instantaneously even at room temperature. This was indicated by the symmetrical and sharp derivative peak which resembled the peaks observed from samples after complete derivatiz-Hewlett-Packard Model 5833A Reporting gas chromatograph. 28 FIGURE 4 : A comparison of the chromatograms obtained from incomplete and complete r e a c t i o n between metoclopramide (MCP) and h e p t a f l u o r o b u t y r i c anhydride (HFBA). a. The d e r i v a t i v e peak at 3.38 minutes i s obtained a f t e r i n c u b a t i n g the sample w i t h 10 y l of hepta-f l u o r o b u t y r i c anhydride (HFBA) at 55°C f o r 10 minutes. Incomplete r e a c t i o n i s i n d i c a t e d by the t a i l i n g of the peak. b. I d e n t i c a l treatment of the sample as described i n a. except the i n c u b a t i o n time i s prolonged to 20 minutes. Note the d e r i v a t i v e at 3.25 minutes i s sharp and symmetrical i n d i c a t i n g the complete-ness of r e a c t i o n . 29 30 ation (Fig.5a). Based on this evidence, HFBA was chosen as the derivatizing agent. For subsequent analysis, 20 u l of HFBA was added. B. Column Selection - Since the derivative synthesized was relatively polar, a non polar dimethylsilicone OV-101 liquid phase was chosen for separation. The 0.6 m, 2 mm i.d., 10% OV-101 column used under the previously discussed GLC conditions was found to be satisfactory. However, the column had a very short l i f e . Extensive deterioration was noted after a month of intensive use. Column bleed at high column temperatures (280°C) was well recognised. Use of the high loading (10%) OV-101 column was subsequently discontinued due to the problems of deterioration (Fig. 5). Other relatively more polar phases were tested, viz., 0V-25 and OV-17. A 1.8 m, 2 mm i.d. glass column packed with 3% OV-25 coated on Chromosorb W (80-100 mesh) was found to be unsatisfactory for separation and quantitation of the derivative (Fig. 6). The following conditions were used: Injection temperature, 250°C; oven temperature, 250°C; detector temperature, 350°C; carrier gas flow rate, 40 ml/min. Tailing of the derivative peak was observed. A 1.8 m, 2 mm i.d. glass column packed with 3% 0V-17 coated on Chromosorb W (80-100 mesh) under the following conditions was found to be suitable (Fig. 7): Injection temperature, 250°C; oven temperature, 250°C; detector temperature, 350°C; carrier gas flow rate, 40 ml/min. Hence, this column was chosen for subsequent GLC analysis. II-9. Summary of the preliminary GLC conditions: A. GLC injection port temperature = 250°C 31 FIGURE 5: Chromatograms obtained from the 0.6 m, 2 mm i . d . glass column packed w i t h 10% OV-101 coated on Chromosorb W before and a f t e r i t s d e t e r i o r a t i o n . a. before: The d e r i v a t i v e peak (M) at 3.38 minutes i s sharp and symmetrical. b. a f t e r : The M peak i s broad and e x h i b i t s excessive t a i l i n g . I i n both chromatograms represents the i n t e r n a l standard, diazepam. 32 33 FIGURE 6: A chromatogram obtained a f t e r e l u t i n g the d e r i v a t i v e (M) and i n t e r n a l standard (I) through the 1.8 m, 2 mm i . d . 3% OV-25 column. The r e t e n t i o n time of the d e r i v a t i v e peak (M) i s 1.50 min. and the i n t e r n a l standard peak i s 5.00 min. Note, the M:.peak t a i l s . M 35 FIGURE 7: A representative chromatogram of the derivative obtained from the 1.8 m, 2 mm i.d. 3% OV-17 packed on Chromosorb W column. 36 i M 37 oven temperature = 250UC detector temperature *=* 350°C carrier gas (95%/5% argon/methane) flow rate = 40 ml/min. column = A 1.8 m, 2 mm i.d. glass column packed with 3% OV-17 coated on Chromosorb W (80-100 mesh). B. Derivatization procedure - Twenty y l of HFBA was added to a known concentration of MCP solution (benzene). The mixtures were incubated at 55°C for 20 minutes. C. Removal of excess HFBA - Excess HFBA was removed by hydro-lysis with 0.5 ml of water and neutralization with 0.5 ml of 1% NH^ OH (v/v). 11-10. Effect of HFBA on diazepam (internal standard) - One-half ml of the diluted diazepam solution (section II-4) was pipetted separately into ten 15 ml centrifuge tubes. To 5 of them, 0.5 ml of the diluted griseofulvin solution (section II-7) was added. Twenty y l of HFBA was added to the remaining 5 samples. They were incubated at 55°C for 20 minutes. After the mixtures were cooled to room temperature, the excess HFBA was removed by 0.5 ml of water and 0.5 ml of 4% NH^ OH. One-half ml of the diluted griseofulvin solution was added to the organic phase. One u 1 of each sample was Injected in t r i p l i c a t e into the GLC-ECD under the following conditions: injection temperature, 250°C; oven temperature, 270°C; detector temperature, 350°C; carrier gas (95%/5% argon/methane); flow rate, 40 ml/minute. A 1.8 m, 2 mm i.d. glass column containing 3% 0V-17 coated on Chromosorb W (80-100 mesh) was used. Comparison of the response of diazepam before and after the reaction with HFBA was accomplished by the 38 determination of the area ratios of the diazepam/griseofulvin. 11-11. Optimization of the GLC conditions - The GLC conditions obtained above (section II-9A) required optimization. GLC operating temperature optimization was accomplished by fixing two of the three temperature variables (inlet, oven and detector) and altering the third. A fixed carrier gas flow rate of 40 ml/min was chosen for the temperature optimization studies. A. Injection temperature 0.4, 0.6 and 0.8 ml of the diluted MCP-HC1 solution was pipetted separately into three 45 ml centrifuge tubes. To each tube, 1 ml of 1 N NaOH solution was added. The volumes were diluted to 2 ml with d i s t i l l e d water. Each sample was then extracted with 8 ml of benzene. After shaking the mixtures for 20 minutes, the two layers were allowed to separate. Four ml of the organic layer was pipetted into a 15 ml centrifuge tube. To each sample, 1 ml of internal standard solution was added. The content was dried under a gentle stream of nitrogen at ambient temperature. The residues were reconstituted with 1 ml of benzene. Derivatization and removal of excess HFBA was carried out in precisely the same manner as described in section II-9B and 9C. Keeping the oven and detector temperature constant, 1 ml of the derivative in benzene was injected separately into the GLC with the following injection temperatures; 200, 250, 275 and 300°C. The response of each sample corresponding to the specified injection temperatures was estimated by the area ratios of the derivative/ internal standard (Fig.8 ). 