ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 10, No. 1 Copyright© 1980, Institute for Clinical Science, Inc. 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 diphenyl- hydantoin) 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 diphenyl- hydantoin. 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 phen- suximide 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.