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CLINICAL TOXICOLOGY 8(3), pp. 311-324 (1975)
Single Extraction GLC Analysis of Six Commonly Prescribed Antiepileptic Drugs*
DENNIS P. RITZ Poisonlab Division of Chemed Corp. San Diego, California C. GERALD WARREN Chemistry Department, Colorado State University F o r t Collins, Colorado
INTRODUCTION The necessity of monitoring blood levels of antiepileptic drugs has resulted in the development of many methods, especially since the utilization of gas-liquid chrornotography [ 1-33]. Earlier methods of thin layer chromography and colorimetry have been supplanted by the much greater resolving power of gas-liquid chrornotography, which has become the method of choice. *Reprint requests to: Daniel T. Teitelbaum, M.D., Poisonlab, Denver, Colorado.
311 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
RITZ AND WARREN
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312
The first synthetic drug used to treat epileptic disorders was phenobarbital in 1912. It was not until 1938 that diphenylhydantoin was used in the treatment of convulsive disorders [ 341. Since 1938 the list of antiepileptic agents has become quite extensive and many patients are on polydrug therapy [ 35-37]. This fact emphasizes the need for multiple, reproducible, and rapid analysis of these drugs. Antiepileptic drugs are rather closely related and may be represented a s the following:
R 2
0
/y 4
\R3
where R1, Rz , and Rs refer to various side chain members, and X refers to the ring-closure components of different drug groupings. Hydantoins -N H -, barbituates , -CO-NH- , oxazolidinediones -0-, succinimides -CH2-, and acelytureas -NH2- [lo]. These classes include all antiepileptic drugs except the benzodiazepines, and all are structurally similar as seen above. Even with the benzodiazepines, Camerman and Camerman [ 381 compared the steric similarities between diazepam and diphenylhydantoin and found the angles of the two phenyl groups and the positioning of the two electron-donating groups in each molecule result in a high degree of configurational similarity. The two ketonic oxygen atoms occupy the same position in relation to the diphenyl groups in diphenylhydantoin as does the ketonic oxygen and trigonal nitrogen in diazepam. The mechanism for suppression of seizures is, however, not known. The procedure reported here is generally applicable to the barbituates, hydantoinn, and succinimides. Quaternary ammonium derivations have been developed for these classes [ 1, 2, 4, 8-11, 13, 15, 211. However they may not be satisfactory for several reasons. Incomplete formation of the derivative will particularly affect day-to-day reproductibility, and
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 13
it has been noted that on-column derivative formation is heavily dependent on injection temperature, injection port design, and injection speed. Indeed some injection port designs preclude the use of on-column derivatization ([lo], pp. 111-125). The presence of other drugs, especially similar ones, and endogenous plasma compounds may produce erroneous results ([lo], p. 99). The methyl derivatives (tetramethylammonium hydroxide--TMH) confuse identification of the barbituates. For example, mephobarbital and phenobarbital methyl derivatives are both 1,3-dimethylphenobarbital, and secobarbital splits into two methyl derivatives ([ 101 pp. 117- 118). Problems encountered with the use of trimethylphenyl ammonium hydroxide (TMPH) include the splitting of phenobarbital derivatives into three peaks, all of which must be identified and quantitated together for accurate results [ 81. The derivation techniques allow relatively low-temperature analysis of the hydantoins, which have been found to tail badly on liquid phases such as OV-17 and SE-30. However, it is shown that the analysis of diphenylhyclantoin on a relatively polar liquid phase (3%0V-225) gives data comparable with the derivation techniques [ 71. It has been previously demonstrated that the use of formic acid saturated c a r r i e r gas reduces tailing and baseline rise ([ 121, p. 254). M A T E R I A L S AND METHODS Equipment A. Special Items 1. Roller extractor: Discussed by Clarke, Isolation and Identification of Drugs [ 391 o r suitable agitator. The Roller Extractor was built bv Poisonlab. Div. of Chemed C o r n 2. Evapo Mix. Buchler, Inc. o r suitable shaker/vacuum evaporator. See Discussion. B. Instrumentation 1. Hewlett-Packard 5750 Research Gas Chromatograph. HewlettPackard, 1501 Page Mill Road, Palo Alto, Cal. 94304. Equipped for dual column FID. 2. Fisher- Victoreen 4400 Gas Chromatograph, Fisher Scientific Company (Pittsburgh, Pa.) Equipped for dual column FID.
