usefulness of these techniques for future more detailed investigations of the disposition of cocaine in man. The EC and nitrogen detection methods provided a high degree of precision, accuracy, and sensitivity and the FID method was rapid, simple, and also highly accurate. Furthermore, in a comparison between the GLC and Radioimmunoassay (RIA) data reported previously from this laboratory (27), full agreement was shown between the two methods in 95.5% of the 200 urine samples analyzed for berizoylecgonine. LITERATURE CITED (1) G. Deneau, T. Yanagita, and M. H. Seevers, Psychopharmacology, 16, 30 (1969). 12) G. R. Gay, D. S. Iraba, C. W. Sheppard, J. A. Newmeyer. and R. T. Rap@, Clin. Toxicol., 8 , 149 (1975). (3) T. Harwood, Drug Enforcement, 1, 25 (1974). (4) S. J. Mul6, Ed., Cocaine: Chemical, Biological, Clinical, Social and Treatmen! Aspects”, CRC Press, Cleveland, Ohio, 1976. (5) F. Fish and W. D. C. Wilson, J . Pharm. Pharmacoi., 21, 135s (1969). (6) M. L. Bastos, D. Jukofsky, and S. J. MuE, J. Ctwomatogr.,89, 335 (1974). (7) N. N. Vaknju. M. M. Baden, S. N. Valanju, D. Mulligan, and S. K. Verma, J . Chromatogr., 81, 170 (1973). (8) M. L. Bastos and D. B. Hoffman, J . Chromatogr. Sci., 12, 269 (1974). (9) P. I. Jatlow, in “Cocaine: Chemical, Biological, Clinical, Social and Treatment Aspects”, S.J. Muli, Ed.. CRC Press, Cleveland, Ohio, 1977, pp. 59-70. (10) J. W. Blake, R. S. Ray, J. S.Noonan. and P. W. Murdick, Anal. Chem., 46, 288 (1974).
J. E. Wallace, H. E. Hamikon, D. E. King, D. J. Bason, H. A. Schwertner, and S. C. Harris, Anal. Chem., 48, 34 (1976). A. P. Graffeo, D. C. K. Lin, and R. L. Foltz, J . Chromatogr., 126 717 (1976). J. I. Javiad, H. Dekirmenjian, E. G. Brunngraber, and J. M. Davis, J . Chromatogr.. 110, 141 (1975). S. Koontz, D.Besemer. N. Mackey and R . Phillips, J . Chromatogr , 8 5 , 75 (1973). N . C. Jain, D. M. Chinn, R. D. Budd, T. S.Sneath, and W. J. Leung, J . Forensic Sci.. 22, 7 (1977). P. I. Jatlow and D. N. Bailey, Clin. Chenl. ( Winston-Saiem, N . C . ) ,21, 1918 (1975). C.Van Dyke, P. G. Barash, P. Jatlow, and R. Byck. Science, 191, 859 (1976). J. B. Brooks, J. A. L i i l e , and C. C. Alley. Anal. Chem.,47, 1960 (1975). C. C. Alley, J. B. Brooks. and G. Choudhary, Anal. Cham., 48. 387 (1976). S. A. Bland, J. W. Bhke, and R. S.Ray, Chromatogr. Sci., 14, 201 (1976). T. W. Walle, J . Chromatogr., 114, 345 (1975). J. M. Moore, Clin. Chem. (Winston-Salem. N.C.), 21, 1538 (1975). F. K. Kawahara, Anal. Chem., 40, 2073 (1968). H. Ehrsson, Acta Pbarm. Suec., 8 , 113 (1971). S. P. Findlay, J . Am. Chem. SOC.,7 6 , 2855 (1954). F. Fish and W. D. C. Wilscn, J . Chromatogr., 40, 164 (1969). S. J. Mul6, D. Jukofsky, M. Kogan, A. DePace. and K. Verebey, Clin. Chem. ( Winston-Salem, N . C . ) ,23, 796 (1977).
RECEIVED for review June 16,1977. Accepted August 4,1977. This investigation was supported in part by NIDA grant DA-01007.