39 FIGURE 8: An i l l u s t r a t i o n to show how the response of the metoclopramide (MCP) h e p t a f l u o r o b u t y r y l (HFB) d e r i v a t i v e i s a f f e c t e d by i n j e c t i o n temperatures. The response i s r e f l e c t e d by the area r a t i o of the HFB d e r i v a t i v e / i n t e r n a l standard. ( A — A ) = 2.27 mcg/ml, ( • — • ) = 1.70 mcg/ml and ( • — • ) = 1.13 mcg/ml of MCP. n = 1, each n i s the average of 3 i n j e c t i o n s . Area ratio of the HFB derivative/ internal standard 41 B. Detector temperature: About 0.2, 0.3 and 0.5 ml of the diluted MCP-HCI solution were pipetted individually into three 45 ml centrifuge tubes and 1 ml of 1 N NaOH was added to each tube. The volume was made to 2 ml with water. Exactly the same GLC and deriva-tization procedures were used as described in section II-9E and C respectively. One y l of the derivative formed was injected into the GLC. A l l GLC conditions were held constant and the detector tempera-ture was varied, viz . , 300 and 350°C. The response was monitored by the area ratio of the derivative/internal standard (Fig. 9). C. Oven temperature: One-half ml of the diluted MCP solution and 0.5 ml of the diluted internal standard solution were separately pipetted into three 15 ml centrifuge tubes. The samples were deriva-tized and cleaned as described in section II-9B and C. One y1 of the derivative formed was injected into the GLC. Fixing a l l other GLC conditions, the response of the samples were studied under the following oven temperatures: 240, 245, 250 and 260°C (Fig. 10). 11-12. Removal of the excess derivatizing agent. A. Evaporation method: Three samples of the mixture of MCP and internal standard were prepared as described in section II-11C. After derivatization (as shown i n section II-9B), the samples were allowed to cool to room temperature. Subsequently, they were dried under a gentle stream of nitrogen at room temperature. The residues were reconstituted with 1 ml of benzene. One y1 of the derivative solution was injected into the GLC with the following optimized GLC conditions (Fig. l i b ) ; Injection port temperature, 250°C; oven temperature, 250°C; detector temperature, 350°C; carrier gas 42 FIGURE 9 : Comparison of the area r a t i o s of the h e p t a f l u o r o b u t y r y l (HFB) d e r i v a t i v e of metoclopramide (MCP) obtained using v a r i o u s d etector temperatures v i z . , ( A ) = 350°C, ( • ) = 300°C. No s i g n i f i c a n t d i f f e r e n c e i s observed ( t - t e s t , p<0.05) between the area r a t i o s at the two detector temperatures t e s t e d , namely 300 and 350°C. Maximum % standard d e v i a t i o n =2.20%. n=3 (each n i s determined i n t r i p l i c a t e ) . 43 • B H D B B D B B B B B C I B B B B B B B B B B B B B B B B B B B B B H B 3 I H B Q B S D B B a H a < < <] < < <3 0 <] < < . ^ „ < < <J<1 < < < < < <i > > t> > > > > t> t> > > > > > > > > > > > > > > > > > > > > > > > > > > > > r> t> t> > > a a a a n a B a B a • a B B1 Q B Q H B a a a B s B a a B? <>< ^ 0 < o , 3 , < <J t> t> > 0 > t> > > > > g g £1 9 D H n B {j t> > >5 o o o o o o o o bJO fl o +-> oi >-( fl CD CJ fl o cu -a s u Cn JD TJ o 44 FIGURE 10: The response of the metoclopramide heptafluorobutyryl (HFB) derivative at different oven temperatures. The response is estimated by the area ratio of the HFB derivative/internal standard. No significant difference is obtained (t-test, p<0.05). The range of column temperature test (240-260°C). Maximum % standard deviation = 5.55%. n=3. (each n is determined in t r i p l i c a t e ) . Area ratio of the HFB derivative /internal standard < o o o 46 FIGURE 11: A comparison between the methods used to remove excess h e p t a f l u o r o b u t y r i c anhydride (HFBA) a. by h y d r o l y s i s of ( water and n e u t r a l i z a t i o n w i t h an aqueous ammonia: s o l u t i o n procedure. The r e t e n t i o n times of the d e r i v -a t i v e and the i n t e r n a l standard are 2.49 and 5.88 minutes, r e s p e c t i v e l y , b. evaporation method. The chromatograms shown c l e a r l y i n d i c a t e that method a. i s superior to method b. This i s due to the comparatively b i g l o s s of the d e r i v a t i v e by evapora-t i o n as shown by the smaller peak height of both the d e r i v a t i v e peak and the i n t e r n a l standard peak ( r e t e n t i o n times are 2.51 and 5.88 minutes r e s p e c t i v e l y ) . 48 (95%/5% argon/methane) flow rate = 40 ml/min and a 1.8 m, 2 mm i,d. glass column containing 3% 0V-17 coated on Chromosorb W (80-100 mesh) was used. B. (i) Hydrolysis with water; Twelve 15 ml centrifuge tubes containing MCP in benzene were prepared in the same manner as described in section II-11C. After derivatization(section II-9B) 0, 0.5, 1.5 and 3.5 ml of distilled water was added individually to 3 of the samples. The hydrolyzed HFB acid was neutralized by 0.5 ml 4% NH^ OH. This created an organic:aqueous ratio of 0.5, 1.0, 2.0 and 4.0 respectively. One y l of the organic solution was injected into the GLC. Quantitative comparison between different extraction samples was accomplished by calculating the area ratio of the HFB derivative/internal standard (Fig. 12). (ii) Neutralization with ammonium hydroxide: 0.5, 1, 2, 3, 4, 6, 8, and 10% solutions of ammonium hydroxide (v/v) were prepared by diluting the concentrated NH^ OH solution (30%). Twenty-four organic solutions were prepared as described in section II-11C. After derivatization (section II-9B), each sample was extracted with 0.5 ml of distilled water, then neutralized with 0.5 ml of either concentration of ammonium hydroxide prepared above. The response of the derivative after the extraction procedures was estimated by the area ratio of the HFB derivative/internal standard. (Fig. 13). Each datum was obtained from the average of 3 samples determined in triplicate by the GLC. 11-13. Reaction time. Solutions containing MCP and internal standard were 49 FIGURE 12: The hydrolysis of excess heptafluorobutyric anhydride by water. The response estimated by the area r a t i