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O P E R A T I N G CONDITIONS AND R E Q U I R E M E N T S
A. Columns 1. Hewlett-Packard 5750: 6 f t X 1/8 in. stainless steel packed with 3%0V-225 on Gas Q 100/120 (Applied Science Laboratories, State College, Pa. 16801). 2. Fisher-Victoreen 4400: 3 1/2 ft X 1/8 in. glass packed with 3% OV-225 on Gas Q 100/120. (Applied Science Laboratories, State College, Pa. 16801). B. Injection port temperatures 1. Hewlett-Packard 5750 280°C 2. Fisher-Victoreen 4400 300°C C. FID detector temperatures 1. Hewlett-Packard 5750: 290°C 2. Fisher-Victoreen 4400: 300°C D. Programmed oven temperatures Program 1. If ethosuximide and methsuximide are known to be absent: 150 to 250°C at 2O0/min. Initial hold-0. Final hold 5 min. Program 2. If ethosuximide and methsuximide are included 125 to 250°C at 2O0/min. Initial hold-0. Final hold 5 min. See Procedure and Results. E. Flow Rates Hewlett-Packard 5750 Helium carrier-20 ml/min Air-400 ml/min Hydrogen-35 ml/min Fisher-Victoreen 4400 Nitrogen carrier-20 ml/min Air-20 ml/min Hydrogen-50 ml/min F. Recorders
Hewlett-Packard 5750: 1 mV, 0.50 in./min Fisher-Victoreen 4400: 1 mV, 0.50 in./min G. Attenuation 28 X 10' REAGENTS A. CHCls Nanograde. Burdick and Jackson "Distilled in Glass" B. MeOH Nanograde. Burdick and Jackson "Distilled i n Glass" C. Blank Blood: Outdated blood bank blood tested for absence of drugs.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
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D. Mixed Antiepileptic Standard Ethosuximide-supplied by Parke-Davis (Detroit, Mich. ) Methsuximide -supplied by Parke- Davis (Detroit, Mich. ) Mephobarbital-supplied by Applied Sciences (State College, Pa. ) Phenobarbital-supplied by Mallinkrodt Chemical Works (St. Louis, Mo.) Primidone-supplied by Ayerst Laboratories (New York, N. Y. ) Diphenylhydantoin-supplied by Parke-Davis (Detroit, Mich.) E. Mixed Standards in MeOH 1. Ethoswimide -50 mg/dl 2. Methsuximide -50 mg/dl 3. Mephobarbituate -50 mg/dl 4. Phenobarbituate -100 mg/dl 5. Primidone -100 mg/dl 6. Diphenylhydantoin - 100 mg/dl F. Internal Standards 1. Alpha, alpha-dimethyl-beta-methylsuccinimide-50 mg/dl in MeOH. Aldrich Chemical Company, 940 West St. Paul Ave., Milwaukee, Wisc. # 16,3 50-3 2. 5,p-MethylY phenyl-5-phenylhydantoin- 100 mg/dl in MeOH. Aldrich Chemical Company, 940 W. St. Paul Ave., Milwaukee, Wisc. #16,145-4 All standards are kept at -20°C and remade every two months. PROCEDURE The blank, standards, and patient extractions a r e prepared according to Table 1 in a 500 ml F 24/40 roller extractor bottle. Add to all bottles 50 m l CHC13 and roller extract for 15 min. Phase Separating Paper* is used to filter the CHC13 into a 60-ml separatory funnel. If ethosuximide and methsuximide are to be analyzed, 25 ml of the CHCl3 is drawn off into a conical centrifuge tube F 19/22 for evaporation under vacuum. To the remaining CHC13 is added 8 m l 0.45 N NaOH. The phases are shaken for 30 sec and allowed to separate. Discard the CHCls phase. Acidify the NaOH phase with 1 ml concentrated HC1 and reextract with two 10 ml portions of CHC13. Filter through Phase Separating Paper into a 19/22 conical centrifuge tube. All fractions are evaporated under vacuum. The succinimide
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TABLE 1. Blank, Standards, and Patient Extraction Scheme ~~
~~~
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Blank
STD 1
STD 2
Patient
Patient blood, ml
0
0
0
4
Blank blood, ml
4
4
4
0
Mixed standard, p1
0
40
120
0
p-Tolyl Dilantin, pl
0
40
120
40
Alpha-alpha dimethyl suc., pl
0
40
120
40
fraction is evaporated at room temperature and the barbituate/ hydantoin fraction in a 50°C water bath. The extracts are reconstituted with 25 pl MeOH, and 1 pl is injected into the gas chromatograph. Concentration of the drugs are determined by standard/internal peak height ratios after demonstration of linearity. RESULTS The use of OV-225 has been found to be significantly better than OV- 17 for the resolution of primidone, diphenylhydantoin, and 5,p-methyl, phenyl-5 phenylhydantoin, and is represented in Figs. 1 and 2. No interfering peaks greater than 0.08 mg/dl equivalent have been found in the succininiide region (125-150°C), even with minimal cleanup. It was, however, necessary to re-extract the barbituates and hydantoins from 0.45 N NaOH to eliminate interfering peaks [Fig. 3(a) and @)I. This procedure gave exceptionally "clean" blanks with no interfering peaks greater than 0.06 mg/dl. Calculations were based on peak response measurements as follows: Unknown drug response Unknown internal response Known drug response Known internal response
pg/ml
=
Drug pg/ml
Our experience shows that absolute retention times may vary with the long temperature Program 2; however, the relative retention times remain constant.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 17
A B
F
FIG. 1. Separation of: A. alpha, alpha-dimethyl-beta-methylsuccinimide, 0.50 pg. B. ethosuximide, 0.50 pg. C. methsuximide, 0.75 pg. D. mephobarbituate, 0.50 pg. E. phenobarbituate, 0.50 kg. F. primidone, 0.50 pg. G. Diphenylhydantoin, 0.50 pg. H. 5,pmethyl, phenyl- 5-phenylhydantoin, 0.50 pg. Full temperature program 1.
