Simultaneous Rapid HPLC Determination of Anticonvulsant Drugs in Plasma and Correlation with EMIT®

RAYMOND S. RYDZEWSKI, M.S., RICHARD H. GADSDEN, P h .D.,* and CONSTANCE A. PHELPS, A.S.M.T.

Division of Clinical Chemistry, Department of Laboratory Medicine, Medical University of South Carolina, Charleston, SC 29403

ABSTRACT

A method is presented for measuring simultaneously five anti-convulsants (primidone, ethosuximide, phénobarbital, carbamazepine and diphenylhydantoin) in serum and plasma by high-performance liquid chroma tography (H PLC) usingalphenal (5 - allyl - 5 - phenyl - barbituric acid) as the internal standard. All five drugs are separated from each other and, in the case of primidone, from its metabolite, 2 - phenyl - 2 - ethyl - malondiamide. Total HPLC time for the separation is seven minutes. The chromatography is performed on a C-8 reverse phase column with a mobile phase consisting of acetonitrile/phosphate buffer (35/65) at 25°C. The eluted drugs are detected at 220 nm and quantitated from their peak heights relative to that of the internal standard. The lower limits of detection for each drug is 200 ng per ml for primidone, 1000 ng per ml for ethosuximide, 200 ng per ml for phéno barbital, 100 ng per ml for carbamazepine and 200 ng per ml for diphenylhydantoin. Analytical recoveries for the five drugs ranged from 97 to 107 percent. Correlation of results for 187 specimens by enzyme-immunoassay (EMIT) was 0.981 for primidone, 0.827 for ethosuximide, 0.975 for phéno barbital, 0.889 for carbamazepine and 0.990 for diphenylhydantoin. In the HPLC assay, ethotoin was found to interfere with phénobarbital and phensuximide with carbamazepine.

Introduction of epileptic patients4,9,10'11 and has placed an increasingly heavier workload on the Continuous monitoring of patients re laboratory which has the capability of per ceiving anticonvulsant drug therapy has forming these assays. Consequently, in become a routine aid in the management addition to accuracy and precision, the demand is increasing for drug assays * Address reprint requests to: Dr. Richard H. which are more convenient, less time con Gadsden, Department of Laboratory Medicine, Medical University of South Carolina, 171 Ashley suming, pertinent to therapeutic monitor Avenue, Charleston, SC 29403. ing and economical. 89 0091-7370/0100-0089 $00.90 © Institute for Clinical Science, Inc. 90 RYDZEWSKI, GADSDEN AND PHELPS

The high-performance liquid chroma specimen. The HPLC analysis was per tography (HPLC) assay that is used in our formed within one week of receipt of the laboratory is more convenient than specim en. analysis by gas-liquid chromatography in that there is no requirement for derivatiza- C hromatography tion. A single set of analytical conditions allows for simultaneous determination of A DuPont 848 Liquid Chromatograph the drugs and the HPLC assay is more equipped with a DuPont Model 837 rapid, specific and less expensive than UV-VIS spectrophotometer, a Universal enzyme immunoassay (EMIT) when more Septumless Injector with a 40 ¿¿1 loop, and than one drug is measured in the same a DuPont CLC 1 reverse phase modified sample. The accuracy of the HPLC assay C-8 liquid chromatography column (25 cm is increased by addition of the internal x 4.6 mm), was used to perform the HPLC standard prior to pretreatment to the analyses. A guard column (3 cm x 2.1 mm) packed with DuPont Permaphase ETH standards, controls and samples.7 was attached to the analytical column to While many HPLC methods have been prevent column blockage and deteriora reported for the assay of anticonvulsants tion.* The columns were eluted at 25°C by reverse phase chromatography using a w ith the m obile phase at a flow rate of 2 ml C-18 column, and with analysis times of per min (2100 psi) and the effluent moni up to 20 minutes,1’2’7,8’9,11'15 the present tored at 220 nm. authors were able to achieve separation of all five drugs, with good baseline reten E nzymeimmunoassay ( E M I T ) tion, recovery and sensitivity in less than seven minutes with a C-8 reverse phase The routine EMIT analyses were per column. In addition, our HPLC assay al formed with a MicroMedic MS-2 spectro lowed us to measure levels of all five photometer equipped with a Digital drugs at both therapeutic and acknowl Kinetic A nalyzer and a T em perature C on edged toxic concentrations.16 troller. f A MicroMedic Automatic A comparative study was conducted be Pipettef equipped with a 100¿il (sample) tween our HPLC assay and the EMIT and 1 ml (reagent) pump was used to di assay with 187 plasma specimens. Statis lute and transfer the EMIT reagents and tical analysis of the study’s data showed samples. Measurements were made at excellent correlation between the assays cuvette tem perature of 30° C. All calibration for primidone, phenobarbital, carba- standards and EMIT reagents were re mazepine and dephenylhydantoin as well constituted as directed by the manufac as a fair correlation for ethosuximide. turer, and all data plotted on the appropri ate graph papers included with each set of Methods and Materials reagents.

