J. Neurol. 213, 41--46 (1976) © by Springer-Verlag 1976

Measurement of Diphenylhydantoin and Phenobarbital by Enzyme Immunoassay and Gas-Liquid Chromatography Dieter Schmidt Abteilung fiir Neurologie (Leiter: Prof. Dr. D. Janz), Klinikum Charlottenburg, l%eie Universit~t Berlin Received March 3, 1976

Summary. A new enzyme-multiplied immunoassay technique (EMIT) was compared with Kupferberg's gas-liquid chromatography (GLC) for the determination of diphenylhydantoin and phenobarbital. 116 plasma samples of patients with epilepsy were examined simultaneously by EMIT and GLC. The precision of repeated determinations was similar for both procedures. There was good agreement between the EMIT and GLC results. The rapid and precise analysis, made possible by the EMIT system, is useful for the treatment of epilepsies. Key words:Enzyme immunoassay-- Gas-liquid chromatography - - Diphenylhydantoin - Phenobarbital - - Epilepsy. Zu~ammen/assunfl. Ein neues enzymimmunologisches Verfahren (EMIT) zur Bestimmung yon Diphenylhydantoin und Phenobarbital wurde mit der gaschromatographischen Methode yon Kupferberg (GLC) verglichen. Es wurden dazu 116 Plasmaproben yon Patienten mit Epilepsie mit beiden Verfahren bestimmt. Die Pr~zision wiederholter Bestimmungen derselben Probe war bei beiden Verfahren ~hnlich. EMIT- und GLC-Resultate zeigten gate ~bereinstimmung. Die schnelle und pr~zise EMIT-Analyse ist fiir die Behandlung yon Epilepsien yon Nutzen. Introduction T h e d e t e r m i n a t i o n o f a n t i e p i l e p t i c d r u g s in blood i m p r o v e s t h e efficacy a n d s a f e t y o f d r u g t h e r a p y for e p i l e p s y [3, 11]. T h e results will be m o s t helpful if t h e y are a v a i l a b l e while t h e p a t i e n t is in t h e office. R e c e n t l y a simple a n d r a p i d e n z y m e - m u l t i p l i e d i m m u n o a s s a y t e c h n i q u e ( E M I T ) b e c a m e a v a i l a b l e for t h e d e t e r m i n a t i o n of d i p h e n y l h y d a n t o i n a n d p h e n o b a r b i t a l in small v o l u m e s o f p l a s m a or s e r u m [6, 9]. This i n v e s t i g a t i o n was d e s i g n e d t o c o m p a r e t h e e n z y m e i m m u n o a s s a y w i t h gas-liquid c h r o m a t o g r a p h y which is a well e s t a b l i s h e d m e t h o d for t h e d e t e r m i n a t i o n o f a n t i e p i l e p t i e drugs.

Methods

Enzyme Immunoassay The assay is a homogeneous enzyme immunoassay technique [8]. An antiepileptic drug is conjugated with the enzyme glucose-6-phosphate dehydrogenase. An antibody to the antiepileptic drug binds with the enzyme-labeled drug in a manner which blocks the active site

42

D. Schmidt

of the enzyme [5]. I f an antiepileptie drug is present in the patient's plasma, the drug will compete with the enzyme-labeled drug for the antibody. The antibody-drug-complex makes the antibody unavailable for binding with the enzyme-labeled drug. As a result, the residual enzyme activity is proportional to the antiepileptic drug concentration in the sample. Apparatus. A Gilford 300 N microsample spectrophotomcter equipped with a thermally regulated flowcell is connected to an EMIT printer calculator. Sample and reagents are measured with an EMIT pipettor dilutor. Reagents. Reagents are commercially available from Syva Corporation. Reagent A contains the antibody and the substrate, Reagent B is the drug-enzyme complex. They arc available in the lyophilized form and are reconstituted in destilled water. The Tris-(hydroxymethyl)-amino-mcthan-hydrochloride buffer (pH 7.9) with added surfaetant, and a series of serum calibrators containing diphenylhydantoin and phenobarbital in concentration ranges from 0--30 and 0--80 ~g/ml respectively, are also commercially available from Syva Corporation. Procedure. 50 ~zl of standard or unknown plasma plus 250 ~l of buffer are transferred with the pipettor-dilutor to a 1 ml disposable beaker. This diluted sample may be used for up to 5 assays. It is best to wait 1 rain before further processing the sample. 50 ~l of the diluted plasma plus another 250 ~l of buffer arc pipetted in a second beaker. Then, 50 ~l of l~eagent A, plus 250 ~zl of buffer, and 50 ~l of Reagent B with 250 ~l of buffer, are added. Immediately after the addition of Reagent B the contents of the second beaker are aspirated into the flowcell of the spectrophotometer. This activates the printer/calculator. Two absorbance readings are made at 340 nm within 80 s. The difference of the absorbance readings is the result. The calibrator curve is prepared with the calibrators, the results being plotted on a modified log paper to obtain a linear curve. The concentration of the sample is read from the calibrator curve.