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H
FIG. 2. (a) Full temperature program 1 with typical blank extraction plus 5,p-methyl, phenyl-5-phenylhydantoin (H). Note artifacts 1 and 2. These occur at the retention time of phenobarbital (1) and primidone (2). (b) Full temperature program l with NaOH back extraction. Note absence of interferences 1 and 2.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 19
E
L r"" FIG. 3. Short temperature program 2 showing blood extraction of phenobarbital (E) 0.5 mg/dl, diphenylhydantoin (G) 1 mg/dl, and 5,p-methyl, phenyl-5-phenylhydantoin(€ 1 mg/di. I)
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TABLE 2. Recoveries of the Drugs Extracted from Whole Blood with CHC13
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1.0 mg/dl
2.0 mg/dl
Ethosuxinimide
93
96
Methsuximide
94
98
Mephobarbital
92
96
Phenobarbital
95
99
Primidone
86
90
Dilantin
99
100
The recoveries and precision are given in Tables 2 and 3. The entire procedure may be performed in 1 hr, with an approximately 10-min p r o g r a . DISCUSSION The evaluation of direct chloroform analysis versus on column methylation of diphenylhydantoin has been reported [ 71. Twenty extracts of Serum Toxicology Control* were analyzed. The results of direct analysis were 2.01 mg/dl f 0.78 (CV = 3.9%). This was in excellent agreement with tetramethyl ammonium hydroxide derivation method which had a value of 1.96 mg/dl f 1.16 (CV = 5.9%). Control value was 2.00 mg/dl. It is necessary to complete the NaOH re-extraction quickly since even at room temperature, prolonged periods in NaOH will degrade the barbituates by reverse condensation. Primidone was poorly re-extracted from the acidified NaOH, and we found that two portions of CHCls were required. This gave recoveries in the 80-90qbrange rather than the 40-50qbrange. Double extraction with two aliquots of 0.45 N NaOH was found to increase primidone recovery only slightly. If the laboratory does not have equipment for the routine evaporation of CHCl3 under vacuum, the ethosuximide/methsuximide fraction may be evaporated under a stream of dry air o r nitrogen *Lederle Diagnostics, Pearl River, N.Y.
WALKIW AND DOUGLAS
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326
possible causative agent. Since arsenic is present in minute amounts in normal diets, in larger amounts in diets consisting of seafoods, and i n some water supplies, this element is a normal constituent of human urine. Current methods for the determination of arsenic in biologic materials cannot distinguish between the more toxic trivalent, and the less toxic pentavalent form of the element. When a patient presents with a neuropathy of unknown etiology, accompanied by an elevated urinary arsenic excretion, it is mandatory for the clinician, with the assistance of the laboratory, to determine the source of the arsenic. In such an investigation, the arsenic excretion of the spouse o r of someone else in the patient's household may s e r v e as a useful control. Recently, two cases of elevated arsenic excretion have come to our attention. The abnormal amounts of arsenic determined in both of these patients resulted from the use of health food supplements in tablet form prepared from kelp. This communication is presented to draw attention to this possibly unsuspected source of dietary arsenic, and also to question the safety of the daily ingestion of appreciable quantities of this element in apparently readily absorbable form over long periods of time, as may occur among health food faddists. METHODS AND M A T E R I A L S Twenty-four hour urine specimens were collected directly in specially washed polythene bottles. The method of Vasak and Sedivec for the determination of arsenic [ 1, 21 was used throughout our investigation. It has been generally accepted as one of the best methods for the determination of this element [ 3 1. CASE R E P O R T S Patient A, a 45-year-old married woman, was admitted to the neurologic ward of The Montreal General Hospital. As part of the neurologic investigation of a foot-drop, a urinary arsenic determination was performed and a value of 38.8 pg/24 h r was found. Six days later, a 24-hr excretion of 138 pg was found. Questioning of the patient by the physician elicited the information that she had been taking a number of health food supplements in tablet o r capsule form; these included alfalfa leaf, desiccated liver with , lecithin-D,
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standard-50/50 mixture. The pattern of peak response will be heightened by the drug(s) present. This method combines simplicity, rapidity, and accuracy in the routine and emergency situations, involving antiepileptic drugs. SUMMARY A simple gas-liquid chromatographic technique for the quantitation of ethosuximide (Zarontin), methsuximide (Celontin), mephobarbituate (Mebaral), phenobarbituate, primidone (Mysoline), and diphenylhydantoin (Dilantin) is described. The drugs and internal standards are extracted from whole uncoagulated blood using an organic solvent, re-extracted to eliminate interferences, and chromatographed using temperature programming on 3%0V-225. The results compare very favorably and eliminate some difficulties associated with derivation methods.