P a t i e n t S a m p l e s Reagents and Reference Materials

Plasma from 187 blood specimens Phosphate Buffer. Prepared with sterile routinely submitted for analysis for anti water, 20 mmol per 1 KH2P 0 4 and 1 mmol convulsant drugs by EMIT was separated per 1 K2H P04, adjusted to pH 5.6 at 20°C into two aliquots, one being stored at 2 to as necessary and vacuum filtered. 6°C for the EMIT analysis and the other frozen until the HPLC analysis could be * All are available from DuPont DeNemours, Inc., Clinical Specialties Group, Wilmington, DE 19898. performed. The EMIT assay was con f Available from MicroMedic Systems, Horsham, ducted within 48 hours of receipt of the PA. HPLC DETERMINATION OF ANTICONVULSANT DRUGS IN PLASMA 91

Mobile Phase. Acetonitrile/phosphate TABLE I buffer mixture at a 35/65 ratio by volume. Peak Heights and Ratios of Aqueous Standard This is vacuum filtered prior to use. Working R a tio Drug/ Drug Standards. The following drugs Standard I n te r n a l Drug N (Mg/ml) H eight Standard were obtained: Ethosuximide and di phenylhydantoin;! Carbamazepine;§ Primidone 48 10.3 54.6 ± 4.6 0.54 ± 0.03 Ethosuximide 48 164.2 • 182.9 ± 5.5 0.86 ± 0.04 primidone;11 phenobarbital;1i and alphenal Phénobarbital 48 25.8 143.2 ± 3.8 1.43 ± 0.03 Alphenal 48 20.0 99.8 ± 3.1 - (5 - allyl - 5 - phenyl - barbituric acid).** Carbamazepine 48 8.1 66.5 ± 1.6 0.71 ± 0.01 48 20.4 84.1 ± 4.4 Stock solutions of each of these drugs Diphenylhydantoin 0.86 ± 0.03 were prepared in 10 percent methanol. A working standard (table I) was prepared under a stream of dry nitrogen at room weekly from the stock solutions at concen temperature. The residue is dissolved in trations consistent with the individual 200 ¡i\ of mobile phase and 50 /u,l injected drug’s therapeutic range. Alphenal, the into the chromatograph. An anticon internal standard, was made to 20 fig per vulsant control serum and the working ml. The stock solutions, when stored in standard were processed in the same brow n glass bottles at 2 to 6°C, w ere found manner within each run. to be stable for at least two months. Standard curves including the internal Control serum. Anticonvulsant control standard (alphenal) were run for each of serum containing the five anticonvulsant the five anticonvulsant drugs studied and drugs stu d ied .! f alphenal, in both spiked plasma and EMIT reagents and calibrator aqueous solutions. The data illustrating standards. The EMIT reagents were pur limit(s) of linearity under analytical condi chased in kit form, as were the EMIT cali tions are summarized in table II. bration standards.^ I Solvents. Acetonitrile and chloroform, Results and Discussion both ultraviolet grade, distilled in glass. § § These were vacuum filtered prior to The total time to chromatograph the five using. anticonvulsants by this system is slightly less than seven minutes. In figure 1 is Procedure shown the chromatogram of a mixture of the drugs and the internal standard, al Chromatography: Two hundred micro phenal. Two additional compounds liters of the alphenal working solution and which were adequately resolved by this 75 jLtl of glacial acetic acid were added to system w ere 2 - phenyl - 2 - ethyl - m alon- 200 ill o f serum o f plasm a and vortexed for diamide (PEMA), the metabolite of 30 seconds. Five milliliters of chloroform primidone, and the anticonvulsant, are added to the mixture and shaken for five minutes, followed by five minutes of T A B L E II centrifugation at 2,000 rpm. The chloro Linearity of Standards form phase is collected and evaporated