Determination of Phenylethylmalonamide by Electron-Capture Gas Chromatography Jack E. Wallace,* Horace E. Hamilton, Eugene L. Shimek, Jr., and Harvey A. Schwertner Department of Pathology, The University of Texas Health Science Center at San Antonjo, San Antonio, Texas 78284
Klaus D. Haegele Department of Pharmacology, The University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284
A method for the determination of phenylethylmalonamide (PEMA) in minute amounts of serum is described. The procedure involves the derivatization of PEMA with trifluoroacetic anhydride to form a product that is extremely sensitive to the eiectron-capture detector of a gas-liquid chromatograph. The extraction process is a one-step operation utilizing 20 % ethyl acetate in benzene as the extracting sohreni and ammonium sutfate to penntt a sattingout of the PEMA from the biologic matrix. Recovery of PEMA is greater than 92 YO and the relative standard deviation between analyses is usually less than 3.0%. Ouantitatlon Is based on the utilization of “p-methyl PEMA” as the internal standard.
Primidone is among the three most frequently utilized anticonvulsanta (1-3), and determinations of serum primidone concentrations constitude a major portion of the workload of laboratories performing therapeutic monitoring services ( 4 ) . A number of methods are available for the determination of primidone including a very sensitive chromatographic procedure recently developed in our laboratory (5). Although a quarter of a century has passed since primidone was introduced for the control of seizures (6), there exists a marked Pack of confidence among clinicians in “Established thera-
peutic concentration ranges” for this important anticonvulsant (7). Some investigators have suggested that the greater variability between plasma drug levels and clinical effect observed for primidone relative to other anticonvulsant drugs is due to the fact that primidone is converted in vivo to two active metabolites (8). Primidone is metabolized by ring cleavage to phenylethylmalonamide (PEhL4) and by oxidation to phenobarbital. The efficacy of phenobarbital as a seizure-control agent has long been established, and numerous methods are available for the quantitation of that compound. Because of available analytical methods, a number of investigators have examined the relative concentrations of primidone and phenobarbital in clinical studies (9-11). PEMA is another major metabolite of primidone, and is normally a t considerably higher concentrations than is the metabolite phenobarbital (3)in the serum of patients receiving primidone therapy. PEMA possesses an inherent anti-seizure activity ( 2 ) and also enhances the activity of phenobarbital ( 2 ) ;it is more toxic than the parent compound (12) and has a much greater half-life leading to potential problems of toxicity (2, 3 ) . I t is important that a more comprehensive understanding of the relationships between dosage. serum drug concentrations, and clinical effects for primidone and its metabolites be established. Although PEMA was identified as a primidone metabolite over two decades ago (13),clinical ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1969
data are limited for this compound because of an absence of adequate analytical methods for its accurate and rapid determination in biologic specimens. Pippenger and Gillen (14) proposed a gas chromatographic procedure for the simultaneous determination of a number of anticonvulsants, including PEMA, based upon flameionization detection of the nonderivatized drugs and their metabolites. Retention data for seven chromatographic liquid phases were obtained, but no clinical data were presented. In 1972, serum concentrations of primidone, PEMA, and phenobarbital in epileptic patients receiving primidone therapy were reported ( 2 , 3 ) ,each measuring the trimethylsilyl ether derivative of PEMA by flame-ionization detection subsequent to separation on an OV-17 column. Baume1 et al. (2)measured primidone as the trimethylsilyl ether and phenobarbital nonderivatized; Gallagher et al. (3) measured the methylated analogues of primidone and phenobarbital formed by flash alkylation with trimethylphenylammonium hydroxide. Schafer (15) studied the flash methylation of PEMA with trimethylanilinium hydroxide and observed that PEMA is not cleanly methylated to N,N-dimethyl-PEMA as might be expected, but that several additional peaks of varying intensity are observed, apparently as the dimethylated product is degraded