o of the d e r i v a t i v e / i n t e r n a l standard at d i f f e r e n t organic: aqueous r a t i o s (1:0.5-4) has no s i g n i f i c a n t d i f f e r -ence ( t - t e s t , p<0.05). Maximum % standard deviation = 14.96%. n=3. (each n i s determined i n t r i p l i c a t e ) . o CTQ o OO ' I o Area ratio of metoclopramide HFB derivative/internal standard X H o 51 FIGURE 13: N e u t r a l i z a t i o n of the h e p t a f l u o r o b u t y r i c a c i d w i t h v a r i o u s concentrations of ammonium hydroxide (% v / v ) . No s i g n i f i c a n t d i f f e r e n c e i n response was observed (p<0.05) as estimated by the area r a t i o of the d e r i v -a t i v e / i n t e r n a l standard. Maximum % standard d e v i a t i o n = 11.40%. n=3. (each n i s determined i n t r i p l i c a t e ) . Area ratio of metoclopramide H F B derivative/internal standard cn cn 53 prepared as described in section II-11C. Twenty y1 of HFBA was added separately to the samples. A set of 3 samples were incubated at 55°C for 0, 5, 10, 15, 20, 25, 35, 45 and 60 minutes respectively. The excess HFBA was hydrolysed with 0.5 ml of water and neutralized with 0.5 ml of 4% NH^ OH. The responses were recorded as the area ratio of the derivative/ internal standard after the injection of 1 y l of the derivative solution (Fig. 14 ). 11-14. Stability of the derivative. Three samples of MCP and internal standard solutions were prepared as described in section II-9C. The samples were incubated at 55°C for 20 minutes after the addition of 20 u1 of HFBA. After the samples were cooled to room temperature, 0.5 ml of water was added to promote hydrolysis of the excess HFBA and 0.5 ml of 4% NH,OH for neutralization of the HFB acid. One y l of 4 the derivative was immediately injected into the GLC (time = 0 hr.). Subsequently, 1 y1 of the same sample was injected into the GLC after 1.5, 3.0, 4.5, 6.0, 20, 40 and 60 hrs. The responses were compared as the area ratios of the derivative/internal standard (Fig.15). 11-15. •Selection of extraction solvent. One ml of the diluted MCP-HC1 solution ( 3 ng/y 1) was separately pipetted into twelve 45 ml centri-fuge tubes. One ml of a 1 N NaOH solution was added to each tube. Four ml of benzene, methylene chloride, chloroform or hexane was transferred separately to each sample. Three identical samples were prepared for the individual solvent extraction. After shaking for 20 minutes, 2 ml of the organic layer was pipetted into a 15 ml centrifuge tube and 1 ml of internal standard added. After drying with nitrogen, the residues 54 FIGURE 14: The k i n e t i c s of the d e r i v a t i z i n g r e a c t i o n w i t h respect to time. The response obtained a f t e r i n c u b a t i n g the samples w i t h 20 u 1 of h e p t a f l u o r o b u t y r i c anhydride at 55°C f o r 0-60 minutes was not s i g n i f i c a n t (n = 3, p<0.05). Maximum % standard d e v i a t i o n = 5.44%. Area ratio of metoclopramide H F B derivative / internal standard 56 FIGURE 15: The sta b i l i t y of the heptafluorobutyryl derivative with time. No significant difference in the area ratio of the derivative/internal standard is observed (p<0.05) up to 20 hours. Maximum % standard deviation = 1.22%. n = 3. (each n i s determined in t r i p l i c a t e ) . H 3 CD Area ratio of metoclopramide HFB derivative / internal standard on oo 4^ on "I O N O to O J 58 were reconstituted with 1 ml of benzene. The derivatization and removal of the excess HFBA as described in section II-9B and 12 were followed. One y l of the derivative was injected into the GLC. Quantitative estimation was accomplished by using the observed area ratios of the HFB derivative/internal standard (Fig. 16). 11-16. Optimal Extraction of MCP-HC1 from aqueous solution. One ml of the diluted MCP'HCl solution (3 mcg/ml) was separately pipetted into fifteen 45 ml centrifuge tubes. To them, 1 ml of a 1 N NaOH solution was added. The aqueous phases were individually extracted by 2, 4, 6, 8, and 10 ml of benzene respectively. Three samples were prepared for each extraction. After shaking for 20 minutes, 1, 2, 3, 4 and 5 ml of the organic phases were pipetted into a separate 15 ml centrifuge tube respectively. One ml of diluted diazepam solution was added to each tube. The contents were dried under a gentle stream of nitrogen. The residue was reconstituted using 1 ml of benzene. Derivatization and .removal of the excess HFBA procedures are followed as described in section II-9B and 12 respectively. One y1 of a sample was injected into the GLC. Quantitative estimation was accomplished by measuring the peak area ratios of the derivative/internal standard (Fig. 17). 11-17. Calibration curve preparation. A. Standard curve of MCP. 0.05, 0.2, 0.25, 0.7, 0.9, 1.5, 2.00 and 3.00 ml of diluted MCP solution were individually pipetted into the 15 ml centrifuge tubes. One ml of diluted internal standard was added to each sample. The contents were dried and reconstituted with 1 ml of benzene. Derivatization and removal of excess HFBA 59 FIGURE 16: The e x t r a c t a b l l i t y of metoclopramide (MCP) w i t h methylene c h l o r i d e , benzene, chloroform and hexane. The r e l a t i v e amount of MCP being extracted i s es t i m -ated by the area r a t i o of the d e r i v a t i v e / i n t e r n a l standard, n = 3. Each n i s the average of 3 i n j e c -t i o n s . The maximum % of standard d e v i a t i o n i s 13.19%. 60 O S O S C/5 OS > • >—( OS > 5-1 1.5 1.0 0.5 OS methylene benzene chloroform hexane chloride SOLVENT 61 FIGURE 17: The extent of e x t r a c t i o n of a f i x e d amount metoclopramide (MCP)(3 meg) i n a f i x e d volume (2 ml) of the a l k a l i n i z e d s o l u t i o n (pH-13) by v a r i o u s volumes of benzene (aqueous: organic 1:1-5). The r e l a t i v e amount of MCP recovered i n the organic phase i s determined by the area r a t i o of the d e r i v a -t i v e / i n t e r n a l standard. n = 3. Maximum % standard d e v i a t i o n = 5.66%.No s i g n i f i c a n t d i f f e r e n c e i s observed i n the range of the aqueous:organic r a t i o t e s t e d (p<0.05). T 63 described in section IT-9B and 12 were followed, One u l of the benzene containing the derivative formed was injected into the GLC, The c a l i -bration curve was obtained by plotting the area ratios of the HFB derivatives/internal standard against the concentration of MCP in benzene. B. Standard curves from extraction. 