yielded by IV are shown in Figure 3 in comparison with that of IV. First, both these compounds are found to have the same molecular weight (Mf = 284). In view of the principle fragmentation of the two geometrical isomers of IV (7), shown below,
r, 133
135J" e107
161~" the mass spectra indicate that the first peak eluate and the second one are regarded as IVa and IVb, respectively. From the results of the infrared and mass spectrometric analysis described above, it is concluded that the two peaks for IV are attributable to the geometrical isomers of IV. With respect to the separation of the 4,4'-disubstituted azoxybenzenes studied, there is no methanol-water mobile phase which permits all of the azoxybenzenes to be sufficiently separated for quantitative determination (as I is not liquid crystal, the separation and determination is discussed to the exclusion of I). The resolution of V and VI are in competition with that of VI11 and IX. However, V and VI can be ade-
quately separated with a methanol mobile phase containing 20% (w/v) of water and on the other hand VI11 and IX can be separated with a methanol mobile phase containing 10% (w/v) of water. Consequently it is concluded that for any mixtures of the azoxybenzenes, qualitative and quantitative determination can be made with two methanol mobile phases containing an appropriate amount of water. The capacity factors of the azoxybenzenes studied were between one and twenty with a methanol mobile phase containing 10% (w/v) of water, while the resolution of V and VI was poor (Figure la). Hence in order to study the reproducibility, calibration curves were established with a methanol mobile phase containing 10% (w/v) of water between peak areas and amounts of the azoxybenzenes injected over the range of 0.5 to 10 pg. The linearity was good and the plots passed through the point of origin. The slope of the calibration curve, the relative standard deviation of the slope, the precision and the detection limit obtained for each azoxybenzene are shown in Table I. In Table I, the two unsymmetrically disubstituted azoxybenzenes, I11 and IV, are treated as a mixture of their isomers. LITERATURE CITED (1) W. R. Edwards, Jr., 0.S. Pascual. and C. W. Tate, Anal. Chem., 28, 1045 (1956). (2) C. C. Jahiatt, An. Fac. Ouim. Farm., Univ. Chile, 18, 282 (1966). (3) F. H. Alvarado. J . Chromatogr., 42, 144 (1969). (4) T. Kawahara, S.Goto, and T. Kashiwa, BunsekiKagaku, 18, 1344 (1969). (5) R. E. Rondeau, U.S.P. 3, 730, 687. (6) G. G. Spence, E. C. T a r n , and 0.&char&, Chem. Rev., 70, 231 (1970). (7) J. H. Bowie, R. G. Cooks, and G. E. Lewis, Aust. J . Chem., 20, 1601 (1967).
RECEIVED for review July 7,1977. Accepted August 22,1977.
Quantitative Determination of Benzoylecgonine and Cocaine in Human Biofluids by Gas-Liquid Chromatography M. J. Kogan,* K. G. Verebey, A. C. DePace, R. B. Resnlck, and S. J. Mu16 New York State Office of Drug Abuse Services, Testing and Research Laboratory, Brooklyn, New York 11217, and New York Medical College, Department of Psychiatry, New York, New York 10029
Cocalne (COC) and Its prlnclpal metabollte In man, benroylecgonlne (BE) were determined by quantitative gas-liquid chromatographlc methods. Nitrogen detectlon was used for COC and electron capture detection for BE following extraction from human plasma. Using flame lonlzatlon detectlon, COC and BE were slmultaneousiy determlned from human urlne. The limits of detection for COC underivatlred and BE as the pentafluorobenzyl bromide derlvative In plasma were 10 and 5 ng/mL, respectlvely. I n urlne the sensltlvlty limits of the sllyl derivatives of COC and BE were 0.5 and 1.0 pg/mL, respectively. The coefflcient of varlation ranged between 0.9-2.2% and the coefficient of determination was 0.99 for these methods. Data on plasma and urine concentration of COC and BE collected over a tlme period of 24 h from three human subjects who recelved 1.0 to 1.9 mg/kg cocalne*HCI i.v. are presented.
