frequency modulated pulses to produce a constant detector current.
ACKNOWLEDGMENT The technical assistance of Larry Burkett and Donald Nickolaus is deeply appreciated. The p-methyl primidone for this particular study was supplied by Kenneth Dudley of the University of North Carolina at Chapel Hill who in turn had received a supply of the material from H. C. Schafer, DestinWerk Carl Klinke GmbH., Hamburg 63 Germany. LITERATURE CITED (I) C. E. Pippenger and H. W. Gillen, Clin. Chem. ( Winston-Salem, N.C.), 15, 582 (1969). (2) B. B. Gallagher and I. P. Baumel, Neurology, 21, 394 (1971). (3) J. C.Rodger, G. Rodgers, Jr., and A. Soo,Clin. Chem. (Winston-Salem, N.C.),19, 590 (1973). (4) F. L. Vandemark and R. F. Adams, Clin. Chem. ( Winston-Salem,N.C.), 22, 1062 (1976). (5) H. J. Kupferberg, Clin. Chim. Acta., 29, 283 (1970). (6) P. A. Toseiand, J. Grove, and D. J. Berry, C/h. Chim. Acta., 36, 321 (1972).
(7) H. R. Schafer, “Some problems concerning the quantitative assay of primidone and its metabdies”, in H. Schnekier, D. Jam,C. Gardner-Thorp, H. Melnardi, and A. L. Sherwin, “Clinical Pharmacology of Anti-Epileptic Drugs”, Springer-Veriag, New York, 1975. (8) J. C. Van Meter and H. W. Giiien, Clln. Chem. ( Winston-Salem, N.C.). 18, 357 (1973). (9) C. E. Pippenger, J. K. Penry, B. G. White, D. D. Daly, and R. Ruddington, Arch. Neurol., 33, 351 (1976). (10) A. Richens, Acta Neurol. Scand., Suppl., 60, 81 (1975). (11) R. E. Beam, Am. J . Med. Techno/.,40,211 (1974). (12) L. Livingston, W. Berman, and L. L. Pauli, J . Am. Med. Assoc., 232,
60 (1975). (13) A. Arbin and P. 0. Edlund, Acta Pharm. Suec., 12, 119 (1975). (14) A. C.Moffat, E. C. Honing, S. B. Matin, and N. Rowland, J. Chromatogr., 66,255 (1972).
RECEIVED for review December 17,1976. Accepted March 4, 1977. This research was supported in part by Grant R01 DA 00729-03 from the National Institute on Drug Abuse, NIH, Bethesda, Md., and in part by Grant R01 MH 26431-0111 from the National Institute of Mental Health, NIH, Bethesda, Md.
Identification and Chemical Classification of Drugs Based on the Relative Response of a Nitrogen Selective Detector and a Flame Ionization Detector in Gas Chromatographic Analysis John K. Baker Department of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University, Mississippi 38677
The use of a gas chromatographic system equipped with dual flame ionlzation and nitrogen Selective rubidium bead detectors in the identificationof drugs is presented. With the method, drugs are chromatographed along with a caffeine standard on a 3 % OW17 column programmed from 100 to 250 OC at 4’/min. I n addition to characterizing the drug in terms of a retention time relative to caffeine, the drug can also be characterized by the ratio of the nitrogen/FID response of the drug to the nitrogen/FID response of the caffeine standard. The response index and relative retention time of 71 nltrogsn-containing drugs commonly encountered In forensic and toxicology applications are presented.