R ange* I Parke Davis and Co., Detroit, MI. Concentration Attentation § Geigy Pharmaceuticals, Div. Ciba-Geigy Drug ]ig per ml AVFS Corp., Summit, NJ. II Ayerst Laboratories, Inc., New York, NY. Primidone 0.5 - 46.4 0.32 H Mallinckrodt Chemical Works, New York, NY. Etho suximide 1.3 - 249.2 0.08 ** Ganes Chemicals, Inc., Carlstadt, NJ. Phénobarbital 0.5 - 52.6 0.32 11 Ortho Diagnostics, Inc., Raritan, NJ. Carbamazepine 0.2 - 11.8 0.16 t } SYVA Corp., Palo Alto, CA. Diphenylhydantoin 0.5 - 50.8 0.16 § § Burdick and Jackson Labs., Muskegon, MI. 92 RYDZEWSKI, GADSDEN AND PHELPS

raphy assay for the five drugs in a pre pared pool of commercial material. The accuracy of the liquid chroma tography assay was determined by measuring the recovery of known amounts of drugs added to pooled plasma which was proved to be free of drugs by EMIT and HPLC assay. Aliquots of the plasma were spiked to contain the concen trations listed in table V for each of the drugs. The plasma was extracted and analyzed by the method described except that 4 - methyl - primidone (4-M-P) was

TIME (HIM)

F IG U R E 1. HPLC chromatogram of five anti convulsant drugs: (1) prim idone; (2) zarotin; (3) phenobarbital; (4) internal standard (alphenal); (5) carbamazepine; and (6) diphenylhydantoin. Chromatographic conditions: flow rate of 2.0 ml per min; pressure of 2,150 psi; wavelength of 220 nm; and chart speed of 2 min per in. mephenytoin (figure 2). In table II are summarized retention times for a number of compounds and drugs chromato graphed on this system. During the study, over 400 samples were injected onto the analytical column. The peak heights of the aqueous working standard and the SYYA EMIT standards remained essentially constant throughout the study, indicating little column de TIRE (MIS) terioration. In table I are shown the varia F ig u r e 2. HPLC chromatogram of (1) PEMA, (6) mephenytoin and the following anticonvulsants: tions in the peak heights and the ratios (2) primodone; (3) zarotin; (4) phenobarbital; (5) between the heights of the drug peaks and internal standard (alphenal) (7) carbamazepine; the internal standards for 48 consecutive and (8) diphenylhydantoin. Chromatographic con ditions: flow rate of 2.0 ml per min; pressure of daily runs. In table IV is shown the day- 2.50 psi; wavelength of 200 nm; and chart speed to-day precision of the liquid chromatog of 2 min per in. HPLC DETERMINATION OF ANTICONVULSANT DRUGS IN PLASMA 93

TABLE III TABLE IV

Retention Times for Compounds and Day-to-Day Precision of Anticonvulsant Drugs Chromatographed on EMIT Control Serum by HPLC

Retention Times Target HPLC Range Compound Tim e (LC m in u te s) Cone. (\xg/m l) C.V. Drug N (\ig /m l) Mean + S .D . %