The euphoric and stimulant effects of cocaine induce a high level of psychological dependence ( 1 ) and subsequent abuse
(2). Current detailed information has recently been published concerning the historical, chemical, physiological, sociological, and treatment aspects of cocaine use and abuse (3, 4 ) . Cocaine, a lipophilic drug, is extensively biotransformed in man to water soluble metabolites (5-7). The various analytic methods for the detection of cocaine and its metabolites have been critically reviewed (8, 9). A major biotransformation product of cocaine in man is benzoylecgonine (BE). Moderately sensitive gas chromatographic techniques have been reported recently for the detection of cocaine and benzoylecgonine in urine (10-15) and the quantitative determination of cocaine, but not benzoylecgonine, in human plasma (16, 17). Until now methods were not available for the quantitation of BE in plasma primarily because underivatized BE did not chromatograph well. A variety of electron capture derivatizing agents have been used to improve the detection and quantitation of trace amounts of carboxylic acids (18-24). Benzoylecgonine, a carboxylic acid, will form an ester with the halogenating reagent pentafluorobenzyl bromide (PFB), thus providing a reliable and sensitive benzoylecgonine derivative for gas chromatography. ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1085
In this communication we describe (1)a sensitive quantitative gas-liquid chromatographic (GLC) method for the determination of benzoylecgonine in human plasma; (2) a clinically applicable, rapid, quantitative method for the simultaneous determination of cocaine and benzoylecgonine in human urine; and (3) a modification of the method of Jatlow and Bailey (16) for the determination of cocaine in human plasma. EXPERIMENTAL Apparatus. Electron capture detection was performed on a Hewlett-Packard 5830A gas chromatograph equipped with a 63Ni electron capture detector. A glass column (1.83 m X 2 mm i.d.) packed with 3% OV-225 on 80/100 Supelcoport was used. Column, injector, and detector temperatures were 250, 285, and 300 "C, respectively. The carrier gas was 10% methane in argon flowing at 50 mL/min. Flame ionization detection was performed on a Tracor MT-220 gas chromatograph. Column packing was 3% SE-30 on 80/100 Gas Chrom Q. Column, injector, and detector temperatures were 200, 260, and 250 "C, respectively. Nitrogen carrier gas, air, and hydrogen flow rates were 50, 200, and 30 mL/min, respectively. Nitrogen detection was performed on a Perkin-Elmer 900 gas chromatograph equipped with a rubidium bead Nitrogen-Phosphorus detector. Column packing was 3% OV-22 on 80/100 Supelcoport. Column, injector, and manifold temperatures were 250,285, and 300 "C, respectively. Helium carrier gas, air, and hydrogen flow rates were 30, 120, and 3 mL/min, respectively. Reagents. Cocaine was obtained from Merck & Co., Rahway, N.J. Benzoylecgonine was prepared from cocaine by refluxing in water for 10 h (25). Chlorproethazine.HC1 (SK&F #4788-A) was a gift of Smith Kline & French Laboratories, Philadelphia, Pa. ScopolamineHCl was purchased from Sigma Chemical Co., St. Louis, Mo. Pentafluorobenzyl bromide (PFB) and bis(trimethylsilyl) trifluoroacetamide (BSTFA) + 10% trimethylchlorosilane (TMCS) were purchased from Pierce Chemical Co., Rockford, Ill., and Regis Chemical Co., Morton Grove, Ill., respectively. All solvents were pesticide grade except ethanol (95%) which was reagent grade. Sample Preparation. (a) Determination of Benzoylecgonine in Plasma. A methanolic solution of the internal standard chlorproethazine.HCl(5.0 mg/100 mL) was prepared and 0.1 mL added to 0.5 mL of plasma. The plasma was made basic (pH 9.5) by the addition of 0.5 mL of sodium carbonate/bicarbonate buffer. Throughout the procedures, extraction and derivitization were carried out in 15-mL siliconized conical glass centrifuge tubes fitted with Teflon lined screw caps. Extractions and washings