The initial identification of drugs in forensic and toxicology laboratories is frequently based on the relative retention time of the drug observed on a single gas chromatographic column. In order to confirm the identity of the drug, it is almost always necessary to use some additional technique such as rechromatographing the drug on a second GLC column, TLC analysis, ultraviolet spectral analysis, or mass spectral analysis, etc. Historically, TLC and ultraviolet spectral analysis have been the most widely used techniques;however, the techniques are limited by low sensitivity and poor selectivity, respectively. Mass spectral analysis is certainly the most reliable of these techniques for confirming the identity of a drug; however, the high cost and mechanical complexity of the equipment have prevented this method from becoming the mainstay of most laboratories. Alkali flame ionization GLC detectors have been used since 1964 to detect organic compounds containing phosphorus or nitrogen (1). Recently, a new version of this detector utilizing an electrically heated rubidium silicate bead as the alkali 908
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
source has been reported in the literature (2). The sensitivity of this detector for nitrogen-containing organic compounds is about 10 to 50 times greater than a standard flame ionization detector and, additionally, the selectivity of the detector is about 5000 times greater for nitrogen-containing compounds than for simple hydrocarbons. Because of these characteristics, this detector is extremely useful in the GLC analysis of nitrogen-containingdrugs such as narcotics, amphetamines, barbiturates, tranquilizers, and most of the other drugs that have a high abuse potential. When the nitrogen detector is used by itself, it is often difficult to determine if a particular chromatographic peak represents a small amount of a nitrogen-containing drug or a large quantity of a hydrocarbon. However, if the chromatogram is obtained using dual FID-nitrogen detectors, the two cases can be readily distinguished by noting the relative response of the compound on the two detectors. The response of the nitrogen selective detector is roughly proportional to the number of nitrogens contained in the compound, but the response is also highly dependent on the type of nitrogen group the compound contains. In general, those compounds having structures that would be expected to be favorable for the formation of cyan radicals following pyrolysis on the surface of the rubidium bead would be expected to give the larger response (2). Thus not only would the dual detector system distinguish between a hydrocarbon and a nitrogen-containing compound with similar retention times, but it could also be used to distinguish between two nitrogen-containing compounds with similar retention times.
EXPERIMENTAL A model 900 Perkin-Elmer gas chromatographwas equipped with a 2 mm X 183 cm glass column packed with 3% OV-17 on 110-120 mesh Anachrom ABS support. Following the injection of the sample, the column temperature was programmed from
100 t o 250 O C at 4O/min, then held at 350 O C for 15 min (52.5 min total). The helium carrier gas (27 mL/min) was split equally between a flame ionization detector (H, = 19 mL/min) and a rubidium bead nitrogen-phosphorus selective detector (H, = 5.1 mL/min). The latter detector was operated in the mode where both nitrogen- and phosphorus-containing compounds would be detected and the rubidium bead was electrically heated (current control was set at 6.0). An air flow of 300 mL/min was used for both detectors. A 1-bg sample of each drug was chromatographed as a mixture with 1 fig of caffeine. The retention time of each drug was measured from the leading edge of the solvent peak obtained from the flame ionization detector and the relative retention times were based on that of caffeine (22 rnin). The ratios of NP-detector and the FID detector peak heights of the drug were measured and compared to the same response ratio for the caffeine standard. By definition, this was taken to be the “response index” of the drug and this parameter was calculated as shown below.
response index = [(NP peak height of drug)/(FID peak height of drug)] / [(NP peak height of caffeine)/(FID peak height of caffeine)]