0.1, 0.25, 0.5, 0.7 and 0.9 ml of diluted MCP'HCI solution were separately pipetted into a 45 ml centrifuge tube containing either 0.1 ml of d i s t i l l e d water, plasma, whole blood or urine. One ml of 1 N NaOH solution was added. The volume was made up to 2 ml with d i s t i l l e d water. This phase was extracted with 8 ml of benzene. Six ml of the organic phase was transferred to a 15 ml centrifuge tube followed by the addition of 1 ml internal standard solution (benzene). After drying, derivatization and removal of the excess HFBA as described in section II-9B and 12. One y l of the derivative solution was injected into the GLC. Quantita-tive calibration was accomplished by plotting the area ratios of the HFB derivative/internal standard. 11-18. Mass Spectrometry (M.S.). A. GLC/MS - A Finnigan model 3200 GLC-Electron Impact (E.I.) mass spectrometer was used to study the fragmentation pattern of the HFB derivative. The following conditions were used: for the GLC, the injection port temperature was 250°C; oven temperature, 250°C; helium (carrier gas) flow rate, 30 ml/min. A 6 f t . 2 mm i.d., glass column packed with 3% 0V-17 coated on Chromosorb W (80-100 mesh) was used. For the mass spectrometer, the ionization beam energy was, 70 eV; 64 e l e c t r o n m u l t i p l i e r v o l t a g e , 2 Kv; analyzer temperature 50"C; separator oven temperature 200°C. B. Chemical I o n i z a t i o n (C.I.) - A Dupont model 21-490B chemical i o n i z a t i o n mass spectrometer was employed to determine the molecular i o n of the HFB d e r i v a t i v e . Samples were introduced by the d i r e c t probe method. The f o l l o w i n g c o n d i t i o n s were used: probe temperature, 200°C; source temperature, 150°C; i o n i z a t i o n v o l t a g e , 70 V; i o n i z a t i o n gas, isobutane. 11-19. Animal Studies - An amount of MCP'HCl equivalent to 10 mg/kg of MCP i n 0.9% NaCI s o l u t i o n was i n j e c t e d i n t o a male Wistar r a t (200-300 gm) through a cannula i n s e r t e d i n t o the r i g h t j u g u l a r v e i n . Approximately 0.1 - 0.2 ml of blood were taken at appropriate time i n t e r v a l s from the cannula which was e x t e r i o r i z e d at the nape of the neck. The samples were immediately c e n t r i f u g e d and f r o z e n u n t i l a n a l y s i s . Twenty to f i f t y p i of plasma was e x t r a c t e d and analysed as described above. I I I . RESULTS AND DISCUSSION 66 III RESULTS AND DISCUSSION I I I - l . Confirmation of Purity - The spectrum from the DSC showed that MCP-HC1 began to decompose at 110°C(Fig. 18). No peaks were observed prior to decomposition indicating that the purity of the HC1 salt was satisfac-tory. After recrystallization of the free base from benzene, i t was found from the DSC spectra that an endothermic peak occurred at 120°C before melting. When the samples were dried at 125°C under vacuum for 2 hrs., the peak disappeared (Fig. 19). It was postulated that benzene, the recrystallizing solvent, was either adsorbed onto or formed a solvate with the free base. The peak at 120°C was confirmed to be a volatile substance by coupling the DSC with effluent gas analysis. No further attempt was made to identify whether the solvent was adsorbed to or incorporated into the crystal l a t t i c e of the free base. III-2. Optimization of the GLC Conditions. A. Column selection. Before the GLC assay was developed, selection of a column capable of separating the materials to be analysed was required. Since the selection of the polarity of a liquid phase to be used is a function of the materials being analysed, (73) a table of Rohrschneider constants was used to help in choosing the ideal liquid phase. I n i t i a l l y , a relatively non-polar OV-101 column was found to be suitable. At the specified GLC conditions (section II-8A), a symmet-r i c a l and sharp peak with a retention time of 3.38 min. was observed (Fig. 5a). In order to confirm that the peak at 3.38 min was 67 FIGURE 18: A d i f f e r e n t i a l scanning c a l o r i m e t r i c spectrum of metoclopramide monohydrochloride. The sample was put i n a m e t a l l i c pan and crimped. The r a t e of increase of temperature i s 10°C/min during each determination. 68 69 FIGURE 19; The d i f f e r e n t i a l scanning calorimetric spectra of the free base before and after drying at 125°C under vacuum. The samples were put i n metallic pans and crimped. The rate of increase of temperature during each analysis i s 10°C/min. 70 D . S . C . 120 130 140 150 Temperature °C 71 resolved and corresponded only to the derivative, repeat analysis was performed at lower column temperatures. No interferences appeared even at 240°C. Therefore, this column was selected for subsequent analysis. Unfortunately, after about a month of intensive use, t a i l -ing began to occur, suggesting column deterioration (Fig. 5b). The condition had come to a point where the column had to be abandoned. This was due to a combination of high operating temperature (280°C) and heavy loading of the column (10%) which led to bleeding. When compared to the OV-101 liquid phase used in this analysis, OV-25 i s a relatively polar phase. The reason for choosing this phase was to obtain more information about the completeness of the separation of the derivative peak from the remaining impurities. Different polarities, viz . from polar to non-polar, of the liquid phase may give a better resolution for certain substances but poorer for others. Thus, impurities may be separated from a drug peak when phases contain-ing different polarities were used. Although no interferences were observed when the OV-25 column was used, confirmation was required as to whether the peak of interest was resolved due to severe t a i l i n g (Fig. 6) which might mask minor peaks contributed by impurities. The less polar OV-17 liquid phase was chosen to solve the ta i l i n g problem. It was found that the 1.8 m 3% OV-17 column at the specified GLC conditions (section II-8B) was suitable (Fig. 7). A symmetrical and sharp peak was observed. Lower loading (3% vs. 10%) and lower oven temperature increase the l i f e of the column to about half a year. Therefore, this column