REFERENCES
[ 11 E. B. Solow, J. M. Metaxas, and T. R. Summers, J. Chromatogr. Sci 12, 256 (1974). [2] %low and J. B. Green, Neurology, 540 (1972). [ 3 ] P. A. Toseland, J. Grove, and D. J. Berry, Clin. Chim. Acta, 38, 321 (1972). B. Solow and J. B. Green, Clin. Chim. Acta, 33, 87 (1971). [4] [ 51 A. S. Ftxpadopoulos, E. M. Baylis, D. E. Fry, and U. Marks, Clin. a i m . Acta, 48, 135 (1973). [ 6 ] D. H. Sandberg, G.L. Resnick, and C. Z. Bacallas, Anal. Chem 9 40(4), - 736 (1968). [ 71 M. J. Barrett, Clin. Chem. Newsletter, 3, 1 (1971). [ 81 R. J. Perchalski, 'K. N. Scott, B. J. Wilder, and R. H. Hammer, J. Pharm. Sci., 62 (lo), 1735 (1973). [ 9 ] J. MacGee, AnalTChem., 2, 421 (1970). [ 101 J. B. MacGee, International Congress Series #286 Methods of Analysis of Antiepileptic Drugs, Excerpta Medica, Netherlands, 1972, pp. 111. Ell] R. H. Hammer, B. J. Wilder, R. R. Streiff, and A. J. Mayersdork, Pharm. Sci., 60, 327 (1971). [ 121 A. J. Deome, Learyxaboratory, Inc., Boston, Mass., personal communication.
TB.
22,
3 23
GLC ANALYSIS O F ANTIEPILEPTIC DRUGS
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[ 131 [ 141 [ 151 [ 161 [ 171 [ 181
H. J. Kupferberg, Clin. Chim. Acta, 29, 283 (1970). H. J. Kupferberg, J. Pharm. Sci., 64-284 (1972).
E. W. Robb and J. J. Westbrook, Anal. Chem., 35, 1644 (1963). G. W. Stevenson, Anal. Chem., 38, 1948 (1966).T. Chang and A. J X L s Clin. Med., 75, 145 (1970). T. Chang, A. Savory, and A m h e m , Biophys. Res. Commun., 444 (1970). [ 191 E. M. Baylis, D. E. Fry, and V. Marks, Clin. Chim. Acta,
F,
e,
93 (1970). [20] G. W. Bock and A. L. Sherwin, Clin. Chim. Acta, 97 (1971). [21] W. Diinges, H. Heinemann, and K. J. Netter, Chromatography 1972, Applied Science Publishers, Barking, 1973. [22] C. Gardner-Thorpe, M. J. Parsonage, P. F. Smethhurst, and 223 (1972). C. Toothill, Clin. Chim. Acta, (231 C. E. Pippenger and H. W. Gillen, Clin. Chem., E, 582 (1969). [24] K. Sabih and K. Sabih, Anal. Chem., 41, 1452 (1969). [25] K. Sampson, I. Harasymiv, and W. TTHensley, Clin. Chem., 17, 382 (1971). Van d e r Kleijn, P, Collste, B. Norlander, S. Agurell, and [ 261 F. Sjaquist, J. Pharm. Pharmacol., 2, 324 (1973). [27] J. C Van Meter, Clin. Chem., 17, 460 (1971). 1281 J. C. Van Meter, H. S. Buckmaster, and L. L Shelly, Clin. Chem -916 135 (1970). -., [29] W. A. Dill, L. Peterson, T. Chang, and A. J. Glazko, American Chemical Society 149th Natl. Meeting, Detroit, Mich., April 59, 1965. Abstracts. p. 3011. [30] A. J. Glazko and W. A. Dill, Antiepileptic Drugs (D. M. Woodbury, J. K. Penry, and R. P. Schmidt, eds.), Raven P r e s s , New York, 1972, pp. 413-415. [31] Reference 30, p. 455. [32] C. A. Miller and L. M. Long, J. Am. Chem. SOC., 77, 4895 (1951). [33] J. E. Huisman, Clin. Chim. Acta, 13, 323 (1966). [34] D. M. Woodbury, J. K. Penry, a n d x P. Schmidt, Antiepileptic Drugs, Raven Press, New York, 1972, pp. 1-5. [35] Reference 34, pp. 497-516. [36] S. W. Rose, L. D. Smith, and J. K. Penry, Blood Level Deter-
e,
-
z,
minations of Antiepileptic Drugs, Clinical Value and Methods, U.S. Dept. of Health, Education, and Welfare, Washington, D. C.,
1971. [37] J. K, Penry, L. D. Smith, and B. G. White, Blood Level Determinations of Antiepileptic Drugs, Clinical Value and Methods, U.S. Dept. of Health, Education, and Welfare, Washington, D.C., 1972.