RESULTS AND DISCUSSION The relative retention times that were obtained (Table I) were found to very closely parallel those reported in a more comprehensive compendium where isothermal chromatographic systems were used ( 3 ) . The values for the response index obtained varied over 100-fold from drug to drug with the largest number having values in the 0.10 to 0.30 range. An example of the utility of the technique in the identification of drugs can be seen through comparisons of the data in Table I in the 1.14-1.19 RRT range. In this range several compounds have either identical or very similar retention times, yet have markedly different response indices. Theophylline has a response index 10 times larger than propoxyphene and would be easily differentiated. However, it should also be noted that similar compounds, such as the isomeric propoxyphene and methadone, have almost identical response indices. The relative retention times of several drugs are also closely grouped in the 0.80-0.92 range. Though the response indices of pentobarbital and secobarbital are very similar, they can be differentiated from the thiobarbiturates and the N-substituted barbiturates in the same area. The response and the selectivity of the NP detector has been reported to be highly dependent on the hydrogen flow rate and the temperature of the alkali source ( 1 , 2 , 4 , 5 ) . In order to determine if the response index was also highly dependent on the hydrogen flow, diazepam was arbitrarily selected as a test compound and it was run under a variety of conditions. The hydrogen flow rate of the flame ionization detector was increased to 136% above the standard conditions given in the Experimental section and the hydrogen flow rate of the NP detector was increased 41% above the standard conditions. Even with the larger changes, the response index of diazepam was within 1% of the original value. Several compounds were run numerous times over a 2month period in order to estimate the short term and long term repeatability of the response index of the drugs (Table 11). The standard deviation for runs made over a period of 1 day was about 6% for the response index and 0.5% for the relative retention time. The values for the short term standard deviation of response index ranged from a low of 1% for nikethamide to a high of 17% for one of the phendimetrazine runs. The long range reproducibility of the response index was about 10% of the value and about 2% for the relative retention times. A crude evaluation of this technique in identifying unknown drugs was made by determining the number of the drugs in
Table I. Relative Retention Times and Response Index of Selected Drugs
Drug
Relative retention time
Cyclopen tamine Propylhexidrine Amphetamine Methamphetamine Mephenteramine Carbromal Acetylcarbromal Nicotine Ephedrine Phenylpropanolamine Ethinamate Phendime trazine Phenmetrazine Metharbital Barbital Salicylamide Nikethamide Methyprylon Allobarbital Aprobarbital Butalbital Butabarbital Amobarbital Methylphenidate Pentobarbital Phenacetin Methohexital Mescaline Secobarbital Meperidine N,N-Dimethyltryptamine Phencyclidine Thiopental Dimenhydrinate Meprobamate Thioamyal Hexobarbital Glu the thimide Carbisoprodol Doxylamine Mephobarbital Caffeine Methapyriline Cyclobarbital Phenobarbital Tybamate Procaine Methadone Dextromethorphan Theophylline Propox y phene Methaqualone Disulfiram Cocaine Pentazocine Promethazine Levallorphan Oxazepam Chlordiazepoxide Oxymetazoline Dih ydrocodeine Codeine Diphenylhydantoin Morphine Diazepam Oxycodone Oxymorphone Fentanyl Phenazocine Heroin Flurazepam
0.10 0.14 0.15 0.18 0.23 0.24 0.24 0.35 0.37 0.38 0.45 0.47 0.48 0.49 0.58 0.61 0.63 0.64 0.69 0.70 0.74 0.74 0.77 0.80 0.81 0.8 3 0.84 0.85 0.86 0.86 0.87 0.88 0.91 0.92 0.95 0.96 0.96 0.96 0.98 0.99 0.99 1.00 1.07 1.09 1.10 1.14 1.14 1.16 1.16 1.18 1.19 1.27 1.28 1.28 1.29 1.30 1.37 1.41 1.41 1.41 1.44 1.44 1.48 1.50 1.51 1.63 1.66 1.66 1.76 1.79 1.91
Response index 0.28 0.27 0.33 0.42 0.23 0.10 0.087 0.36 0.34 0.31 0.017 0.20 0.24 0.24 0.11 0.22 0.42 0.1 2 0.036 0.044 0.036 0.043 0.035 0.16 0.037 0.098 0.16 0.23 0.033 0.15 0.30 0.12 0.21 0.14 0.0090 0.18 0.18 0.019 0.20 0.28 0.15 1.00 0.47 0.066 0.070 0.15 0.33 0.10 0.11 0.95 0.098 0.24 0.48 0.13 0.093 0.24 0.093 0.24 0.23 0.26 0.13 0.13 0.081 0.12 0.27 0.11 0.12 0.19 0.096 0.11 0.29