Bilirubin Not detected Meperidine Not detected Primidone 48 7.5 7.1 0.3 4.2 Meprobamate Not detected Ethosuximide 48 50.0 47.5 3.0 6.3 Methamphetamine Not detected Phénobarbital 48 28.3 29.1 0.6 2.1 Methylphenidate Not detected Carbamazepine 48 3.7 3.2 0.3 9.4 Nicotine Not detected Diphenylhydantoin 48 10.8 10.3 0.5 4.9 Sodium valproate Not detected Acetaminophen Less them 1.5 Acetazolamide Less than 1.5 Caffeine Less than 1*5 Dramamine Less than 1.5 These were calculated as follows: propoxyphene Less than 1.5 Theophylline Less than 1.5 2-Phenyl-2-ethyl-malondaimide 1.9 plasma drug peak height plasma 4-M-P peak height Primidone 2.0 Absolute Recovery % =-*■------— — ——— -a-.— Barbital 2.3 aqueous drug peak height Ethosuximide 2.3 aqueous 4-M-P peak height Phenylethylhydantoin 3.0 Carbamazepine epoxide 3.3 Ethotoin 3.5 The absolute recoveries in table V were Phénobarbital 3.5 Codeine 3.9 calculated from comparison of the drugs to Metharbital 3.9 the internal standards in plasma samples N-desmethyl-methsuximide 4.1 Phenacetin 4.2 to the results obtained from aqueous so Alphenal 4.4 lutions. Calculation of absolute recoveries Chloridiazepoxide 4.5 Mephenytoin 5.1 is a measure of analytical efficiency and Carbamazepine 5.9 Phensuximide 5.9 essentially eliminates variances owing to Diphenylhydantoin 6.3 Pentobarbital 6.5 Cortisol 6.6 T A B L E V Amobarbital 6.7 Hexobarbital 7.1 Recovery Data of Drugs Added to Plasma Nitrazepam 7.6 Glutethimide 8.2 Mephobarbital 8.2 R e l a t i v e A b s o lu te Methsuximide 8.2 Cone. R e c o v e ry R e c o v e ry Secobarbital 8.6 Drug N (M g/ml) P e r c e n t P e r c e n t Methaqualone More than 10 Amphetamine More than 10 Diazepam More than 10 Primidone 16 1 0 .9 1 05 103 21.8 99 99 32.7 97 97 43.6 101 100

Ethosuximide 16 124.6 108 107 added to the 200 ju, 1 of mobile phase used 186.9 105 105 to dissolve the drug residue prior to injec 249.2 101 101 tion. The 4 - methyl - primidone served as Phénobarbital 16 12.7 98 98 25.4 99 98 an external standard for calculation of the 38.1 96 96 absolute recovery data. 50.8 99 99

The percent absolute recovery listed in Carbamazepine 16 3.0 107 107 table V was derived from comparison of 6.0 102 102 9.0 98 98 the ratio of the peak height of each drug in 12.0 100 101 plasma to the height of the 4 - methyl - Diphenylhydantoin 16 12.5 102 102 primidone peak (20 ¿¿g per ml), to the ratio 25.0 99 99 37.5 99 100 of the peak heights of the drug in aqueous 50.0 104 102 solution to the 4 - methyl - primidone. RYDZEWSKI, GADSDEN AND PHELPS 9 4

TABLE V I 2. A t w e l l , S., G r e e n , V., and H a n e y , W.: D e Statistical Results for Comparison Study velopment and evaluation of a method for Regression Line simultaneous determination of phénobarbital and diphylhydantoin in plasma by high-