324
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168,
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[38] A. Camerman and N. Camerman, Science, 1457 (1970). [39] E. G. C. Clarke, Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1969, p. 24.
Note added in proof: Methsuximide metabolite-N-desmethyl F methsuximide is available from Parke-Davis, Inc. and may be included.
CLINICAL TOXICOLOGY 8(3), pp. 311-324 (1975)
Single Extraction GLC Analysis of Six Commonly Prescribed Antiepileptic Drugs*
DENNIS P. RITZ Poisonlab Division of Chemed Corp. San Diego, California C. GERALD WARREN Chemistry Department, Colorado State University F o r t Collins, Colorado
INTRODUCTION The necessity of monitoring blood levels of antiepileptic drugs has resulted in the development of many methods, especially since the utilization of gas-liquid chrornotography [ 1-33]. Earlier methods of thin layer chromography and colorimetry have been supplanted by the much greater resolving power of gas-liquid chrornotography, which has become the method of choice. *Reprint requests to: Daniel T. Teitelbaum, M.D., Poisonlab, Denver, Colorado.
311 Copyright 0 1975 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
RITZ AND WARREN
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312
The first synthetic drug used to treat epileptic disorders was phenobarbital in 1912. It was not until 1938 that diphenylhydantoin was used in the treatment of convulsive disorders [ 341. Since 1938 the list of antiepileptic agents has become quite extensive and many patients are on polydrug therapy [ 35-37]. This fact emphasizes the need for multiple, reproducible, and rapid analysis of these drugs. Antiepileptic drugs are rather closely related and may be represented a s the following:
R 2
0
/y 4
\R3
where R1, Rz , and Rs refer to various side chain members, and X refers to the ring-closure components of different drug groupings. Hydantoins -N H -, barbituates , -CO-NH- , oxazolidinediones -0-, succinimides -CH2-, and acelytureas -NH2- [lo]. These classes include all antiepileptic drugs except the benzodiazepines, and all are structurally similar as seen above. Even with the benzodiazepines, Camerman and Camerman [ 381 compared the steric similarities between diazepam and diphenylhydantoin and found the angles of the two phenyl groups and the positioning of the two electron-donating groups in each molecule result in a high degree of configurational similarity. The two ketonic oxygen atoms occupy the same position in relation to the diphenyl groups in diphenylhydantoin as does the ketonic oxygen and trigonal nitrogen in diazepam. The mechanism for suppression of seizures is, however, not known. The procedure reported here is generally applicable to the barbituates, hydantoinn, and succinimides. Quaternary ammonium derivations have been developed for these classes [ 1, 2, 4, 8-11, 13, 15, 211. However they may not be satisfactory for several reasons. Incomplete formation of the derivative will particularly affect day-to-day reproductibility, and
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 13
it has been noted that on-column derivative formation is heavily dependent on injection temperature, injection port design, and injection speed. Indeed some injection port designs preclude the use of on-column derivatization ([lo], pp. 111-125). The presence of other drugs, especially similar ones, and endogenous plasma compounds may produce erroneous results ([lo], p. 99). The methyl derivatives (tetramethylammonium hydroxide--TMH) confuse identification of the barbituates. For example, mephobarbital and phenobarbital methyl derivatives are both 1,3-dimethylphenobarbital, and secobarbital splits into two methyl derivatives ([ 101 pp. 117- 118). Problems encountered with the use of trimethylphenyl ammonium hydroxide (TMPH) include the splitting of phenobarbital derivatives into three peaks, all of which must be identified and quantitated together for accurate results [ 81. The derivation techniques allow relatively low-temperature analysis of the hydantoins, which have been found to tail badly on liquid phases such as OV-17 and SE-30. However, it is shown that the analysis of diphenylhyclantoin on a relatively polar liquid phase (3%0V-225) gives data comparable with the derivation techniques [ 71. It has been previously demonstrated that the use of formic acid saturated c a r r i e r gas reduces tailing and baseline rise ([ 121, p. 254). M A T E R I A L S AND METHODS Equipment A. Special Items 1. Roller extractor: Discussed by Clarke, Isolation and Identification of Drugs [ 391 o r suitable agitator. The Roller Extractor was built bv Poisonlab. Div. of Chemed C o r n 2. Evapo Mix. Buchler, Inc. o r suitable shaker/vacuum evaporator. See Discussion. B. Instrumentation 1. Hewlett-Packard 5750 Research Gas Chromatograph. HewlettPackard, 1501 Page Mill Road, Palo Alto, Cal. 94304. Equipped for dual column FID. 2. Fisher- Victoreen 4400 Gas Chromatograph, Fisher Scientific Company (Pittsburgh, Pa.) Equipped for dual column FID.
