standard dwiation of 0.3 %. Data from individual standard curves, one of which is shown in Figure 3, indicate that 95% confidence h i t s of the assay are 5-10 pmoles/2 ml sample. The results of a dose plasma level study in rats are shown in Table 11. Two-ml aliquots of plasma'taken from rats given 0.25 to 10 mg/kg amphetamine were extracted and analyzed as described.
ACKNOWLEDGMEW The advice and assistance of Margareth Roch and R. W. Silverman are gratefully acknowledged. RECEIVED for review September 25, 1972. Accepted November 16, 1972. This work was supported by USPHS Grants: No. MH20473 and MH17691.
Rapid Determination of Barbiturates by Gas Chromatography- Mass Spectrometry Ronald F. Skinner,*t2Edward G . Gallaher, and David B. Predmore Washington State Toxicology Laboratory, Unicersity of Washington, Seattle, Wash. 98195
THEWIDESPREAD AVAILABILITY of barbiturates obtained both by prescription and from illicit sources has caused a rapid increase in accidental and deliberate overdosages from these drugs. Over 15,000 persons are hospitalized each year for treatment of acute barbiturate poisoning ( I ) . In order to properly treat these overdose cases, it is necessary to rapidly establish the identity and amount of the barbiturate present. In cases of lethal intoxication, this information is essential for establishing the cause of death, since the different barbiturate derivatives have a wide range of lethal levels. Gas chromatography and thin-layer chromatography have been used in most laboratories to provide identification of the particular barbiturate involved in a n overdose case. Neither of these techniques can unquestionably identify the barbiturate or barbiturates present in a sample, because of the large number of structurally and chromatographically similar barbiturates. Other compounds in the sample may occasionally give rise to results which could be misinterpreted as evidence of barbiturates; therefore, a back-up method such as ultraviolet analysis must be used to verify the presence of the barbiturate. The recent advent of less expensive and less complex gas chromatograph-mass spectrometer combinations (GC-MS) has made this powerful tool a reasonable one for use in laboratories doing routine analyses. The GC-MS provides rapid, accurate qualitative and quantitative identification of barbituric acid derivatives. The advantages of and conditions for chromatographing the barbiturates as their 1,3-dimethyl derivatives have been discussed by Brochmann-Hansen and Oke (2) and others (3-6). The procedure presented here is a modification of the Brochmann-Hansen and Oke methodology and makes use of triPresent address, Finnigan Instrument Corp., 595 N. Pastoria Ave., Sunnyvale. Calif. 94086. Mass spectra of the methylated barbiturate derivatives are available from this author. ( I ) N. M.Simon and F. A. Krumlovsky, Ratioiiul Drug Tllerupy, 5, l(1971).
Brochmann-Hansen and T. 0. Oke, J . PIiurrn. Sci., 58, 370 (1969). (3) G. W. Stevenson. ANAL.CHEM., 38, 1948 (1966). (4) J. G. H. Cook, C. Riley, R . F. Nunn, and D. E. Budgen, J . Clirornurogr., 6, 182(1961). (5) H. F. Martin and J. L. Driscoll, ANAL.CHEM., 38, 345 (1966). (6) G. A. Neville, ibid.. 42, 347 (1970).
(2) E.
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ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
2
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4
V
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10
iL'es
Figure 1. Chromatogram of 200 ng each of (1) barbital, (2) probarbital, (3) aprobarbital, (4) butabarbital, (5) amobarbital, (6) pentobarbital, (7) secobarbital, (8) hexethal, (9) glutethimide, (10) phenobarbital, (11) heptabarbital, (12) diphenylhydantoin methylanilinium hydroxide both to extract the barbiturate from the organic phase and to methylate it. This considerably shortens the procedure necessary for the preparation of an injectable fraction for GC-MS ideritification.
EXPERIMENTAL Apparatus. A Beckman GC-5 gas chromatograph equipped with a hydrogen flame detector, a n optical follower temperature programmer, and a Beckman 10-inch strip chart recorder was used for the initial scanning of samples. Mass spectrometry was done with a Finnigan Model 3000 gas chromatograph-mass spectrometer equipped with a glass jet separator, a Heath Model EU-205-11 recorder, and a Honeywell Visicorder. The electron ionization energy was 70 eV. Both gas chromatographs were equipped with 6-ft x '/,-inch stainless steel columns packed with 3% OV-1 on 1001120 mesh Gas Chrom Q. During analysis, the column temperature was programmed from 120-220" C at 10' C,/minute. The flow rate of the helium carrier gas was 30 m l h i n u t e . The column was kept at 240 "C when not being used for analysis, as this resulted in less column background.