were accompanied by agitation for 10 min on a mechanical shaker. Organic and aqueous phases were separated by centrifugation at 850 X g for 5 min. Evaporation of organic solvents was achieved by heating in a water bath at 55 "C under a gentle stream of nitrogen gas. The samples were extracted with 10 mL of 20% (v/v) ethanol in chloroform. The organic phase was transferred to a clean tube and evaporated to dryness. The dry residue was dissolved in 0.2 mL pentafluorobenzyl bromide (PFB) solution (5 pL PFB in 10 mL of methylene chloride) and 5 pL of 1%(v/v) pyridine in benzene solution. Derivatization was performed by heating the mixture for 1 h at 80 "C in a constant temperature block. The solvent and excess PFB were evaporated and the residue was partitioned between 0.2 mL of 0.15 M NH40H and 2 mL benzene. Following shaking and centrifugation, the benzene layer containing the PFB-benzoylecgonine derivative was transferred to a clean tube containing 0.5 mL of 0.03 N H2S04 and the derivative reextracted into acid and the benzene layer aspirated. Approximately 5 mg of solid sodium bicarbonate was added to the remaining aqueous phase and the derivative was extracted into 2 mL of benzene. The final benzene extract was transferred into a clean tube (10 X 75 mm) and evaporated. The residue was dissolved in 20 WLof methanol and 0.5 to 2.0 pL injected into the gas chromatograph equipped with electron capture detector. ( b ) Determination of Cocaine in Plasma. Forty microliters of the internal standard chlorproethazineHC1 solution used in the previous procedure were added to each 0.5-mL aliquot of plasma and then made basic with 0.5 mL of pH 9.5 sodium 1986
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
carbonatejbicarbonate buffer. The plasma was extracted with 10 mL of a 2% (v/v) isoamyl alcohol in n-heptane. Following extraction and centrifugation, the organic phase was transferred to a clean tube containing 1 mL of 0.1 N &SO4 and extracted into the acid. The organic phase was aspirated and the aqueous phase washed once with 3 mL of the extracting solvent. The aqueous phase was made basic by saturation with a solid mixture of sodium carbonate/sodium bicarbonate (1.14:1, w/w) and extracted into 2 mL of benzene. The benzene layer was transferred to a clean tube and evaporated to dryness at room temperature (20 f 2 "C). The final extract was dissolved in 20 p L of methanol and 1.0 to 2.5 pL were injected into the gas chromatograph equipped with nitrogen detector. ( c ) Simultaneous Determination of Benzoylecgonine and Cocaine in Urine. An aqueous solution of the internal standard scopolamine-HCl(4 mg/lO mL) was prepared and 25 WLadded to each 1-mL aliquot of urine. The urine was made basic by the addition of solid sodium bicarbonate and extracted once with 7 mL of 20% (v/v) ethanol in chloroform. After centrifugation, the aqueous phase was aspirated and the organic phase decanted into a clean tube and washed with 1 mL of 0.01 N NaOH. The mixture was centrifuged and the aqueous phase aspirated. The organic phase was evaporated and 40 pL of BSTFA + 10% TMCS added to the tube. Derivatization was carried out for 30 min at 80 "C in a constant temperature block. After cooling and centrifugation 1.0 to 2.0 pL were injected into the gas chromatograph equipped with flame ionization detector. Estimation of Drug Concentration. Following chromatography, the baseline was drawn and peak heights were measured. The ratio of cocaine or benzoylecgonine to that of the internal standard was calculated. Quantitation was based on standard curves constructed from peak height ratios of known concentrations of benzoylecgonine or cocaine (0.1 to 20.0 pg/mL) to the internal standard. Standards in cocaine-free plasma or urine were determined along with each set of unknown samples. Samples containing less than 0.5 mL plasma or 1mL urine were corrected for volume after calculation of the concentration based on 0.5 pL plasma or 1 mL urine. All values for