314
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O P E R A T I N G CONDITIONS AND R E Q U I R E M E N T S
A. Columns 1. Hewlett-Packard 5750: 6 f t X 1/8 in. stainless steel packed with 3%0V-225 on Gas Q 100/120 (Applied Science Laboratories, State College, Pa. 16801). 2. Fisher-Victoreen 4400: 3 1/2 ft X 1/8 in. glass packed with 3% OV-225 on Gas Q 100/120. (Applied Science Laboratories, State College, Pa. 16801). B. Injection port temperatures 1. Hewlett-Packard 5750 280°C 2. Fisher-Victoreen 4400 300°C C. FID detector temperatures 1. Hewlett-Packard 5750: 290°C 2. Fisher-Victoreen 4400: 300°C D. Programmed oven temperatures Program 1. If ethosuximide and methsuximide are known to be absent: 150 to 250°C at 2O0/min. Initial hold-0. Final hold 5 min. Program 2. If ethosuximide and methsuximide are included 125 to 250°C at 2O0/min. Initial hold-0. Final hold 5 min. See Procedure and Results. E. Flow Rates Hewlett-Packard 5750 Helium carrier-20 ml/min Air-400 ml/min Hydrogen-35 ml/min Fisher-Victoreen 4400 Nitrogen carrier-20 ml/min Air-20 ml/min Hydrogen-50 ml/min F. Recorders
Hewlett-Packard 5750: 1 mV, 0.50 in./min Fisher-Victoreen 4400: 1 mV, 0.50 in./min G. Attenuation 28 X 10' REAGENTS A. CHCls Nanograde. Burdick and Jackson "Distilled in Glass" B. MeOH Nanograde. Burdick and Jackson "Distilled i n Glass" C. Blank Blood: Outdated blood bank blood tested for absence of drugs.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
315
D. Mixed Antiepileptic Standard Ethosuximide-supplied by Parke-Davis (Detroit, Mich. ) Methsuximide -supplied by Parke- Davis (Detroit, Mich. ) Mephobarbital-supplied by Applied Sciences (State College, Pa. ) Phenobarbital-supplied by Mallinkrodt Chemical Works (St. Louis, Mo.) Primidone-supplied by Ayerst Laboratories (New York, N. Y. ) Diphenylhydantoin-supplied by Parke-Davis (Detroit, Mich.) E. Mixed Standards in MeOH 1. Ethoswimide -50 mg/dl 2. Methsuximide -50 mg/dl 3. Mephobarbituate -50 mg/dl 4. Phenobarbituate -100 mg/dl 5. Primidone -100 mg/dl 6. Diphenylhydantoin - 100 mg/dl F. Internal Standards 1. Alpha, alpha-dimethyl-beta-methylsuccinimide-50 mg/dl in MeOH. Aldrich Chemical Company, 940 West St. Paul Ave., Milwaukee, Wisc. # 16,3 50-3 2. 5,p-MethylY phenyl-5-phenylhydantoin- 100 mg/dl in MeOH. Aldrich Chemical Company, 940 W. St. Paul Ave., Milwaukee, Wisc. #16,145-4 All standards are kept at -20°C and remade every two months. PROCEDURE The blank, standards, and patient extractions a r e prepared according to Table 1 in a 500 ml F 24/40 roller extractor bottle. Add to all bottles 50 m l CHC13 and roller extract for 15 min. Phase Separating Paper* is used to filter the CHC13 into a 60-ml separatory funnel. If ethosuximide and methsuximide are to be analyzed, 25 ml of the CHCl3 is drawn off into a conical centrifuge tube F 19/22 for evaporation under vacuum. To the remaining CHC13 is added 8 m l 0.45 N NaOH. The phases are shaken for 30 sec and allowed to separate. Discard the CHCls phase. Acidify the NaOH phase with 1 ml concentrated HC1 and reextract with two 10 ml portions of CHC13. Filter through Phase Separating Paper into a 19/22 conical centrifuge tube. All fractions are evaporated under vacuum. The succinimide
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TABLE 1. Blank, Standards, and Patient Extraction Scheme ~~
~~~
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Blank
STD 1
STD 2
Patient
Patient blood, ml
0
0
0
4
Blank blood, ml
4
4
4
0
Mixed standard, p1
0
40
120
0
p-Tolyl Dilantin, pl
0
40
120
40
Alpha-alpha dimethyl suc., pl
0
40
120
40