to a-phenylbutyramide or its N-methyl derivative. Schiifer thus proposed that PEMA be assayed nonderivatized on a mixed phase consisting of SE 52 and Poly A 103 utilizing temperature programming a n d 5-p-methylphenyl-5phenylhydantoin as an internal standard. No clinical data were reported. In a recent letter to the editor, Soldin and Hill (16)pointed out that a previously described high performance liquid chromatography procedure ( I 7) failed to separate PEMA from primidone, and that data reported as primidone were in reality a combination of primidone and PEMA. It was suggested that the combined. value may be more valuable for clinical purposes than the value of primidone alone, but this has not been validated. T h e present paper describes a sensitive and specific procedure for the quantitative determination of PEMA. It does not possess the product stability problems of previously described methods, nor does i t require temperature programming. A chromatographic liquid phase more commonly available than that proposed by Pippenger and Gillen is used, as is an internal standard which is a structural analogue of PEMA, thus affording a closer similarity in extraction and derivatization characteristics. A highly specific extracting solvent is utilized which provides a higher recovery than that normally reported for primidone or PEMA, but one which co-extracts considerably less endogenous materials than does the highly polar solvent system proposed by Schiifer. Clinical data, including primidone as well as PEMA values, are presented for epileptic patients receiving primidone therapy. EXPERIMENTAL Apparatus and Operating Conditions. A Hewlett-Packard Model 5830A gas chromatograph (a keyboard-controlled instrument with a multifunctional digital processor) equipped with a 63Ni-electron-capture detector and a 1 m X 4 mm i.d. glass column packed with 3% OV-1 on 100/120 mesh gas Chrom Q (Applied Sciences Laboratories, State College, Pa.) was utilized for gas-liquid chromatographic analysis. Column, injector, and detector temperatures were 135, 300, and 350 OC, respectively. Five percent methane in argon was used as the carrier gas at a flow rate of 60 mL/min. A Hewlett-Packard gas chromatograph-mass spectrometerdata system (Models 5710A, 5980A, and 5933A, respectively) was used for investigating the nature of the products formed. Electron-impact ionization was performed at 70 eV; chemical ionization, utilizing methane as reagent gas, was carried out at a pressure of 0.5 Torr. Ion source temperature in both modes of ionization was 195 OC. The gas chromatograph was interfaced via a membrane separator to the mass spectrometer. 1970
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
Reagents. Ammonium sulfate (granular), ACS grade, was obtained from Mallinckrodt Chemical Works, St. Louis, Mo. (No. 3512). A saturated solution was prepared in deionized water. Ethanol was obtained from Chemical Solvents Corporation, Terre Haute, Ind. Ethyl acetate, methanol, and toluene were obtained from Fisher Scientific Company, Fair Lawn, N.J. (E-191pesticide grade, A-412 ACS grade, and T-324 ACS grade, respectively). p-Methyl phenylethylmalonamide (p-Methyl PEMA) was obtained originally from Kenneth Dudley, University of North Carolina, Chapel Hill, N.C. It is currently available from Aldrich Chemical Company, Inc., Milwaukee, Wis., 53233, catalogue No. 19,496-4. A stock solution of 1mg/mL was prepared in ethanol. The stock solution was diluted to 100 pg/mL with ethyl acetate, which was used to prepare the extracting solvent that contained 0.5 pg/rnL p-methyl phenylmethylmalonamide in ethyl acetate/ benzene (20180). Trifluoroacetic anhydride (TFAA) was purchased from Pierce Chemical Company, Rockford, 111. Phenylethylmalonamide (PEMA) was obtained from Ayerst Laboratories, Inc., New York, N.Y. A stock solution of 1mg/mL was prepared in ethanol, which was diluted with ethyl acetate to the desired concentration for the working standard solution. Procedure. Specimens for the standard curve are prepared by diluting 1 mg/mL PEMA in ethanol with ethyl acetate and placing appropriate amounts of the diluted PEMA solution into a 16 X 150 mm tube, bringing the contents of the tube to dryness under a stream of dry nitrogen, adding plasma, and mixing well with a vortex mixer (vortexing). For analysis, 0.5 mL of saturated ammonium sulfate solution is added to 0.5 mL of plasma or serum in a 16 X 150 mm tube, and the contents of the tubes briefly vortexed. The ammonium sulfate is added as a