benzoylecgonine and cocaine were calculated as the free base. Human Study. One hundred mg cocaine.HC1 was administered intravenously to three human subjects. No restrictions were placed on food or water intake before or during the experiment. Blood samples (15 cm3)were obtained at 5,15,30,and 45 min and 1,2, 3,4,5,6,and 24 hours after drug administration. Blood was transferred to a clean glass test tube containing heparin and a sodium fluoride solution (3.5 mL supernatant of a saturated solution). Sodium fluoride was added to inhibit in-vitro hydrolysis of cocaine by serum esterases. After gentle mixing and cooling on ice for 5 min, the blood was centrifuged for 10 min, the plasma removed and stored at -20 "C until assayed. Urine samples were collected at @2, 2-4, and 4-6 h following cocaine administration. The total volumes of fractional urine samples were recorded and aliquots stored at -20 "C until analyzed. RESULTS A N D D I S C U S S I O N The quantitative determination of benzoylecgonine in plasma utilizing electron capture detection of benzoylecgonine-PFB derivative (method a) was both sensitive and precise. The relationship between peak height ratio and plasma concentration was linear over the range of 0.05 to 4.0 pg/mL plasma (r2 = 0.999, mean CV = 0.9%) indicating excellent accuracy and precision (Table I) for this method. The maximum sensitivity for benzoylecgonine detection was 5 ng/mL of plasma. The method was unique because following a crude extraction of the polar metabolite benzoylecgonine it was immediately reacted with PFB. The more lipid soluble derivative was partitioned into a nonpolar solvent leaving the more polar interfering substances behind. In most other methods, elimination of interfering substances from the initial extract precedes derivatization. The method described for plasma may also be used for the quantitative determination of benzoylecgonine in urine. However, a shortcoming of this procedure was that in drug-free urines (controls), a small chromatographic peak closely
Moreover, the presence of other drugs such as cocaine or methadone in patient samples did not react with pentafluorobenzyl bromide nor interfered with the derivatization of benzoylecgonine. Cocaine, although extracted with benzoylecgonine from plasma does not interfere with the GC analysis, since it provided only a minimal EC detedor response and eluted from the column earlier than either benzoylecgonine-PFB or the internal standard. The standard curve data for the simultaneous determination of benzoylecgonine and cocaine in urine (method c) is given in Table 11. With this method, the sensitivity limit for benzoylecgonine and cocaine was 1.0 and 0.5 rg/mL urine, respectively. The mean coefficient of variation (CV), a measure of precision, for cocaine and benzoylecgonine was 2.2% and the coefficient of determination (r’), a measure of accuracy, was 0.99, nearly perfect. Although this method was only as sensitive for cocaine and benzoylecgonine as previously reported FID methods (5, 14, 15,26), it does provide for a rapid, simple simultaneous determination of both benzoylecgonine and cocaine when a large number of samples need to be analyzed. Under more idealized conditions of HPLC solvent programming followed by GC-MS selected ion monitoring and using a pentadeuterated benzoylecgonine internal standard, the sensitivity of a benzoylecgonine-BSTFA derivative was reported as 1 ng/mL urine (12). Obviously, a rapid single extraction FID method cannot attain this degree of sensitivity. The utility of methods (a), (b), and (c) was validated with urine and plasma samples obtained from three human subjects who received intravenous doses of 100 mg cocaineHC1. The results of these analyses appear in Table 111. Method (b) represents a very slight modification of the technique published by Jatlow and Bailey (16) (elimination of a drying step and use of chlorproethazine as an internal standard). In our laboratory, the sensitivity limit