was chosen for subsequent analysis. B. Choice of internal standard. An ideal internal standard for quantitative analysis of a substance should possess the following properties: a) i t should have a chemical structure similar to the compound being analyzed, b) i t should have a similar partition coefficient to the compound of interest such that both substances are extracted optimally, c) the internal standard peak, under the optimal GLC conditions, should have a retention time similar to the compound peak but totally resolved from i t , d) no potential chemical reactions between the two, e) i f derivatization was required before gas chromatography, the two compounds should react to the derivatizing agent. For optimal results, these two chemical species should take similar time for completion of reaction. To find an internal standard possessing a l l of the above proper-ties was sometimes d i f f i c u l t especially when the last step was involved. A chemical analog of the compound to be analysed may possess similar physical properties. However, after derivatization, the v o l a t i l i t y and polarity of the compound may change dramatically. This directly affects the GLC behavior of the compound (81). Furthermore, the time for complete reaction during derivatiziation may vary several fold even for similar chemical analogs (82). •It was noted in the literature (83) that procainamide, a procaine derivative, has similar GLC properties to MCP before derivatization. It was observed in our laboratory that the time for derivatization of procainamide was at least 2 fold longer than MCP. In addition, the retention time of the procainamide derivative was almost identical to the derivative of MCP. The two peaks were not resolved under the varied GLC conditions examined. Similar situations happened when the GLC-ECD assay was developed for tocainide (83), another procaine derivative. These compounds were shown to be inadequate as internal 73 standards because when the 1.8 m 3% OV-17 column was used, the retention time of the derivatives of these compounds were short even at low column temperatures (180°C) as compared to the derivative of MCP which eluted optimally at a much higher temperature (250°C). Therefore, no further attempt was made to search for the ideal internal standard. Diazepam, although chemically unrelated to MCP, under the given set of GLC conditions Csection 11-10), was shown to elute 2.5 minutes after the derivative of MCP (Fig. 7). Baseline resolution between the peaks were observed. This compound was later chosen as the internal standard after the test with HFBA. C. The influence of HFBA on diazepam - the internal standard. It i s shown in Fig. 20 that the area ratio of diazepam/griseofulvin before and after HFBA derivatization were found to be identical. No change in the retention time of the diazepam peak was observed after the "reaction" suggesting that this compound is inert to the deriva-tizing agent. Even though diazepam does not f u l f i l l a l l of the requirements mentioned above (section III-2B), i t nevertheless renders a sharp and symmetrical peak which is completely resolved from the derivative peak. Quantitation of MCP using diazepam i s highly consistent (see later sections) and adequate. D. Injection temperature. The response and the peak shape of a sample are partially dependent on the temperature at which the sample is vaporized in the injection port. If the injection temperature is too low, i t w i l l cause the samples to be introduced in a broad band (84), thus, resulting i n peak broadening. Incomplete vaporization of a sample may cause condensation onto the walls of the injection port. 74 FIGURE 20: Chromatograms to show the difference obtained before and after incubation of diazepam with 20 u1 of heptafluorobutyryl anhydride at 55°C for 20 minutes. 76 Alternatively, ah-"excessively high injection temperature may cause decomposition of the compound. These factors w i l l eventually decrease the response. An injection temperature which is high enough to vapor-ize the sample but low enough to avoid decomposition is necessary. Conventionally, the injection temperature is set 50°C higher than the oven temperature ( 85 ). In the range studied i n this experi-ment, i t was found that the variations in response (area ratios) of the derivative from 240 °C to 300 °C were insignificant (Fig. 8 ). But in certain instances, i t was observed that the injection temperature above 275 °C caused slight decomposition. Hence, the subsequent injection temperature chosen was 250 °C. E. Oven temperature. One of the major factors affecting the efficiency and sensitivity of an assay is the column temperature. A direct effect i s observed on the peak shape and the time required for each GLC analysis ( 85) . Higher oven temperatures result in shorter analysis time and sharper peaks, but the separation efficiency may be lowered (85). In addition, shorter column l i f e is anticipated. There-fore, one has to balance a l l factors such that a temperature is chosen to optimize a l l the factors mentioned above. As the column temperature was increased from 240, 245, 250 to 260 °C, the retention times of the derivative peak was observed to be 3.47, 2.92, 2.50, and 1.83 minutes respectively (Fig. 21). In a similar fashion,the retention time of the diazepam peak increased from 4.20 to 8.27 minutes. The area ratios of the derivative/internal stnadard did not change significantly, (t-test, p< 0.05) (Fig.10 ). 