Simultaneous analysis of phenobarbital and p-hydroxyphenobarbital in biological fluids by GLC–chemical-ionization mass spectrometry

Simultaneous Analysis of Phenobarbital and p -Hydroxyphenobarbital in Biological Fluids by GLC-Chemical-Ionization Mass Spectrometry INDRAVADAN H. PATEL *$ RENB H. LEVY **., WILLIAM ,F. TRAGER *

JOHN M. NEAL *, and

Received June 11,1979, from the *Department of Pharmuceutical Sciences, School of Pharmacy, and the :Department of Neurological surgery, !Present address: Department Accepted for publication April 1,1980. School of Medicine, University of Washington, Seattle, WA 98195. of Pharmacokinetics and Biopharmaceutics, Hoffmann-La Roche Inc., Nutley, NJ 07110. Abstract A sensitive and specific GLC-chemical-ionization mass spectrometric method was developed for the simultaneous assay of phenobarbital (I) and p-hydroxyphenobarbitaal(I1)in biological fluids (urine and plasma) using stable isotope analogs of the compounds as internal standards. After extraction, the compounds were methylated with diazomethane and quantitated by CLC-chemical-ionization mass spectrometry. The detection limit of the method was 0.1 pg/ml for both compounds. The intraday precision (RSD) for 0.4-2.4 pglml was <2% for I and <4%for II.The interday precision for 0.55 and 2.11 p g l d of each compound was 5.5 and 2.996 for I and 7.3 and 5.0%for II,respectively.This method has been applied in several pharmacokinetic studies. Keyphrases 0 Phenobarbital-simultaneous analysis with p-hydroxyphenobarbital by CLC-chemical-ionization mass spectrometry in biological fluids 0 p-Hy oxyphenobarbital--simultaneoue analysis with phenobarbital by CL -chemical-ionization mass spectrometry in biological fluids 0 Antiepileptic drugs-phenobarbital, simultaneom andpis with p-hydroxyphenohbid by GLC-chemical-ionizatiOXlmass spectrometry in biological fluids

t

Numerous methods are available for the quantitation of phenobarbital (I)alone or in the presence of other antiepileptic drugs in plasma. In contrast, relatively few methods have been reported for the simultaneousanalysis of I and its known major metabolite, p-hydroxmhenobarbital (11). Butler (1) reported a spectroscopicmethod, which depends on the initial separation of I from I1 by countercurrent distribution. Subsequently, GLC prmedures requiring on-column methylation were reported for the simultaneous analysis of I and I1 in urine (2, 3). A thin-layer densitometric method was proposed recently by Levin et al. (4). However, all of these methods have a detection limit of -1 pg/ml. This report describes a GLGchemical-ionization,stable isotope assay that is sensitive (the detection limit is 0.1 pg/ml or 0.1 pg using a 1-ml sample for I and 11) and can measure I and I1 simultaneously in urine and plasma. METHODS Chemicals-Phenobarbital', p-hydroxyphenobarbitaP, (1,3-I6N (gg??,), 2 . W (90%)]phenobarbital3 (internal standard for I), [1,3-IbN (m), 2-1% (9096)]-p-hydroxyphenobarbital~ (internal standard for XI), p-~lylsulfonylmethylnitrosamide2, reagent grade n -hexane and ether, and nanograde methylene chloride' were obtained commercially. Solutions-The drug standard and internal standard solutions were prepared in methylene chloride. The concentrations of I and I1 were 2, 4 8 , and 12 pglml. The internal standards for I and I1 were prepared a t 10 pg/ml. Phthalate (0.14 M ,pH 5) and borate (0.05 M ,pH 8.5) buffers were used. Plasma Extraction-Two hundred microliters of a standard solution Chemical Co., St.Louie Mo. *1 Sigma Aldrich Chemical Co., Milwaukee, Wis.

Isotepes, Cambridge M a . Mallinckrodt,St.Louis, ho.

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1218 1 Journaiof PharmaceuticalSciences Vol. 69, No. 10. October 1980

Table I-Calibration Curve Data for the Analysis of Phenobarbital (I)in Plasma and for the Simultaneous Analysis of I and p-Hydroxyphenobarbital(I1) in Urine Biological Compound Fluid

Concentration,pg/ml

Ion Ratio, mean", pg (fRSD)

Linear Regression Parameterb ~

Plasma

I

Urine

I

Urine

I1

a

n = 6.