for cocaine with this method was 10 ng/mL plasma. However, methadone interfered with cocaine quantitation when the column packing was OV-17 as used in the original method (16). A 3% OV-22 column packing resolved this problem allowing methadone to be separated from cocaine (relative retention 0.45). In our study, this was important because two of our experimental subjects were on methadone maintenance. Cocaine concentrations in plasma peaked a t 5 min and then declined in a multiexponential fashion over the next 5-6 h. The distributional half-life of cocaine in plasma was 20 to 40 min. This time period corresponded well with the psychologic effects (“high”) observed following cocaine administration. The mean biologic half-life of cocaine in plasma was 2.8 h, a value which is consistent with the 2.5 h apparent half-life of cocaine in the plasma reported by VanDyke et al. (17)following topical intranasal application of cocaine.HC1 (1.5 mg/kg) to patients undergoing dental surgery. Therefore, the biologic half-life values for cocaine were not markedly different following either intravenous or intranasal administration indicating rapid absorption of
Table I. Standard Curve Data for Benzoylecgonine in Plasma Using Electron Capture Detection Benzoylecgonine/ Benzoylecchlorproethazine gonine added, peak height P@ ratio t SDb 0.00 0.05 0.125 0.50 2.00 4.00
Benzoylecgonine cal-
culated, p g ‘
0.00 i. 0.00 0.17 t 0.00 0.36 f 0.01 1.24 i. 0.01 4.55 t 0.03 8.98 f 0.04
0.00 0.05 0.13 0.53 2.01 3.99
a Micrograms ( p g ) of benzoylecgonine added to 0.5 mL Mean of human plasma in triplicate samples each. Calculated from coefficient of variation (CV) = 0.9%. the least-squares line ( r z = 0.999).
corresponded in retention time to the benzoylecgonine-PFB derivative equivalent to 0-25 ng benzoylecgonine/mL urine. Therefore, the limit of benzoylecgonine sensitivity in urine with this method was approximately 50 ng/mL urine, or twice the highest background level of the contaminant. A 2-pg benzoylecgonine standard extracted from plasma and derivatized decreased only 3% in peak height ratio after storage for 24 h at -20 “C, indicating that the PFB derivative of benzoylecgonine was stable under these conditions. A similar derivative stored at room temperature (20 f 2 “C) for 24 h, however, decreased 20%. Samples, before evaporation of the final benzene extract, may also be stored overnight at 3 “C or lower without serious loss of benzoylecgonine. A spuriously high detector response was observed if some of the derivatizing agent and solvent escaped during the reaction. Thus, care must be taken to use tight fitting closures on the reaction vessel during derivatization. Various compounds were evaluated as potential internal standards in connection with method (a). These were in sequence: tropine derivatives; alkaloids of approximate molecular weight of benzoylecgonine but containing carboxylic or phenolic functions; smaller molecules with either of these functional moieties; molecules containing halogen atoms only; and alkaloids containing a halogen atom but not carboxylic or phenolic functions. In all, about thirty compounds were tested. The reasons for eliminating most compounds were: failure to react with pentafluorobenzyl bromide; failure to form a single derivative; failure to back-extract into acid. Chlorproethazine, although dissimilar in structure to benzoylecgonine, proved to be a very reliable internal standard because it posed no problem with derivatization; back-extracted easily into acid; was linear over a 100-fold concentration range; and was easily resolved on the gas chromatograph. A comparison of duplicate aliquots of chlorproethazine before and after derivatization indicated that the internal standard does not react with the derivatizing reagent.
Table 11. Standard Curve Data for Benzoylecgonine and Cocaine in Human Urine Cocaine/
Benzoylecgonine and cocaine added, pp 0.0 2.5 5.0 10.0 15.0 20.0
scopolamine peak height ratio
i.