Gaschromatography The methylating gas chromatographic procedure of Kupferberg with 5-(p-methylphenyl)5-phenylhydantoin, MPPH, as internal standard was used as previously described [1, 12J. For quantitation of phenobarbital p-teluol-phenobarbital, PtPhb, was compared to MPPtt as internal standard [2, 7].

Design o/ the Study 116 plasma samples, most of them from patients on several antiepileptic drugs, were determined by EMIT and GLC simultaneously in double determinations. Pooled specimens of diphenylhydantoin and phenobarbital were processed ten times by EMIT and seven times by GLC to check the precision of repeated determinations.This design allowed for the evaluation of the reproducibility and agreement of EMIT and GLC in common samples. For statistical analysis, the correlation coefficient and the coefficient of variation were determined.

Results

Precision T h e r e p r o d u c i b i l i t y of E M I T and GLC was similar for b o t h drugs (Table 1). T h e precision o f r e p e a t e d GLC d e t e r m i n a t i o n s of p h e n o b a r b i t a l i m p r o v e d w h e n P t P h b was e m p l o y e d as i n t e r n a l s t a n d a r d instead of M P P H (Table 1).

Agreement between E M I T and GLC P l a s m a levels assayed b y E M I T and GLC were e v a l u a t e d for differences bet w e e n b o t h procedures (Table 2). F o r d i p h e n y l h y d a n t o i n d e t e r m i n a t i o n s , b o t h E M I T a n d GLC results were in excellent a g r e e m e n t as i n d i c a t e d b y t h e small difference b e t w e e n E M I T and GLC p l a s m a levels and t h e high coefficient o f corr e l a t i o n (r --~ 0.982) as shown in F i g u r e 1.

Measurement of Diphenylhydantoin and Phenobarbital

43

Table 1. Precision of repeated determinations Sample

Mean concentration (y.g/ml)

Coefficient of variation (%)

DPH EMIT GLC MPPH

14.35 14.25

5.3 1.9

Phb EMIT Phb GLC MPPH GLC PtPhb

30.10 21.00 23.00

5.3 8.7 2.4

Emit 40" lig/m I

30

20

10

llm

O

.

. 10

.

.

2'0

.

:30

40

GLC lig/ml

Fig. 1. EMIT and GLC results of diphenylhydantoin samples; EMIT ~ 0.97 GLC + 0.35; r = 0.982, P < 0.001, ~ = 88

For quantitative determination of phenobarbital, EMIT and GLC with PtPhb as i n t e r n a l s t a n d a r d p r o d u c e d e x c e l l e n t a g r e e m e n t in c o r r e l a t i o n a n a l y s i s (Fig. 2) a n d m e a n results (Table 2). A g r e e m e n t o f gas c h r o m a t o g r a p h i c results w i t h E M I T d a t a was b e t t e r w h e n P t P h b was chosen as i n t e r n a l s t a n d a r d i n s t e a d o f M P P H (Fig. 3).

Discussion T h e r e is r e a s o n a b l e consensus t h a t w i t h r e p e a t e d d e t e r m i n a t i o n s o f d i p h e n y l h y d a n t o i n a n d p h e n o b a r b i t a l n o single v a l u e s h o u l d differ f r o m t h e m e a n b y m o r e t h a n 6 ° . T h i s is t h e case for e n z y m e i m m u n o a s s a y a n d gas c h r o m a t o g r a p h i c d e t e r m i n a t i o n s o f b o t h drugs.

44

D. S c h m i d t GLC PTIPHb ~glml

80-

60-

504030O•o

•:

2010W

~b

o

2b

ab

~

5b

~

/a

8b

~o

jug/ml EMIT

Fig. 2. EMIT and GLC resuIts of phenobarbital samples with PtPhb as internal standard for GLC; GLC = 0.99 EMIT % 0.1; r -~ 0.991, P < 0.001, n ~ 28 GLC MPPH pg/ml

90-

8070605040300o •

20-

• 8

10V

0 ~ug/ml EMIT

Fig. 3. E M I T a n d GLC r e s u l t s of p h e n o b a r b i t a l w i t h M P P H as i n t e r n a l s t a n d a r d for G L C ; GLC = 1.16 E M I T - - 2.9; r = 0.983, P < 0.001, n = 28

Agreement between GLC and EMIT was very satisfactory as indicated b y the high correlations and low differences in mean results of identical samples. The difficulties of the gas chromatographic analysis of phenobarbital are well known [2, 4]. The choice of the internal standard is critical. Pippenger and Kupfer-

Measurement of Diphenylhydantoin and Phenobarbital

45

Table 2 Mean diphenylhydantoin (DPH) and phenobarbital (Phb) concentrations of common samples Method

DPH n ----88

Phb n = 28

EMIT (~g/ml) GLC (~g/ml) Difference between EMIT and GLC (~g/ml)