Standard pressure liquid chromatography. J. Pharm. Sci. E rro r 64:806, 1975. Correlation Inter o f 3. B a u m e l, 1. P., G allagher, B. B., and Drug N Coefficient cept S lo p e E s t in a t e MATTSON, R. H.: Phenyl ethyl malonamide (PEMA), an important metabolite of primidone. Primidone 40 0.981 -0.798 1.102 0.664 Arch. Neurol. 2 7 :34, 1972. Ethosuximide 40 0.827 -9.636 1.094 14.097 Phénobarbital 40 0.975 0.137 1.018 1.719 4. B u c h t a l , F. and Lennox-Buchtal, M. A.: Carbamazepine 40 0.889 -0.420 1.054 1.279 Relation of anticonvulsant effect to concentra Diphenyldantoin 40 0.990 0.104 0.972 0.994 tion in serum. Antiepileptic Drugs. Woodbury, D. M., Penny, J. K., and Schmidt, R. P., eds. New York, Raven Press, 1972. injection volume, column temperature 5. Castro, A., Ihanez, J., D iC e s a r e , J. L., and elution pressure. A dam s, R. F., and M a lk u s , H.: Comparative determination of phenytoin by spectro The data from the comparison study in photometry, gas chromatography, liquid volving clinical specimens assayed in chromatography, enzyme immunoassay, and duplicate by both EMIT and HPLC and radioimmunoassay. Clin. Chem. 24:710, 1978. 6. “ E M IT -ead ” Instruction Booklet are presented in table VI. The statistical 6A164-5. SYVA Corporation, Palo Alto, CA, analysis indicates relatively good correla 1978. tion between the EMIT and HPLC assays 7. K ab ra, P. M., G otelli, G., S tanfill, R., and M artO N , L. J.: Simultaneous measurement of for all the drugs except ethosuximide. phénobarbital, diphenylhydantoin and This poorer correlation may be due to the primidone in blood by high-pressure liquid EMIT ethosuximide assay having less chromatography. Clin. Chem. 22:1070, 1976. 8. K ab ra, P. M. and M a r t o n , L. J.: D eterm in precision than the other EMIT anticon ation of carbamazepine in blood or plasma by vulsant drug assays and the liquid high-pressure liquid chromatography. Clin. chromatography assay being less sensi Chem. 22:1070, 1976. tive for ethosuximide compared to the 9. K ab ra, P. M., Stafford, B. E., and M a r t o n , L. J.: Simultaneous measurement of phéno other drugs. barbital, phenytoin, primidone, ethosuximide, It has been observed that severe and carbamazepine in serum by high-pressure liquid chromatography. Clin. Chem. 23:1284, hemolysis and lipemia interfere with the 1977. EMIT assay, producing erroneous re 10. K u tt, H. and P e n r y , J. K.: Usefulness of blood sults.1 No differences have been found by levels of antiepileptic drugs. Arch. Neurol. us in recovery between severely 31 :283, 1974. 11. N a n d e d k a r , A. K. N ., Ku t t , H ., a n d F a ir - hemolyzed or lipemic specimens, spiked C L O U G H , G. F.: Correlation of the EM IT with a with known amounts of drugs, and normal gas-liquid chromatographic method for deter plasma, thus concluding that they are not mination of antiepileptic drugs in plasma. Clin. Tox. J2:483, 1978. interferences in the HPLC assay. Also, 12. Pippenger, C. E. and G illen, H. W.: Gas simultaneous determination of the drugs chromatographic analysis for anticonvulsant in less than seven minutes and the larger drugs in biological fluids. Clin. Chem. 15:582, 1969. assay range for the liquid chromatography 13. Pippenger, C. E., P e n r y , J. K., and K u lt , H.: method tend to make the analysis of a pa Antiepileptic Drugs: Quantitation Analysis and tient receiving more than one anticon Interpretation. Raven Press, New York, 1978. 14. R o s e , S. W., S m ith , L. D., and P e n r y , J. K.: vulsant drug less expensive and time con Blood level determinations of antiepileptic suming than the EMIT assay and other drugs. Clinical Value and Methods. Bethesda, cited HPLC assays. National Institute of Neurological Diseases and Stroke, National Institutes of Health, 1971. 15. SOLDIN, S. J. and HILL, J. G.: Rapid micro References method for measuring anticonvulsant drugs in serum by high-pressure liquid chroma 1. A d a m s , R. F. and VANDERMARK, F. L.: Simul tography. Clin. Chem. 22:856, 1976. taneous high-pressure liquid chromatographic 16. WlNEK, C. L.: Tabulation of therapeutic, toxic, determination of some anticonvulsants in and lethal concentrations of drugs and chemi serum. Clin. Chem. 22:25, 1976. cals in blood. Clin. Chem. 22:832, 1976.