SDb
0.00 I 0.00 0.44 i. 0.01 0.91 i. 0.01 1.88 i. 0.02 2.87 + 0.09 4.00 t 0.14
Benzoylecgonine/
Calculated amount of cocaine, pgc 0.0 2.5 4.9 9.7 14.7 20.4
scopolamine peak height ratio
f
SDb
0.00 i 0.00 0.19 i. 0.00 0.38 f 0.01 0.78 i 0.01 1.20 t 0.04 1.68 + 0.06
Calculated amount of benzoylec-
gonine, pgd 0.0 2.6 4.9 9.6 14.7 20.4
M e a n coefficient of variation Micrograms of benzoylecgonine and cocaine added to 1.0 mL urine in six samples each. Calculated from least-souares line f r 2= 0.998). Calculated from least-sauares line ( r 2 = 0.999).
(CV) = 2.2%.
ANALYTICAL CHEMISTRY, VOL. 49, NO. 13, NOVEMBER 1977
1967
0 0 0
0 0
000
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co cDco* a???
ln
Figure 1. GLC detection of cocaine and benzoglecgonine extrac J from plasma and urine. Chromatogram A represents electron capture detection of 5.0 and 0.05 kg of chlorproethazine-HCI (IS) and benzoylecgonine (BE), respectively, extracted from 0.5 mL plasma by method (a). Chromatogram B represents nitrogen detection of 0 . 2 5 and 2.0 kg of cocaine (COC) and chlorproethazineHC1(IS),respectively, extracted from 0.5 mL plasma by method (b). Chromatogram C represents flame ionization detection of 2.5, 2.5. and 10.0pg of cocaine (COC), benzoylecgonine (BE), and scopolamineHCI (IS),respectively, extracted from 1.0 mL urine by method (c)
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ANALYTICAL CHEMISTRY, VOL. 49,NO. 13,NOVEMBER 1977
cocaine from the nasal mucosa. Benzoylecgonine was detected in the plasma of each of the three subjects 5 min after intravenous administration of 1 to 1.9 mg/kg cocaineSHC1. Plasma concentrations of benzoylecgonine increased rapidly within the first hour, plateaued for a period of 2 to 4 h, and then declined slowly but were still detectable at 24 h (0.09-0.16 pg/mL). The rapid appearance, magnitude, and persistence of benzoylecgonine in plasma suggests that cocaine was rapidly biotransformed after administration and that benzoylecgonine was strongly bound to plasma proteins. In the f i t 6 h following cocaine administration, 14.5 to 45% of the dose was recovered in the urine and of that amount 6 to 20% was cocaine. The amount of cocaine recovered in the urine (1-21 % ) was comparable to that previously reported (5). The cumulative 0-6 h mean f SD urine concentration for benzoylecgonine and cocaine was 29.8 f 29.1 and 3.5 f 1.6 pg/mL urine, respectively. These values were consistent with the 0-8 h urine values of 47 f 38 hg/mL for benzoylecgonine plus cocaine and 1.0 f 1.0 kg/mL for cocaine reported by Wallace et al. (11)in adult patients receiving cocaine as a local anesthetic (250 mg cocaineHC1) applied topically to nasal mucosa for rhinoplastic or septoplastic surgery. A composite panel of typical chromatographic tracings of extracted standards from the representative biofluids for methods (a), (b), and (c) are shown in Figure 1. The upper case letter at the top of each panel refers to the corresponding method of the same letter described under methods. Peaks corresponding to benzoylecgonine, cocaine, and the internal standard [chlorproethazine.HCl, methods (a) and (b) or scopolamine-HC1 method (c)] are indicated by the symbols BE, COC, and IS, respectively. The retention times for the internal standard and benzoylecgonine (panel A) were 2.65 and 3.49 min, respectively. The retention times for cocaine and the internal standard (panel B) were 1.60 and 4.47 min, respectively. T h e retention times for cocaine, benzoylecgonine, and the internal standard (panel C) were 0.78,0.93, and 1.17 min, respectively. The three procedures described in this report represent comprehensive methodology for the analysis of benzoylecgonine and cocaine in human biofluids. Validation of these methods with human biological materials indicates the
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 ) . It 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
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