77 FIGURE 21: Representative chromatograms showing the change of r e t e n t i o n time of the sample components i n r e l a t i o n to the column temperature (CT.) change. a. C T . = 240°C b. C T . = 245°C c. C T . = 250°C d. C T . = 260°C Note: the r e t e n t i o n time of the d e r i v a t i v e decreases from 3.45 to 1.84 minutes and the i n t e r n a l standard from 8.27 to 4.23 minutes as the column temperature increases from 240 to 260°C 78 79 It i s obvious that as the retention time of a peak increases, the time for each GLC analysis w i l l increase (Fig.21 ). Under these circumstances, there is a trade-off point between the time of analysis, the l i f e of the column and analyst time expenditure. To make a compro-mise, the column temperature was chosen to be 250°C. F. Detector temperature. The process of ionization, recombina-tion and decomposition in the detector i s temperature dependent (86 ). To obtain the maximum response, one has to adjust the detector to the right temperature. There is a narrow range of temperature that one can 63 work with using the Hewlett Packard Ni detector(87). It is not rec-commended to use the detector over 350°C because higher temperature may overheat the detector and hence shortens detector l i f e . Detector cleanliness is insured i f the operating temperature is above 300°C. A paired t-test was performed on the area ratios obtained from the samples at different detector temperatures. No significant d i f f e r -ences were observed (p<0.05)(Fig. 9 ). However, the peak areas of both the derivative and internal standard were significantly lower when the detector temperature was set at 300°C (Table 1 ). This indicated that the detector was less sensitive at 300°C than 350°C. The detector temperature of 350°C was adopted for subsequent analysis. G. Carrier gas flow rate. The most efficient carrier gas flow rate is estimated by the height equivalent theoretical plates. The smaller the height of a theoretical plate, the more efficient is the column at that flow rate. Currently, a flow rate from 30 to 60 ml/min is found to be adequate (88 ). In this analysis, a flow rate of 40 ml/min is employed. 63 TABLE 1. Effect of temperature on the sensitivity of the Ni electron capture detector. Metoclopramide Detector temperature ~C (MCP) ^ 300 350 concentration n area counts (MCP) area counts (diazepam) area counts (MCP) area counts (diazepam) ( rig/ml) x 10 3 x 10*+ x 10 3 x 10^  140.00 1 31.64±1.03 24.09±0.20 37.09±1.07 29.76+0.19 430.00 1 123.0312.35 25.33±0.18 146.47±4.25 31.00±0.25 700.00 1 234.70±1.73 25.34±0.28 275.60±4.30 30.41+0.26 * Each n i s the average of three injections. 81 III-3. Reaction Time - Evaluation of the optimum d e r i v a t i z i n g time was accom-plished by incubating samples containing equivalent amounts of the base with HFBA for various times at 55°C. The y i e l d of the d e r i v a t i v e as observed by the area r a t i o of the d e r i v a t i v e / i n t e r n a l standard was monitored. As shown i n Fig.14 , no s i g n i f i c a n t d i f f e r e n c e i n response was found throughout the range of re a c t i o n time studied (0 - 60 min.). To ensure complete d e r i v a t i z a t i o n , samples were incubated at 55°C for 20 minutes during the current analysis. III-4. Removal of Excess HFBA - It was reported by Walle'et a l . ( 89) that the presence of trace amounts of HFBA residue produced chromatographic problems, v i z . , spurious peaks and a broad solvent front. It was also confirmed i n our laboratory that excess HFBA caused a huge solvent front which masked the HFBA d e r i v a t i v e peak (Fig.22). Therefore,a method which could remove the excess reagent without diminishing the response of the d e r i v a t i v e was necessary. Walle (89) suggested two methods: a) Hydrolyze excess HFBA with water and n e u t r a l i z e i t with aqueous ammonia, and b) dry the re a c t i o n mixture af t e r incubation by a gentle stream of nitrogen. A. ( i ) Optimal aqueous/organic r a t i o i n removing the excess  HFBA: The goal was to achieve optimal removal of the excess HFBA from the organic layer without diminishing the response of the d e r i v a t i v e . In Fig.12, i t i s shown that with the d i f f e r e n t extraction r a t i o s [(0.5 - 4):1], the response expressed as the peak area r a t i o s of the d e r i v a t i v e / i n t e r n a l standard was not s i g n i f i c a n t l y changed ( t - t e s t , p<0.05). For convenience sake, the r a t i o of 1:1 was adopted, i . e . the excess HFBA was removed 82 FIGURE 22: A chromatogram to show the incomplete removal of heptafluorobutyric anhydride from the sample. The solvent peak becomes so broad that i t makes the integration of the derivative peak area d i f f i c u l t and potentially erroneous. 83 84 by h y d r o l y s i s w i t h 0.5 ml of water and n e u t r a l i z a t i o n w i t h 0.5 ml 4% NH.OH. 4 ( i i ) Aqueous ammonia: When ammonium hydroxide was used to remove the excess HFBA a f t e r the d e r i v a t i z a t i o n of t o c a i n i d e (82 ), i t was observed that the response of the compound decrea-sed as the ammonium hydroxide co n c e n t r a t i o n increased. An i n d i -c a t i o n of the e f f e c t of ammonium hydroxide on the response of the d e r i v a t i v e of MCP i s shown i n Fig.13 . No s i g n i f i c a n t d i f f e r e n c e ( t - t e s t , p<0.05) i n response of the d e r i v a t i v e was observed, (concentration range from 0.5 to 10% v / v ) . In some instances ,Lt i s shown that a l o w c o n c e n t r a t i o n of ammonium hydroxide (< 2% v/v) caused a broad solvent f r o n t i n the chroma-togram (Fig.22 ). This i s an i n d i c a t i o n of the incomplete n e u t r a l i z a t i o n of the hydrolyzed HFBA. Hence, to ensure the maximal removal of HFBA, 0.5 ml of 4% NH.OH s o l u t i o n was used 4 i n a l l subsequent analyses. B. Removal of the excess HFBA by d r y i n g . This method i s found to be e f f e c t i v e and was adopted by Venkataramanan and Axelson (82 ). However, the response of the d e r i v a t i v e was decreased by at l e a s t two-thirds when compared to the method employed i n s e c t i o n III-4A. (Fig.11 ). The decrease i n response when the d r y i n g method was ap p l i e d was probably due to v o l a t i l i t y of the d e r i v a t i v e (89 ), whereas the l o s s of the d e r i v a t i v e from evaporation was minimal when the method described i n s e c t i o n III-4A was used. I t i s obvious that the h y d r o l y s i s and subsequent n e u t r a l i z a t i o n method was adopted to 85 remove the excess HFBA a f t e r d e r i v a t i z a t i o n , III-5. S t a b i l i t y of the Derivative - As shown i n Fig.15 , the area r a t i o s of the d e r i v a t i v e / i n t e r n a l standard did not drop s i g n i f i c a n t l y a f t e r 20 hrs. of storage. It was also observed that the areas under the two peaks did not change perceptibly during that time. This property of the d e r i v a t i v e made the preparation of large numbers (> 100) of samples possible. Hence, manual handling of the samples was reduced to a