0.4 0.8 1.6 2.4 0.4 0.8 1.6 2.4 0.4 0.8 1.6 2.4

0.457 (f0.44b) 0.882 (f0.596) 1.730 (f1.270) 2.533 (f0.7%) 0.419 (&0.9%) 0.827 (f0.7W) 1.636 (f0.890) 2.442 (24.4%) 0.495 (f2.0%) 0.992 (f3.4%) 1.963 (i1,6%) 2.902 (i1.7%)

r = 0.9999 rn = 1.0394 b = 0.0493 r = 1.0000 rn = 1.0113 b = 0.0160

r = 0.9999

rn = 1.2034 b = 0.0233

r m the correlation coefficient,m is the slope, and b is the intercept.

of I and 100 pl of the internal standard solution for I were transferred to a 12-ml screw-capped tube and evaporated under a nitrogen stream at 40°. Then 1.0 ml of drug-free plasma, 2 ml of phthalate buffer, and 8 ml of methylene chloride were added to the residue. The tube was capped with a polytef-lied screw cap, and the contents were shaken at a moderate speed for 15 min on a reciprocating shaker. After centdugation (2000 rpm, 10 min). the methylene chloride extract (-5 ml) was recovered and evaporated under a nitrogen stream at 40°. The residue was dissolved in 2 ml of borate buffer and washed with 8 ml of n-hexane. The borate buffer (-1.8 ml) then was acidified with 1 ml of phthalate buffer, and the compounds were extracted into 8 ml of methylene chloride. The methylene chloride (-6 rnl), separated after centrifugation, was methylated with diazomethane (5) and evaporated to dryness. The residue waa dissolved in 40 jd of methanol, and 5-10 pl was injected onto the gas chromatograph for the CLC-chemical-ionization maas spectrometric analysis. Unknown plasma samples (1 ml) were analyzed in an identical fashion except for the addition of I. Urine Extraction-To a 12-ml screw-capped tube were added 0.2 ml of the standard solutions of I and I1 and 0.1 ml of their internal standard solutions. After the evaporation of the solvent under nitrogen at 40°, 1 ml of drug-free urine (or water) and 1 ml of phthalate buffer were added to the residue. The drugs were analyzed using the extraction scheme outlined for plasma except that ether was used as an extracting solvent instead of methylene chloride. This change was necessary because ether extracts both I and I1 whereas methylene chloride selectively extracts I (6,7). Unknown urine samples (1 ml)were analyzed in an identical fashion except that standard solutions of I and I1were not added. GLC-Chemical-Ionization Mass Spectrometry-The prepared analytical samples were assayed for I and I1 using a chemical-ionization quadrapole mass spectrometer6 interfaced to a gas chromatographe having an eight-channel, digital readout, multiple-ion detectorP. The gas chromatograph was equipped with a 121.9-cm (4-ft) X 6.4-mm 0.d. X 2-mm i.d. glass column packed with 10%OV-7 on Gaa Chrom Q7(80-100 mesh). -

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*ScientificResearch Cop., Baltimore, Md. 6 Vuinn Aerpgraph 1400, V u h n Aesodatee, Walnut Creek. Cali. Applied hence Laboratones,State College,Pa. 0022-3549/80/1000-1218$01.0010

0 1980, American RmrmEceuticalAssocktion

For optimal retention times, the column was maintained at 210° for 200 sec and then was temperature programmed from 210 to 280° at 20°/min. The injection port was maintained at 250".The carrier gas was helium (20 ml/min). Under these conditions, the retention times for I and I1 were 3 and 5 min, respectively. Methane (0.5 torr) was the reagent gas. The source temperature was maintained at 250O. A mass window 0.75 amu wide was used, and the intensity of the ions a t m/e 261 and 264 for I and 291 and 294 for I1 were recorded. Absolute Recovery-The absolute recovery was determined by spiking plasma samples with 0.4 or 2.4 pg of I and spiking urine samples with either 0.4 pg of I or 2.4 pg of both I and 11. Quantitative analyses were conducted as described under their respective extraction procedures except for the omission of the internal standard before the fiit extraction step. Instead, the internal standard was added just prior to methylation. These spiked samples were compared to a calibration curve prepared by adding 0.1 ml of the internal standard solution (10pg/ml in methylene chloride) to each 0.2 ml of the drug standard solution (2,4,8,and 12p g / d in methylene chloride). The resulting solutions were methylated and injected onto the gas chromatograph. RESULTS AND DISCUSSION Calibration curve data for the analysis of I in plasma and for the simultaneous analysis of I and I1 in urine are shown in Table I. Compound 11was not assayed simultaneously with I in plasma because plasma levels of 11 in the unknown samples were below the assay limit. The calibration curves were linear over the range of 0.4-2.4 pg/ml for both compounds in both media. The intraday precision, reflected by the relative standard deviations, was <1.5% for the analysis of I in plasma and urine and <4% for the analysis of 11 in urine. The absolute recovery was determined from plasma sample^ containing 0.4or 2.4 pg of I and urine samples containing either 0.4 pg of I or 2.4pg of bothI and I1 (Table 11). The absolute recovery of I when extracted from plasma was 28 and 31% at 0.4 and 2.4pg, respectively. The recovery of I from urine was quite similarto that from plasma. The recoveryof I1 from urine was comparable to the recovery of I from plasma and urine. These low absolute recoveries can be accounted for mainly by the volumetric loss during extraction; the corresponding relative recoveries ranged from 78 to 100% (Table 11). The relative recovery ofI from plasma and urine was lower a t 0.4 pg than at 2.4 pg (Table II), which suggests adsorption of I onto the glass surface. However, the linearity of the calibration curve was not affected by the adsorption for the followingreason. In the preparation of samples for the calibration curve, as well as unknown samples, the internal standard (isotope) was added at the beginning and compensatory adsorption of the internal standard occurred (since it was expected to possess an identical adsorption isotherm). For both biological fluids, the extraction procedure was reproducible, e.g., the relative standard deviation was <5% (Table 11). The interday precision of the method was determined by repeatedly assaying two reference standards prepared for each biological fluid over 4 months (Table 111). The relative standard deviations for plasma reference standards were 6% at 0.525 pg/ml and 4% a t 2.10 pg/ml of I. The relative standard deviations for urine reference standards were 2 4 % for I and 5-71 for 11. As expected, the interday precision for both compounds was better a t higher concentrations. The reproducibility of the method also was evaluated using slopes of 15 plasma and urine calibration curves obtained over 4 months; the means & RSD of the slope were 1.13 f 5.6% for I in plasma and 1.04 f 4.4 and 1.20 i 7.6% for I and I1 in urine, respectively. The proposed method offers several advantages in terms of sensitivity (the detection limit for I and 11was 0.1pg/d), specificity, and ability to assay I and I1 simultaneously in biological fluids. The major disadvan-