fraction is evaporated at room temperature and the barbituate/ hydantoin fraction in a 50°C water bath. The extracts are reconstituted with 25 pl MeOH, and 1 pl is injected into the gas chromatograph. Concentration of the drugs are determined by standard/internal peak height ratios after demonstration of linearity. RESULTS The use of OV-225 has been found to be significantly better than OV- 17 for the resolution of primidone, diphenylhydantoin, and 5,p-methyl, phenyl-5 phenylhydantoin, and is represented in Figs. 1 and 2. No interfering peaks greater than 0.08 mg/dl equivalent have been found in the succininiide region (125-150°C), even with minimal cleanup. It was, however, necessary to re-extract the barbituates and hydantoins from 0.45 N NaOH to eliminate interfering peaks [Fig. 3(a) and @)I. This procedure gave exceptionally "clean" blanks with no interfering peaks greater than 0.06 mg/dl. Calculations were based on peak response measurements as follows: Unknown drug response Unknown internal response Known drug response Known internal response
pg/ml
=
Drug pg/ml
Our experience shows that absolute retention times may vary with the long temperature Program 2; however, the relative retention times remain constant.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 17
A B
F
FIG. 1. Separation of: A. alpha, alpha-dimethyl-beta-methylsuccinimide, 0.50 pg. B. ethosuximide, 0.50 pg. C. methsuximide, 0.75 pg. D. mephobarbituate, 0.50 pg. E. phenobarbituate, 0.50 kg. F. primidone, 0.50 pg. G. Diphenylhydantoin, 0.50 pg. H. 5,pmethyl, phenyl- 5-phenylhydantoin, 0.50 pg. Full temperature program 1.
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H
FIG. 2. (a) Full temperature program 1 with typical blank extraction plus 5,p-methyl, phenyl-5-phenylhydantoin (H). Note artifacts 1 and 2. These occur at the retention time of phenobarbital (1) and primidone (2). (b) Full temperature program l with NaOH back extraction. Note absence of interferences 1 and 2.
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GLC ANALYSIS OF ANTIEPILEPTIC DRUGS
3 19
E
L r"" FIG. 3. Short temperature program 2 showing blood extraction of phenobarbital (E) 0.5 mg/dl, diphenylhydantoin (G) 1 mg/dl, and 5,p-methyl, phenyl-5-phenylhydantoin(€ 1 mg/di. I)
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TABLE 2. Recoveries of the Drugs Extracted from Whole Blood with CHC13
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1.0 mg/dl
2.0 mg/dl
Ethosuxinimide
93
96
Methsuximide
94
98
Mephobarbital
92
96
Phenobarbital
95
99
Primidone
86
90
Dilantin
99
100
The recoveries and precision are given in Tables 2 and 3. The entire procedure may be performed in 1 hr, with an approximately 10-min p r o g r a . DISCUSSION The evaluation of direct chloroform analysis versus on column methylation of diphenylhydantoin has been reported [ 71. Twenty extracts of Serum Toxicology Control* were analyzed. The results of direct analysis were 2.01 mg/dl f 0.78 (CV = 3.9%). This was in excellent agreement with tetramethyl ammonium hydroxide derivation method which had a value of 1.96 mg/dl f 1.16 (CV = 5.9%). Control value was 2.00 mg/dl. It is necessary to complete the NaOH re-extraction quickly since even at room temperature, prolonged periods in NaOH will degrade the barbituates by reverse condensation. Primidone was poorly re-extracted from the acidified NaOH, and we found that two portions of CHCls were required. This gave recoveries in the 80-90qbrange rather than the 40-50qbrange. Double extraction with two aliquots of 0.45 N NaOH was found to increase primidone recovery only slightly. If the laboratory does not have equipment for the routine evaporation of CHCl3 under vacuum, the ethosuximide/methsuximide fraction may be evaporated under a stream of dry air o r nitrogen *Lederle Diagnostics, Pearl River, N.Y.