Table I. Gas Chromatographic and Mass Spectrometric Properties of Barbituric Acid Derivatives Mass Mass Fragmentation Compound Mol wtC RETd R1 R2 Ri R2 routese Allobarbital 236 0.87 allyl allyl 41 41 C,A Alphenal 272 1.03 allyl phenyl 41 77 D,B Amobarbital 254 0.90 ethyl isoamyl 29 71 A,C Aprobarbital 238 0.87 allyl isopropyl 41 43 CAE Barbital 212 0.83 ethyl ethyl 29 29 A,C Butabarbital 240 0.88 ethyl sec-butyl 29 57 A,C Butalbital 252 0.89 allyl isobutyl 41 57 A,C Butethal" 240 0.89 ethyl butyl 29 57 A,C Cyclobarbital 264 1.01 ethyl 1-cyclohexen-1-yl 29 81 D,B Cyclopal" 262 0.96 allyl 2-cyclopenten-1-yl 41 67 D,A Diphenylhydantoin 280 1.15 ... ... ... ... ... Glutethimide 231 0.99 ... ... ... ... Heptabarbital 278 1.05 ethyl 1-cyclohepten-1-yl 29 95 B,D HexethaP 268 0.96 ethyl hexyl 29 85 A Hexobarbitala,* 250 0.99 methyl 1-cylohexen-1-yl 15 81 CAD Pentobarbital 254 0.91 ethyl 1-methylbutyl 29 71 A Phenobarbital 260 1.00 ethyl phenyl 29 77 D,B Probarbital 226 0.85 ethyl isopropyl 29 43 A,C Secobarbital 266 0.93 allyl 1-methylbutyl 41 71 A,C sec- butyl 41 57 C,A Tal butal 252 0.90 allyl Vinbarbital 252 0.92 ethyl 1-methyl-1-butenyl 29 69 D,B a Not listed in "Physicians' Desk Reference," 25th ed., 1971, Medical Economics, Inc., Oradell, N.J. N-methylated. e Molecular weight of methylated derivative. Relative Elution Temperature = elution temperature in "C of compound/elution in "C of phenobarbital. e Listed in order of importance. Listed in order of magnitude. 0 R CHSNCO. t
.
.
f
Reagents and Solutions. ACETATE BUFFER. Add 12 grams of sodium acetate to 60 ml 1N acetic acid and dilute to one liter with distilled water. EXTRACTION SOLVENT. Prepare by mixing equal volumes of reagent grade ether and reagent grade toluene. Add 5-allyl-5-(2-cyclopenten-l-yl)barbituric acid (not currently marketed) in a concentration of 2 pg/ml to serve as a n internal standard. TRIMETHYLANILINIUM HYDROXIDE (TMAH) 25 %. Dissolve 43 grams of trimethylanilinium iodide (Eastman No. 4423) in 100 ml of methyl alcohol by gently heating the solution. Add 29 grams of silver oxide and stir for one-half hour or until a test for iodide is negative. This solution is stable for several months when stored under refrigeration. BARBITURATE STANDARDS.The barbiturates were obtained in the acid form from their respective manufacturers and from Applied Science Laboratories, State College, Pa. Procedure. One milliliter of whole blood, serum, or plasma, 1 ml of acetate buffer, and 5 ml of ether-toluene are placed in a 15-ml centrifuge tube fitted with a Teflon (Du Pont)-lined screw cap. The mixture is shaken for 3 minutes a t 60 rpm o n a Fisher Roto-rack and then centrifuged for 1 minute at 2000 rpm. Four and a half milliliters of the organic phase is placed in a Hopkins Vaccine Tube (Kimax No. 45225) which has been modified to take a glass stopper. T M A H solution (0.2 ml) is added to the organic phase and the stoppered tube shaken on a Vortex Jr. mixer for 15 seconds. It is then centrifuged for 30 seconds at 1000 rpm. The supernatant phase is decanted and 1 p1 of the lower T M A H phase, approximately 50 p l , is injected. Powders and tablets suspected of containing barbiturates can be dissolved directly in the 25% T M A H solution in appropriate concentrations, and chromatographed. Standards may also be prepared in this manner from barbituric acids or their salts. Mass spectra are recorded on peaks of interest. RESULTS AND DISCUSSION This extraction technique provides quantitative extraction of all barbiturates tested and is equally effective in the deter-
0
,
2
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4
6
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8
9
IC'
Mlnutes
Figure 2. Chromatogram obtained from a secobarbital overdose. Peaks are secobarbital (1) and the internal standard (2). The level was calculated to be 1.3 mg mination of diphenylhydantoin and glutethimide. Standard deviations of less than are obtained for all barbiturates Experience in our laboratory in the range of 0.2-10 mg and in allied laboratories indicates that about 5 false positives are obtained when it is used as a GC method without the benefit of a mass spectrometer. Relative elution temperatures (7) to phenobarbital are given in Table I for all the barbiturates studied. This has proven to be a reliable index of their elution order. Figure 1 shows a typical chromato-
&5z
z.