saturated solution to prevent repetitive weighing of the salt prior to each addition. Extraction is accomplished by adding 10 mL of 20% ethyl acetate in benzene containing the internal standard to the previously diluted serum, vortexing, and centrifuging the mixture at 2000 rpm for 2 min. The organic layer is decanted into a 16 X 125 mm screw-cap tube or equivalent. Since a highly effective internal standard is utilized, it is not necessary to know the exact volume of solvent removed after each extraction. The contents of the tubes are subsequently dried under a stream of dry nitrogen at 55 OC. Fifty p L of ethyl acetate and 100 pL of trifluoroacetic anhydride (TFAA)are added, the tubes capped, vortexed briefly, and incubated for 30 min at 120 "C in a heating block. After the tubes have returned to near room temperature, the contents are dried under a stream of dry nitrogen. The residue is reconstituted in 1mL of ethyl acetate and vortexed briefly. Five pL of the resulting solution are injected into the electron-capture equipped gas-liquid chromatograph. RESULTS The reaction of trifluoracetic anhydride (TFAA) with PEMA yielded a product which was detected by the electron-capture detector with a high sensitivity. Mass spectra data indicated that the product, rather than containing one or more trifluoroacetic groups, had a molecular weight less than that of the original molecule, indicating the reaction to be essentially a removal of one molecule of water per molecule of PEMA (or p-methyl-PEW). Figure l a shows the chemical ionization mass spectra of the reaction produ& of PEMA and p-methyl-PEMA obtained through the reaction with trifluoroacetic anhydride. They confirm the molecular weight of these products to be 188 for PEMA and 202 for the homologue through the series of adduct ions observed (PEMA derivative: MH+ = 189, MC2H6+ = 217, MC3H6+ = 229; Methyl-PEMA derivative: MH+ = 203, MCzHb+ = 231, MC3H5+= 243). The fragment ions at m f e 162 and m / e 176 are due t o the elimination of HCN from the corresponding MH+ ions. Fragment ions at m l e 145 and m l e 159, respectively, correspond to the loss of OEC-NH~ from the MH+ ions. The ions a t m l e 144 and m l e 158, respectively, arise through the elimination of OCH-NH2from the MH' ions. The electron impact mass spectrum of the PEMA product did not show a molecular ion (Figure l b ) , whereas the spectrum
II 1 4 5
i 89
zoo
:5 c
100
ZG3
, I 5‘!
I
i
1
I
,247 i:c
Chemical ionization mass spectra of the TFAA-reaction products of PEMA and p-methyl PEMA Figure l a .
Flgure lb. Electron impact mass spectra of the TFAA-reactiin products of PEMA and p-methyl PEMA LD
of its methyl homologue shows a weak molecular ion at m l e 202. Loss of O=C=NH from the molecular ions yields the base peak of these spectra at m l e 145 and m J e 159, respectively. Loss of -CH3from these ions then leads to the peaks a t m l e 130 and m l e 144, which indicates that the lost CH3 originates from the ethyl group of the molecule. The mass spectral data suggest the reaction products of PEMA and p-methyl-PEMA with trifluoroacetic anhydride have the structures indicated in Figure 2. T h e compound “p-methyl PEMA” is an ideal internal standard for the analysis of PEMA. The two compounds are identical in chemical structure except for the methyl group a t the para position on the phenyl ring of the internal standard. As illustrated in Figure 3, PEMA and p-methylPEMA are well resolved ( R T T on OV-1, 1.49). Figure 3 further demonstrates that even with significant concentration differences between PEMA and the internal standard, excellent resolution of the chromatographic peaks is obtained. T h e linearity of the derivatization reaction is substantiated by the chromatograms of Figure 3 and the data of Table I. Equivalent concentrations of PEMA and p-methyl-PEMA (Table I) resulted in approximately a 0.700 f 0.009 ratio of
LD -,-
7
H’
0
H’
a
,
b
Structures of PEMA and p-methyl-PEMA products after reaction with TFAA. (a) PEMA product, (b) p-methyl-PEMA product (verified by GUMS) Flgure 2.
the PEMAlp-methyl-PEMA peak areas for the respective derivatives obtained with TFAA. The proposed extraction procedure, utilizing a saturated aqueous solution of ammonium sulfate as a salting-out agent and a solvent (ethylacetate in benzene)-to-aqueous ratio of 10 to 1, resulted in exceptionally clean chromatograms, as indicated in Figure 3. Mean recoveries of PEMA from plasma were 92.4 f 4.1%;in the absence of the ammonium sulfate, mean recoveries were 74.0 3.1%.