minimum when the samples were introduced into the GLC equipped with' _an- automatic sampler. III-6. Identity of the d e r i v a t i v e - The fragmentation patterns of the d e r i v a t i z e d free base and plasma extracted from metoclopramide dosed animals were found to be i d e n t i c a l using GC-MS i n d i c a t i n g MCP was being analyzed from the plasma samples. Like MCP (70 ) and- i t s procaine analogs ( 82), cleavage occurred at the amine bond (m/e 423), as well as the carbonyl-amide bond (m/e 99, and m/e 380). The base peak, m/e 86, was a r e s u l t of the cleavage at the carbon-carbon bond £ to the amine nitrogen (scheme II). Although a mono-substitution reaction was postulated (scheme III),the molecular ion was not r e a d i l y discernable (Fig.23a) from the E.I. mass spectrum. This may be due to the high i o n i z a t i o n energy of the source. Since E.I. mass spectro-metry was not conclusive, chemical i o n i z a t i o n mass spectrometry was employed to reveal the molecular ion. From the chemical i o n i z a t i o n mass spectrum (Fig.23b), a very intense m/e 496 peak which corresponded to the [MH ] + peak was observed. The other two peaks m/e 478 and 446 were postulated to be [MH - water]"*" and [MH - water - methanol]"1" 86 FIGURE 23: Mass spectra of the heptafluorobutyryl d e r i v a t i v e : a. electron impact (E.I.) b. chemical i o n i z a t i o n (C.I.) ( i o n i z a t i o n gas: isobutane) In E.I., the base peak observed was m/e 86. The other most intensive peaks are m/e 423, 380, 99 and 87. However, the molecular ion i s not r e a d i l y discernable. A complementary spectrum from the CI-MS showed the protonated molecular ion [MH] + at m/e 496 and the [MH-water]+ and [MH-water-methanol]+ peaks. Plasma extract after metoclopramide "admin-: //;38 -32 100 > 420 X 5 10 l; n , , |HI | f"1!1'! ••i"-VT-Jf"i-"i,,T i 1 f i i f i ' r r i i"'f i r'l'i T i "i 1 1 1 1 1 1 1 1 1 1 1 1 20 50 100 150 200 250 100 CK \ . O / C 2 H 5 N H — ( 7 x > — C — N H — C H j — C H 2 — N ) C H , 10 M.W. = 495 i i i ^ l i H i l i i ,|_L I l I i ' ! 1 i i ' 1 1 1 1 1 1 1 ' A i ' 1 1 4'5 100 ) 1 1 1 1 1 1 1 1 ' i 111111. I .L 111 / / — 1 1 1 1 1 1 1 1 1 1 1 i^ 11 r i \ S 11 n i 20 50 100 400 *50 10 30 88 SCHEME I I : The postulated fragmentation pattern of the deriva-t i v e of metoclopramide. (GLC-MS electron impact). The molecular ion i s m/e 495; the base peak i s m/e 86; the other most intensive fragments are m/e 423, 380, 99 and 87. 89 — N H — C H C H 2 —N / i \ C 2 H 5 C 2 H 5 OCH, m i/e 495 HN •I c=o I f CF, O II C^ m/e 3 80 CF, — C H , — C H -CH 2N(C 2H 5) 2 CHCH 2 N(C 2 H 5 ) 2 m/e 86 m / e 9 9 CH 3 N(C 2 H 5 ) 2 m/e 87 90 SCHEME I I I : The postulated d e r i v a t i z a t i o n reaction between metoclopramide and heptafluorobutyric anhydride. Monosubstitution i s postulated to occur at the amino group attached to the r i n g . CI \ o H2N—{f N^>—C—NH-—CHj—CH 2 OCH, Metoclopramide C I CF 2 Metoclopramide HFB derivative 11 CF 3—CF 2—CF 2— O CF,—CF2 CF.—C^ 2 II O Heptaf 1 uorobutyryl anhydride CF 3—CF 2—CF 2— COOH Heptaf luorobutyryl acid 92 r e s p e c t i v e l y , I I I - 7 . E x t r a c t i o n . Four s o l v e n t s , namely, methylene c h l o r i d e , benzene, chloroform and hexane, have been used to e x t r a c t MCP from the aqueous phase. Aqueous samples c o n t a i n i n g i d e n t i c a l amount of MCP-HC1 were prepared as described i n s e c t i o n 11-5. These samples (2 ml each) were extr a c t e d w i t h 4 ml of the above mentioned s o l v e n t s . I t was observed from the GLC r e s u l t s that methylene c h l o r i d e gave the highest e x t r a c t -i o n e f f i c i e n c y , followed by benzene and chloroform (Fig.16 ). Hexane gave the l e a s t e x t r a c t a b i l i t y as i l l u s t r a t e d by GLC response. Methylene c h l o r i d e and chloroform have been reported to give i n t e r f e r e n c e peaks a f t e r d r y i n g (82 ). Benzene was shown to be the cleanest solvent f o r the ECD a n a l y s i s . Despite the higher e x t r a c t a b i l i t y of methylene c h l o r i d e , benzene was chosen to be the e x t r a c t i n g s o l v e n t . To achieve maximal e x t r a c t i o n , v a r i o u s organic:aqueous r a t i o s were t e s t e d (1-5 : 1). The extent of e x t r a c t i o n was q u a n t i t a t e d by using the GLC. I t i s found that maximal e x t r a c t i o n was a t t a i n e d at the r a t i o of 1:1. This i s i n d i c a t e d by the area r a t i o s of the d e r i v a -t i v e / i n t e r n a l standard (Fig.17 ). No s i g n i f i c a n t d i f f e r e n c e i n ( t - t e s t , p< 0.05) e x t r a c t i o n was observed from r a t i o s of 1:1 to 5:1. But to ensure optimal e x t r a c t i o n , 6 ml of benzene was used to e x t r a c t 2 ml of the aqueous phase (3:1). I I I - 8 . GLC of plasma, whole blood and u r i n e samples and the c a l i b r a t i o n curve  of the plasma e x t r a c t s - Representative chromatograms from the e x t r a c t of the plasma, blood and u r i n e samples are shown i n Fig.24 . A peak at 8.38 min was observed i n the chromatograms of the blank plasma and 93 FIGURE 24: Sample chromatograms from the e x t r a c t s of b i o l o g i c a l specimens. a. blank plasma e x t r a c t , b. blank whole blood e x t r a c t . c. blank u r i n e e x t r a c t . d. plasma e x t r a c t (91.6 p e g / i n j e c t i o n ) . e. whole blood e x t r a c t (229 p e g / i n j e c t i o n ) . f. u r i n e e x t r a c t (91.6 p e g / i n j e c t i o n ) . 95 the whole blood (Fig.24a and 24b). The u n i d e n t i f i e d impurity did not i n t e r f e r e with either the d e r i v a t i v e of MCP or the i n t e r n a l standard peak (Fig.24d and 24e). No endogenous disturbances were found from the urine extract (Fig.24c and 24f) . Base l i n e r e s o l u t i o n was achieved between the peaks when the 3% OV-17 column was used. Chromatographic response was l i n e a r i n the range studied (91.6 -824.7 ng/ml). Quantitative estimation was achieved by analyzing a s e r i a l d i l u t i o n of known concentration of the plasma extracts (Fig. 25). From the l i n e a r regression a n a l y s i s , the best f i t through the data points was described by: y = .0020x - 0.019 2 with r = 0.999. The minimun detectable amount i s 1 peg. III-9. Recovery - A standard curve was prepared by d e r i v a t i z i n g a s e r i a l d i l u t i o n of the free base i n benzene (Fig. 26). After extracting known quantities of MCP-HCI from the b i o l o g i c a l f l u i d s , the amount of free base recovered i n the organic phase was determined by using the free base c a l i b r a t i o n curve. The average percentage of MCP recovered from plasma, whole blood and urine were, 87.02, 83.50,and 84.73 re s p e c t i v e l y (Table 2 )• The respective percentage standard deviations were, 4.49, 6.06, and 8.00. 