WALKIW AND DOUGLAS
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326
possible causative agent. Since arsenic is present in minute amounts in normal diets, in larger amounts in diets consisting of seafoods, and i n some water supplies, this element is a normal constituent of human urine. Current methods for the determination of arsenic in biologic materials cannot distinguish between the more toxic trivalent, and the less toxic pentavalent form of the element. When a patient presents with a neuropathy of unknown etiology, accompanied by an elevated urinary arsenic excretion, it is mandatory for the clinician, with the assistance of the laboratory, to determine the source of the arsenic. In such an investigation, the arsenic excretion of the spouse o r of someone else in the patient's household may s e r v e as a useful control. Recently, two cases of elevated arsenic excretion have come to our attention. The abnormal amounts of arsenic determined in both of these patients resulted from the use of health food supplements in tablet form prepared from kelp. This communication is presented to draw attention to this possibly unsuspected source of dietary arsenic, and also to question the safety of the daily ingestion of appreciable quantities of this element in apparently readily absorbable form over long periods of time, as may occur among health food faddists. METHODS AND M A T E R I A L S Twenty-four hour urine specimens were collected directly in specially washed polythene bottles. The method of Vasak and Sedivec for the determination of arsenic [ 1, 21 was used throughout our investigation. It has been generally accepted as one of the best methods for the determination of this element [ 3 1. CASE R E P O R T S Patient A, a 45-year-old married woman, was admitted to the neurologic ward of The Montreal General Hospital. As part of the neurologic investigation of a foot-drop, a urinary arsenic determination was performed and a value of 38.8 pg/24 h r was found. Six days later, a 24-hr excretion of 138 pg was found. Questioning of the patient by the physician elicited the information that she had been taking a number of health food supplements in tablet o r capsule form; these included alfalfa leaf, desiccated liver with , lecithin-D,
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standard-50/50 mixture. The pattern of peak response will be heightened by the drug(s) present. This method combines simplicity, rapidity, and accuracy in the routine and emergency situations, involving antiepileptic drugs. SUMMARY A simple gas-liquid chromatographic technique for the quantitation of ethosuximide (Zarontin), methsuximide (Celontin), mephobarbituate (Mebaral), phenobarbituate, primidone (Mysoline), and diphenylhydantoin (Dilantin) is described. The drugs and internal standards are extracted from whole uncoagulated blood using an organic solvent, re-extracted to eliminate interferences, and chromatographed using temperature programming on 3%0V-225. The results compare very favorably and eliminate some difficulties associated with derivation methods.
REFERENCES
[ 11 E. B. Solow, J. M. Metaxas, and T. R. Summers, J. Chromatogr. Sci 12, 256 (1974). [2] %low and J. B. Green, Neurology, 540 (1972). [ 3 ] P. A. Toseland, J. Grove, and D. J. Berry, Clin. Chim. Acta, 38, 321 (1972). B. Solow and J. B. Green, Clin. Chim. Acta, 33, 87 (1971). [4] [ 51 A. S. Ftxpadopoulos, E. M. Baylis, D. E. Fry, and U. Marks, Clin. a i m . Acta, 48, 135 (1973). [ 6 ] D. H. Sandberg, G.L. Resnick, and C. Z. Bacallas, Anal. Chem 9 40(4), - 736 (1968). [ 71 M. J. Barrett, Clin. Chem. Newsletter, 3, 1 (1971). [ 81 R. J. Perchalski, 'K. N. Scott, B. J. Wilder, and R. H. Hammer, J. Pharm. Sci., 62 (lo), 1735 (1973). [ 9 ] J. MacGee, AnalTChem., 2, 421 (1970). [ 101 J. B. MacGee, International Congress Series #286 Methods of Analysis of Antiepileptic Drugs, Excerpta Medica, Netherlands, 1972, pp. 111. Ell] R. H. Hammer, B. J. Wilder, R. R. Streiff, and A. J. Mayersdork, Pharm. Sci., 60, 327 (1971). [ 121 A. J. Deome, Learyxaboratory, Inc., Boston, Mass., personal communication.
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22,
3 23
GLC ANALYSIS O F ANTIEPILEPTIC DRUGS
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[ 131 [ 141 [ 151 [ 161 [ 171 [ 181
H. J. Kupferberg, Clin. Chim. Acta, 29, 283 (1970). H. J. Kupferberg, J. Pharm. Sci., 64-284 (1972).
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-
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minations of Antiepileptic Drugs, Clinical Value and Methods, U.S. Dept. of Health, Education, and Welfare, Washington, D. C.,
1971. [37] J. K, Penry, L. D. Smith, and B. G. White, Blood Level Determinations of Antiepileptic Drugs, Clinical Value and Methods, U.S. Dept. of Health, Education, and Welfare, Washington, D.C., 1972.
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[38] A. Camerman and N. Camerman, Science, 1457 (1970). [39] E. G. C. Clarke, Isolation and Identification of Drugs, The Pharmaceutical Press, London, 1969, p. 24.
Note added in proof: Methsuximide metabolite-N-desmethyl F methsuximide is available from Parke-Davis, Inc. and may be included.
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