*
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1971
Table I. Standard Curvea and Stability of PEMA Productb Concn, MmL 0 1 5 10 20 30
Ratio of PEMAIp-methyl-PEMA peak areas Day 1 Day 2 Day 3 Day 5 0.000 f 0.000c 0.078 i 0.005 0.356 i 0.008 0.700 f 0.009 1.288 f 0.024 1.781 f 0.018
0.000 f 0.000 0.077 f 0.005 0.374 i 0.003 0.717 f 0.018 1.343 i 0.022 1.875 i 0.030
000'0 7 0.078 i 0.394 i 0.710 f 1.322 It 1.871 i
000'0
0.001 0.005 0.020 0.044 0.026
0.000 i 0.072 i 0.386 I 0.741 f 1.309 i 1.815 i
0.000 0.003 0.009 0.039 0.040 0.098
Withinrun variationd
Mean
Between-run variation SD RSD, % Meanlconcn
i
0.000 f 0.000 0.077 i 0.009 0.382 f 0.041 0.717 f 0.026 1.315 i 0.036 1.836 i 0.062
0 4.6 1.7 3.0 2.5 2.4
0 11.9 10.8 11.2 2.7 3.4
0.000 i 0.000 0.0770 0.0764 0.0717 0.0658 0.0612
a 0.5-mL plasma standards. Derivatized products kept in ethyl acetate in capped tubes at room temperature; triplicate determinations performed on four separate days as indicated. Mean i standard deviation for triplicate determinations. Mean % relative standard deviation.
Table 11. Stability of PEMA and Primidone in Serum PEMA, pg/mL Primidone, pg/mL MBTFA' TFAAb PFBCl' PFBCl Patient (11/9/76)d (5/6/77)d (11/9/76)d (5/6/77)d 1-F
2 -P 3 -K 4 -I 5 -H 6-Q a
2.8 10.4 12.8 17.5 15.0 9.2
2.6 9.9 13.0 18.2 14.6 10.1
17.3 20.3 22.8 6.8 13.9 10.6
18.3 20.5 23.6 6.8 14.1 10.8
N-Methyl-bis-trifluoroacetamide. Trifluoroacetic Pentafluorobenzoyl chloride. Date of
anhydride. analvsis. 3
,
2
3
4
5
mnutes
Flgure 3. Concentrations of phenyiethylmalonamide (PE) relative to 10 pg/mL of p-methyl-phenylethylmalonamide(MPE) in ethylacetate. I, 0; 11, 1.0 gg/mL; 111, 10 pg/mL; IV, 20 hg/mL
Table I demonstrates the linearity and reproducibility of the procedure for a wide range of serum concentrations (1-30 pg/mL) of PEMA. The within-run precision of the peak area ratio for triplicate determinations was a 4.6% relative standard deviation a t a concentration of 1bg/mL, and 2.4% over the range of 5 to 30 gg/mL. The between-run variation in estimated concentrations over the range 5 t o 30 pg/mL as estimated by determinations performed over an interval of five days was 6.4%. The stability of PEMA in serum is depicted in Table 11. Serum specimens from patients receiving primidone therapy were divided into two aliquots, one of which was analyzed and the other stored a t -5 " C for over six months prior to analysis. T h e mean difference between the values obtained over the six months interval was 1.0 f 0.7 pg/mL. Primidone was similarly determined by the procedure recently proposed by these investigators (5),and the mean difference was 0.4 h 0.4 pg/mL. The greater difference observed for PEMA relative t o primidone may in part be due to the use of different derivatization reagents. The initial PEMA studies were conducted with n-methyl-bistrifluoroacetamide which produced the same product as that obtained with trifluoroacetic anhydride; the latter reagent is preferred, however, based upon precision and interference studies. Sera (0.5 mL) obtained from seven adult epileptic patients receiving primidone therapy was extracted, utilizing ammonium sulfate, into 10 mL of ethylacetatebenzene containing p-methyl-PEMA and p-methyl-primidone as internal standards. Two mL of the solvent extract were then analyzed by the previously described method for primidone (5), and the remaining extract was assayed for PEMA. The mean relative standard deviation for triplicate determinations was 0.6% (mean 7.8 g / m L ) for primidone and 3.7% (mean 7.5 1972