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Table 11. Short Term and Long Term Reproducibility of the Relative Retention Time and Response Index Relative Response Drug retention time index Nikethamide Period 1 0.622' 0.448' 0.634 i 0.004b 0.422 i O.OOgb Period 2 0.621 i 0.002 0.395 t 0.005 Period 3 Av 0.627 0.422 Bar bi t a1 Period 1 0.579 0.166 0.580 2 0.005 0.089 I0.003 Period 2 0.107 -i 0.010 Period 3 0.588 f 0.003 0.576 * 0.002 0.082 I0.004 Period 4 Av 0.581 0.111 Phendimetrazine 0.467 0.216 Period 1 Period 2 0.480 i 0.007 0.170 I0.017 0.461 t 0.001 0.202 t 0.034 Period 3 Av 0.469 0.196 a Only a single measurement was made during the initial period. Standard deviation of the measurements made over a one-day period. Table I that had a relative retention time within 2% of any other drug. This was found to be true for 59% of the drugs; however, this dropped to 16% if one also required that the response index of the drugs be within 20%. If one were to measure these two parameters of an unknown drug within several hours of an authetic standard, considerably narrower windows could be used and the number of uncertain identifications could be greatly reduced. It has been proposed that rubidium bead nitrogen selective detectors produce an electron current following the conversion of the compound to a cyan radical under the pyrolytic conditions on the surface of the rubidium bead (2). If this were the case, there should be a fairly good correlation between the type of nitrogen that the compound contained and the response index of the compound. As anticipated, the response index of various groups of drugs were found to fall within fairly narrow groups (Figure 1). Nonsubstituted amides had the lowest response index and were considerably lower than the corresponding N-substituted amides. In order to produce a cyan radical from the nonsubstituted amides, it would have been necessary to break the carbon-oxygen bonds, while in the case of the alkyl substituted amides, the cyan radical could have been derived from the alkyl group and the nitrogen. As also observed in earlier reports (2, 61, the N-substituted barbiturates were found to have a much higher response index than the nonsubstituted barbiturates. It was also found that the response index generally increased if the number of nitrogens in the compounds increased (Figure 1). However, this was found to be only a general trend and many examples can be found in Table I where a compound containing two nitrogens had a lower response index than a compound with only one nitrogen. It was also observed that tertiary amines had a much lower response index than secondary or primary amines (Figure 1). A specific example of this difference was noted in comparing the response index of methamphetamine (0.42) and phendimetrazine (0.20). These two drugs had essentially the same structure except the former was a secondary amine and the latter was a tertiary amine. A possible explanation for the
908
ANALYTICAL CHEMISTRY, VOL. 49, NO. 7, JUNE 1977
+
U
RENH 7
-
0 R ~ H X 0 ,CH3
m
*
o n HETEROCYCLICS WITH 2 NITROGENS
m
HETEROCYCLICS WITH 3 NiTROGENS XANTHINES
1 1 , , , 1 0.01
0.05
0.1
0
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, , I l l l l 0 5
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8
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RESPONSE INDEX
Figure 1. Effect of chemical structure on the response index. The vertical bars represent the mean and the standard deviation
observed differences may be that primary and secondary amine readily lost hydrogen under the pyrolytic conditions, then were rapidly converted to a cyan radical, while with tertiary amines a carbon-nitrogen bond would have to be broken first. In summary, it was found that the response index was characteristic of a given drug and had considerable exclusionary value in reducing the possible candidates in the identification of drug unknowns. It was also found that response index of drugs with the same type of nitrogen structure had very similar values. Conversely, when a drug unknown or drug metabolite is chromatographed,the observed response index could be used to narrow down the possibilities of the chemical class of the drug. However, because of the overlap of the response index ranges, it is unlikely that the chemical class of the unknown could be uniquely determined. It should also be emphasized that the response index data reported are preliminary in nature and it has not been demonstrated that these values are reproducible in other laboratories using the same equipment or using detectors of different manufacturing design.
ACKNOWLEDGMENT The technical assistance of Shellie Rothstein in this project is most gratefully acknowledged. LITERATURE CITED (1) A. Karmen and L. Guiffrlda, Nature (London), 201, 1204 (1964). (2) B. Kolb and J. Bischoff, J. Chromatogr. Sci., 12, 825 (1974). (3) B. S. Finkle, E. J. Cherry, and D. S. Taylor, J . Chromatogr. Scl., 9, 393 (1971). (4) B. H. Dvorchik, J . Chromatogr., 105, 49 (1975). ( 5 ) P.I. Jatlow and D. N. Bailey, Clin. Chem. ( Wlnston-Salem. N . C . ) .21, 1918 (1975). (6) M. Donike, L. Jaenicke, D. Stratmann, and W. Hollmann, J. Chromatcgr., 52, 237 (1970).
RECEIVED for review December 27,1976. Accepted March 17, 1977. Parts of this investigation were supported by the Research Institute of Pharmaceutical Sciences.