(7) J. A. Schmit and R. B. Wynne, J . Gas Cliromatogr., 4, 325
(1966). ANALYTICAL CHEMISTRY, VOL. 45, NO. 3, MARCH 1973
575
m*
CH3-N
s Dz
R2
0
m/e= MR2t 97
m*
, CH3-N@
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m/e=M~,+97
free acids. These derivatives were not extensively examined, however, as they are rarely found in the body because of their rapid metabolism. For the barbiturates examined, blood levels of 0.2 mg or higher were sufficient to give a complete identifiable mass spectrum. Lower levels could be identified by extracting larger volumes of blood. The postulated fragmentation patterns of the methylated barbituric acid derivatives are presented in Figure 3, and the major fragments for a given compound are listed in Table I according to the scheme presented in Figure 3. The fragmentation patterns are very similar to those previously obtained for unmethylated barbituric acid derivatives (8-10). They reflect an even higher stability of the ring system than shown by the unmethylated barbiturates. Only when R1or RP is aromatic does significant cleavage of the ring system occur.
n
CONCLUSION
m/e = M ~ , t 1 5 5
m / e 169
m / e 112
Figure 3. Fragmentation patterns of methylated barbituric acid derivatives gram for barbiturate standards. Figure 2 shows a chromatogram from a seconal overdose. N-Methyl barbiturates could not be distinguished from their unmethylated homologs by this procedure. This was not considered a serious deficiency, since they are rapidly metabolized to their N-demethylated derivatives in the body and would be found as such. Preliminary studies with the thiobarbituric acid derivatives indicated that their methylated derivatives are more stable in gas chromatography than the
The above study shows that the GC-MS can be used to provide rapid identification of barbiturates in overdose cases. However, the methodology presented here requires a skilled operator to interpret the spectra obtained. Computerization of the mass spectrometer output and the use of computer search programs would alleviate the need for skilled interpretation. Work in this direction is in progress. RECEIVED for review May 22, 1972. Accepted October 16, 1972. (8) A. Costopanagiotis and H. Budzikiewicz, Moiiatsh. Cliem., 96, 1800 (1965). (9) H. Grutzmacher and W. Arnold, Tetrukedro/7 Lett., 13, 1365 (1966). (10) R. T. Coutts and R. A. Locock, J . Plzarm. Sci., 57,2096 (1968).
Effect of Oxygen on Response of the Electron-Ca pture Detector Francis W. Karasek and David M. Kane Department of Chemistry, Unicersity of Waterloo, Waterloo, Ontario, Canada SINCEITS CONCEPTION by Lovelock ( I ) , the electron-capture (EC) detector for gas chromatography has occupied an important position in analytical use because of its unique sensitivity and selectivity, particularly for compounds of biomedical and environmental significance. The importance of the detector has led to many studies of its characteristics, both to improve performance ( 2 ) and to understand its basic mechanism (3). These early studies and their conclusions were necessarily made from indirect experimental data. The recent development of the new technique of plasma Chromatography for (1) J. E. Lovelock and S. R. Lipsky, J . Amer. Chem. Soc., 82, 431 ( 1960). (2) P. G. Simmonds, D. C. Fenimore, B. C . Pettitt, J. E. Lovelock, and A. Zlatkis, ANAL,CHEM., 39, 1428 (1967). (3) W. E. Wentworth and E. Chen, J . Gas Chromatogr., 5 , 170 (1 967).
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the first time permits one to obtain direct experimental data to observe the charged species being formed in the EC detector and their response and mobility under changing parameters. The plasma chromatograph, operating at atmospheric pressure, involves generation of charged particles in a carrier gas by a radioactive 63Ni source and an ion-molecule reactor whose products are subsequently separated in a coupled iondrift spectrometer. Both positive and negative species can be observed. The instrumentation is shown in Figure 1. Ions formed by the 63Ni source in the flowing carrier gas are moved by a n electrical field through the ion-molecule reactor section toward the drift spectrometer. A pulse of these charged species is injected into the drift spectrometer, where separation of ionic species occurs because of their different mobilities as they move through an inert gas at 760 Torr to reach the detector in a series of ion peaks recorded as a plasmagram. A variable delay gating technique on the scan grid permits recording of