9.27 9.23 0.04

33.84 33.81 0.03

berg reported better results with P t P h b as internal standard t h a n with M P P H [2, 7]. These reports are confirmed here. The precision of repeated determinations and the agreement between enzyme immunoassay and gas chromatography improved when P t P h b was employed as internal standard. As a consequence, P t P h b should be prefered to M P P H as internal standard for gas chromatographic determinations of phenobarbital. A major advantage of enzyme immunoassay is the speed of the measurement which requires only 3 rain after the daily calibration curve has been established. This will, in contrast to GLC, result in the rapid analysis of antiepileptic drugs. I t will be useful for immediate detection of intoxications. For intravenous drug treatment, e.g. in status epilepticus or loading therapy it will be of advantage to have early results for the correction of t r e a t m e n t [10]. The plasma concentration can be determined during a visit of the patient in the office. The physician can correct the medication immediately if necessary [11]. A sample of 0.05 ml is sufficient which is an advantage for the analysis in children and when frequent sampling is necessary. There is no need for a highly skilled laboratory operator as the actual procedure is rather simple. Close supervision b y an experienced laboratory technician is mandatory. Currently earbamazepine, dipropylaeetate and ethosuximide cannot be determined b y EMIT. The system has no screening ability for unsuspected drugs or metabolites. For a research laboratory the GLC will still be the method of choice due to its high versatility and excellent screening ability for studies of new drugs or metabolites of well known drugs. For a clinical laboratory the E M I T is the method of choice due to its simple, rapid and precise analysis in small volumes of blood. The cost of the apparatus is similar for GLC and EMIT, while the cost of the E M I T reagents is higher, especially for measurement in samples with several antiepfleptic drugs. I n conclusion, the E M I T system produces accurate and rapid quantitative determinations of dipheny]hydantoin and phenobarbital for effective clinical management of the patient with epilepsy.

Acknowledgements. For drug level detmrminations the author gratefully thanks I. Einicke. Reagents were partly provided by Syva Corporation, Palo Alto, California, USA. The study w a s supporimd by the Bundesministerium f/ir Jugend, Familie und Gesundheit. References 1. Kupferberg, H. J.: Quantitative estimation of diphenylhydantoin, primidone and phenobarbital in plasma by gas-liquid chromatography. Clin. ehim. Acta 29, 283--288 (1970)

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2. Kupferberg, H. J., Yonekawa, W.: Antiepileptic drug determination: the needs of the clinical chemistry laboratory in comparison to the research laboratory. Submitted for publication 3. Kutt, H., Penry, J. K.: Usefulness of blood levels of antiepileptie drugs. Arch. Neurol. 31, 283--288 (1974) 4. Osiewicz, R., Aggarwal, V., Young, R. M., Sunshine, I.: The quantitative analysis of phenobarbital with trimethylauilinium hydroxide. J. Chromatogr. 88, 157--164 (1974) 5. Pippenger, C. E., Bastiani, R. J., Schneider, R. S. : Evaluation of an experimental homogeneous enzyme immuno-assay for the qantitation of diphenylhydantoin and phenobarbital in serum or plasma. In: Clinical pharmacology of antiepileptic drugs (eds. H. Schneider et al.), pp. 331--335. Berlin-Heidelberg-New York: Springer 1975 6. Pippenger, C. E., Kutt, H. : Clinical applications of a homogeneous immunoassay system (EMIT) for the detection of diphenylhydantoin and phenobarbital. Epilepsia 16, 197 (1975) 7. Pippenger, C. E. : Personal communication, 1975 8. Rubenstein, K. E., Schneider, R. S., Ullman, E. F.: Homogeneous enzyme immunoassay - - A new immunochemical technique. Biochem. biophys. Res. Commun. 47, 846--851 (1972) 9. Schmidt, D., Goldberg, V., Guelen, P. J. M., Johannessen, S., Kleijn, E. v. d., Meijer, J. W. A., Meinardi, H., Richens, A., Schneider, H., Stein-Lavie, Y., Symann-Louette, N. : Comparison of gasehromatography and immunoassay for determination of diphenylhydantoin and phenobarbital - - Results of an European Collaborative Control Study. Presented at the Seventh International Symposium on Epilepsy, 19--21 June, Berlin (West), 1975. Stuttgart: Thieme (in press) 10. Schmidt, D., Vogel, A.: Plasmakonzentrationen nach Injektion und Infusion yon Phenytoin. Klin. Wschr. (in press) 11. S c h m i d t , D . : Blutspiegelbestimmungen yon Antiepileptika. Eine neue Methode der Epilepsie-Therapie. Aktuelle l~eurologie (in press) 12. Schmidt, D., Kupferberg, H. J.: Diphenylhydantoin, phenobarbital and primidone in saliva, plasma and eerebrospinal fluid. Epilepsia 16, 735---741 (1975) Dr. D. Schmidt Abteilung fiir Neurologie Klinikum Charlottenburg Freie Universit~t Berlin Spandauer Damm 130 D-1000 Berlin 19