111-10. Animal Data - The a p p l i c a b i l i t y of t h i s method was demonstrated by studying the elimination k i n e t i c s of MCP i n rats a f t e r an i . v . dose (10 mg/kg). Due to the extremely high s e n s i t i v i t y of t h i s assay method, only small volumes of plasma (20-50 p i ) were required. This method permits s e r i a l blood sampling from the same ra t over a s u f f i c i e n t 96 FIGURE 25: A c a l i b r a t i o n curve of the plasma e x t r a c t s . The curve was obtained by p l o t t i n g the area r a t i o of the d e r i v a t i v e / i n t e r n a l standard, n = 5. L i n e a r i t y i s observed between 90-750 peg per p1 i n j e c t i o n ( y 2 = 0.999). Standard d e v i a t i o n i s + 0.018. Area ratio of the HFB derivative internal standard L6 .98 FIGURE 26: A c a l i b r a t i o n curve of metoclopramide (MCP). The curve i s obtained by p l o t t i n g the area r a t i o of the MCP d e r i v a t i v e / i n t e r n a l standard. L i n e a r i t y i s observed between 50-700 peg per i n j e c t i o n . r 2 = 0.999. n = 5. Standard d e v i a t i o n = 0.0038. 200 400 600 800 metoclopramide f amount per injection (peg Table 2 % recovery after extraction from biological fluids. Metocloprami de base equivalent added (ng) n* Plasma % recovered extract % deviation Whole blooc % recovered extract % deviation Urine % recovered extract % deviation 229.09 5 92.84 + 14.23 90.79 + 0.84 94.16 + 2.10 458.18 5 85.09 + 6.35 82.83 + 3.35 82.08 + 6.55 641.45 5 85.56 + I.65 81.06 + 5.54 84.42 + 5.54 824.72 5 84.57 + 7.75 79-33 + 4.40 78.27 + 1.04 average 87.02 + 4.49 83.50 + 6.06 84.73 + 8.00 . " each n is the average of 3 determinations. 101 period of time to allow adequate c h a r a c t e r i z a t i o n of the pharmaco-k i n e t i c s of MCP i n the r a t . A t - j ^ a °^ minutes was observed i n d i c a t i n g rapid d i s t r i b u -t i o n within the f i r s t minutes a f t e r i n j e c t i o n . An apparent d i s t r i b u -t i o n equilibrium was established at around 16 minutes. From the semi-log p l o t of the plasma p r o f i l e ( F i g . 27), a l i n e a r elimination phase was seen. The terminal h a l f - l i f e c a l c u lated was 49.75 * 8 minutes. When these r e s u l t s were compared to the pharmacokinetic data obtained by Tunon (74), i t was found that the "elimination phase" previously reported was a c t u a l l y part of the d i s t r i b u t i o n a l phase. Therefore, the h a l f - l i f e (13-20 min) calculated was erroneous. This was due to the use of a r e l a t i v e l y i n s e n s i t i v e assay method which was unable to detect any MCP a f t e r 16 minutes. The assay method reported here o f f e r s s i g n i f i c a n t s u p e r i o r i t y over Bakke's (75) TLC -photodensitometry method and Teng's HPLC assay (78). A smaller volume of plasma i s required f o r ana l y s i s , thereby obviating the need for the s a c r i f i c e of an i n d i v i d u a l r a t to obtain a datum. I I I - l l . The f i n a l i z e d standard procedure - About 10 mg/kg of metoclopramide was injec t e d i . v . into a white Wistar r a t (200-300 gm) cannulated i n the r i g h t jugular vein. Approximately 0.1-0.2 ml of blood was taken at appropriate time i n t e r v a l s . The blood samples were immediately centrifuged and stored i n a freezer u n t i l a n alysis (-20°C). Twenty to f i f t y y l of plasma was added to a 45 ml centrifuge tube and to i t 1 ml of 1 N NaOH was added. The contents were made up to 2 ml with d i s t i l l e d water. To the aqueous sample was added 6 ml of benzene. The sample was shaken with a B u r r e l l wrist action shaker for 20 min.. 102 FIGURE 27: A representative semi-log plot of the plasma profile of metoclopramide in a rat after an i.v. dose (10 mg/kg). The elimination kinetics after an i.v. dose into the rat can be described by a bi-exponential curve. The terminal h a l f - l i f e is 49.75+ 8 minutes. Plasma concentration of metoclopramide (mcg/ml ) o 104 After the two layers have separated, 5 ml of the organic phase was pipetted into a 15 ml centrifuge tube. One ml of internal standard solution was added. The content was dried under a.gentle stream of nitrogen at room temperature. The residue was reconstituted with 1 ml of benzene. Twenty u1 of HFBA was added to the reconstituted solution. The reaction mixture was incubated at 55°C for 20 minutes. After the sample was cooled to room temperature. Excess HFBA was removed by vortexing f i r s t with 0.5 ml of water, then, 0.5 ml 4% NH^ OH. One u l of the derivative in benzene was injected into the GLC with the following conditions: Injection temperature: 250°C Column temperature: 250°C Detector temperature: 350°C Carrier gas (95%/5% argon/methane) flow rate: 40 ml/min. Column: A 1.8 m, 2 mm i.d. glass column packed with 3% OV-17 coated on Chromosorb W(mesh 80-100) was used. Quantitation of the drug in biological f l u i d was accomplished by using the calibration curve. The recovery from the biological fluids was determined by comparing the area ratios of a known amount of MCP being extracted and the value obtained from the calibration curve of the pure substance. 111-12. Conclusions 1. The lowest detectable amount is 1 peg. 2. Linearity is observed between 90-750 pcg/ul. 3. The derivative i s stable up to 20 hrs. Recovery of MCP from biological samples is about 85%. Small volumes (0.1 ml) of blood samples can be analysed to study the elimination kinetics of the drug in the rat. 106 REFERENCES 1. E i s n e r , M.: G a s t r o i n t e s t i n a l e f f e c t s of metoclopramide i n man. In v i t r o experiments w i t h human smooth muscle p r e p a r a t i o n . B r i t . Med. J . 4: 679, 1968. 2. 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