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
Patient C
-
01 2 3 4 5
-
0 1 2 3 4 5
Flgure 4. Patient concentration of primidone and PEMA in serum: Patient A: primidone, 6.2 pglmL; PEMA, 4.6 pg/mL. Patient B: primidone, 7.4 pglmL; PEMA, 8.0 pg/mL. Patient C: primidone, 6.5 bg/mL; PEMA, 4.8 pg/mL. p-Methyl-primidone and p-methyl-PEMA concentrations are 10 pg/mL in serum
bg/mL) for PEMA. The mean primidone/PEMA concentration ratio was 1.3 h 0.6 (Table 111). Chromatograms for both primidone and PEMA analyses are exceptionally clean (Figure 4), even though each of these patients were receiving
Table 111. Serum Rimidone and PEMA Concentrations in Epileptic Patients Patient Primidone“ PEMA“ Primidone/PEMAb A 10.7 i 0.1 7.8 k 0.2 1.37 B 6.4 i 0.0 6.5 k 0.4 0.98 13.8 i 0.2 0.72 C 9.9 i 0.0 D 6.3 i 0.0 4 . 3 k 0.1 1.47 E 7.4 * 0.2 5.9 i 0 . 2 1.25 1.07 F 6 . 2 i 0.0 5.8 i 0.1 5.8 f 0.5 2.48 G 14.4 i 0.1 1 . 3 3 * 0.56
“ pg/mL, mean i standard deviation for triplicate determinations. Ratio of concentrations other medications. We have not observed interfering peaks in the chromatograms of patients concurrently receiving phenobarbital, carbamazepine, phenytoin, ethosuximide, dianox, thiamine, oxazepam, imipramine, amitriptyline, digoxin, erythromycin, mebral, diazepam, synthroid, aldomet, or trichloromethiazide. DISCUSSION The method described in this report provides a single stable product for each of PEMA and p-methyl-PEMA, in contrast to the several variable peaks achieved through flash alkylation, or by moisture-labile trimethylsilylation procedures. The method provides a significant enhancement in sensitivity over flame-ionization detection of the nonderivatized metabolite; also, studies in our laboratory have demonstrated that PEMA, like most amides, is not detected by the nitrogenfphosphorous specific detector with an adequate sensitivity. The proposed method possesses the important characteristic of precision over a wide range of PEMA concentrations. The synthetic analogue, p-methyl PEMA, meets all requirements of an internal standard for it is identical to PEMA in extractability, derivatization site and rate, stability, and chromatographic properties. Additionally, other anticonvulsants and their metabolites, including the primidone metabolite phenobarbital, do not interfere. T h e derivatization reaction, in addition to being quantitative, linear, and precise, has the important characteristic that excess amounts of the reagent are easily removed after derivatization. The similarity of the analytical procedures proposed for primidone and PEMA allows for a single extraction for both parent and active metabolite, with the extracting solvent divided into separate aliquots prior to derivatization. The electron-capture capability of primidone after reaction with PFBCl and PEMA after reaction with TFAA provides sufficient sensitivity that both compounds may be quantitated from a single finger-stick specimen of approximately 100 pL. It appears that the routine application of the proposed methods should provide more valid therapeutic drug concentrations for this important antiepileptic agent and its active metabolite, and thus resolve some of the current controversy pertaining to the relation existing between serum concentrations and clinical effects. I t has been reported that for tricyclic antidepressants, the combined concentrations of drug and active metabolite, or the ratios thereof, are of more clinical significance than the serum concentrations of the parent drug