Table 11-Recovery of Phenobarbital (I)from Plasma and Urine and of pHydroxyphenobarbital(I1) from Urine .

~~

Amount Mean" Amount Absolute Relative Biologi- Com- Added, Recovered, Recovery, Recovery*, calFluid pound pg pg (&SD) % % Plasma

I

0.4 ... 2.4 0.4 2.4 2.4

0.11 (*3%\

28 __

0.74 iIZ%j

18 ._

88 85 100 Urine I1 88 a n = 6. * After correcting for the volumetric loss of compound during extraction

Urine

I

31 30 35 31

0.12 (f5%) 0.84(&2%) 0.74 (zt42)

(absolute recovery X 8/5 X 2/1.8 X 8/5).

Table 111-Interday Precision of the GLC-Chemical-Ionization Mass Spectrometer Assay with Phenobarbital (I) and p-Hydroxyphenobarbital (11) in Plasma and Urine Estimated over 4 Months Biological Fluid Plasma

Reference Standard"

Compound

M e a d Assayed Concentration, pg/ml

(fRSD)

I

0.55 (f6.0%) 2.11 (i4.0%) Urine I 0.56 (&4.9%) I1 0.56 (f7.3%) Urine 2 I 2.18 (i1.8%) I1 2.07 (15.0%) a Reference standardswere prepared to obtain the following concentrations of I and I1 in biological fluids:Standard 1,0.525 pg/ml; and Standard 2,2.10pg/ml. 1

2 1

*n

-

I

10-14.

tages are the time-consuming extraction procedure and the use of an expensive instrument as a detector. To date, over 200 human and monkey plasma and urine samples have been assayed with this method to delineate the pharmacokinetic mechanism responsible for the phenobarbital-valproic acid interaction (8). REFERENCES (1) T. C. Butler, J. Pharmacol. Exp. Ther., 116,326 (1956). (2) N.Kallberg, S.Agurell, 0. Ericsson, E. Bucht, B. Jalling, and L. 0.Boreus, Eur. J. Clin. Pharmacol., 9,161 (1975). (3) M. P. Whyte and A. S. Dekaban. Drug Metab. Disp., 5, 63 (1977). (4) S.S.Levin, M. F. Schwartz, D. Y.Cooper, and J. C. Touchstone, J. Chromatogr., 154,349 (1978). (5) M. J. Levitt, Anal. Chem., 45,618 (1973). (6)J. Alvin, T.McHone, A. Hoyumpa, M. T. Bush, and S.Schenker. J. Pharmucol.Exp. Ther., 192,224 (1975). (7) C. T. Viswanathan, H. E. Booker, and P. G. Welling, Clin. Chem., 23,873 (1977). (8) I. H. Patel, R. H. Levy,and R. E. Cutler, Clin. Pharmacol. Ther., 25,241 (1979).

ACKNOWLEDGMENTS Supported in part by National Institutes of Health Research Contract N01-NS-6-2341awarded by the National Institute of Neurological and Communicative Disorders and Stroke, U.S.Public Health Service, and in part by National Institutes of Health Research Career Development Award NOlK04C MU0211 from'the Institute of General Medical Sciences (W. F. Trager).

Journalof Pharmaceutical Sciences I 1219 Vol. 69, No. 10, October 1980