alone; this may well be the case with primidone and its metabolites. Current quality control programs should consider adding phenylethylmalonamide t o spiked serums t o provide more realistic specimens for primidone determinations. The primidone and PEMA concentrations observed in this report are comparable to those reported by Baumel et al. (2) and Gallagher et al. (3) (both papers are from the same research group, reporting different facets of the same study). These data are presented for a limited number of specimens to demonstrate both the applicability of the proposed procedure to the analysis of patient sera and the lack of interference from concurrently administered drugs and their metabolites. The authors are currently engaged in an extensive clinical study in which primidone, PEMA, and phenobarbital serum concentrations are being determined on a large population of patients. It is anticipated t h a t a correlation of observed serum concentrations with clinical effect, dosage regimen, and duration of epileptic therapy will provide useful information concerning the therapeutic efficacy of primidone and its major metabolites. ACKNOWLEDGMENT The authors deeply appreciate the professional liaison with John P. Allen, Director of the Bexar County Epileptic Clinic, who supplied specimens from patients receiving primidone therapy. The technical assistance of Kenneth Palmer, Jr., and Donald Nicholas is also appreciated. The p-methyl phenylethylmalonamide for this particular study was synthesized and supplied by Kenneth Dudley of the University of North Carolina a t Chapel Hill. LITERATURE CITED (1) H. J. Kupferberg, Clin. Chim. Acta, 29, 283 (1970). (2) I. P. Baumel, B. B. Gallagher, and R. H. Mattson, Arch. Aleurn/. (Chicago), 27, 34 (1972). (3) B. B. Gallagher, I. P. Baumel, and R. H. Mattson, Neurology, 22, 1186 (1972). (4) R. E. Beam, Am. J. Med. Techno/.. 40, 211 (1974). (5) J. E. Wallace, H. E. Hamilton, E. L. Shimek, Jr., H. A. Schwertner, and K. Blum, Anal. Chem., 49, 903 (1977). (6) R. Handley and A. S. R . Stewart, Lancet. 1, 742 (1952). (7) L. Livingston, W. Berman. and L. L. Pauli, J. Am. Med. ASSOC.,232, 60 (1975). (8) B. B. Gallagher and I. P. Baumel, Neurology, 21, 394 (Abstract) (1972). (9) H. H. Frey and I. Hahn, Arch. E x p . Pathol. Pharmakol. NaunynSchmiedebergs, 240, 20 (1960). (IO) J. Bogan and H. Smith, J. Pharmacol., 20, 64 (1968). (11) P. A. Toseland, J. Grove, and D. J. Berry, Clin. Chim. Acta, 38. 321 (1972). (12) S. Livingston, “Camprehenslve Management of Epilepsy in Infancy, Childhood and Adolescence”, Charles C Thomas Publishers, Springfield, . Ill.,1972, pp 181-182. (13) J. Y. Bogue, H. C. Carrington, and S. Bentiey, Acta Nsuroi.
[email protected], 640 (1956). (14) C. E.’Pippenger and H. W. Gilien. Clin. Chem. ( Winston-Salem, N.C.), 15, 582 (1969);. (15) H. R. Schafer, Some problems concerning the quantltative assay of primidone and its metabolites”, H. Schneider, D. Janz, C. Gardner-Thorpe, H. Meinardi and A. L. Shorwin, Ed., “Clinical Pharmacobgy of AntLEpileptic Drugs”, Springer-Verlag, New York, N.Y., 1975. (16) S. J. Soidin and J. G. Hill, Clln. Chsm. (Winston-Salem, N.C.), 23, 782 (1977). (17) S. J. Soldin and J. G. Hill, Clin. Chem. ( Winston-Salem, N . C . )22, 856 (1976).
RECEIVED for review June 8,1977. Accepted August 23,1977. This research was supported in part by Grant R 0 1 DA 00729-03 from the National Institute of Drug Abuse, NIH, Bethesda, Md., and in part by Grant R01 MH 26431-02 from the National Institute of Mental Health, NIH, Bethesda, Md.
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