Journal of Analytical Toxicology, Vol. 14, July/August 1990
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Cocaine Metabolism in Man! Identification of Four Previously Unreported Cocatne Metabolites in Human Urine* Ji Y u e Z h a n g and R o d g e r L. F o l t z * *
Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84108
I Abstract
Experimental
I
Cocaine and 11 of Its metabolites were Identified In a urine specimen from e cocaine user. Four of the metabolltes are reported for the first time: ecgonidine, norecgonidlne methyl ester, norecgonine methyl ester, and m-hydroxybenzoylecgonine. The structures of the newly identified metabolitea were confirmed by comparison of their gas chromatographic retention times and their electron Ionization and chemical Ionization mass spectra with the corresponding data obtained on synthesized standards. Other metabolitea present were benzoylecgonine, ecgonlne methyl ester, ecgonine, ecgonidine methyl ester, norcocaine, p~ cocaine, and m-hydroxycoceine.
Introduction In the body, cocaine is rapidly converted to metabolites by enzymatic and chemical processes, so very little cocaine is usually excreted in the urine (1). The two major urinary metabolites of cocaine are benzoylecgonine (2) and ecgonine methyl ester (3). In a recent study, Ambre et ai. reported that 14-17% of a cocaine dose was excreted in the urine as benzoylecgonine, and 12-21% as ecgonine methyl ester (4). Other cocaine metabolites reported in human urine specimens include norcocaine (5), ecgonine (2), benzoylnorecgonine (6), ecgonidine methyl ester (7), cocaine hydroxylated on the benzoyl ring (8), and hydroxymethoxy-substituted benzoylecgonine (9)~, Here we report the results of an extensive GC/MS analysis of a urine specimen received as part of a drug testing program and initially found to be positive for benzoylecgonine. A total of 11 metabolites of cocaine were identified, including four that had not been previously reported. Identifications were based on comparison of the GC retention times and electron ionization (EI) and chemical ionization (C1) mass spectra of the derivatized metabolites with the corresponding data obtained on purchased or synthesized standards. The metabolites were derivatized by treatment with hexafluoroisopropanol and pentafluoropropionic anhydride (10). 9Presented In part at the 37th ASMS Conference on Mass Spectrometryand AlUedTopics, Mtamt Beach, Flonda, May 22-26, 1989. "* Author to whom correspondence should be addressed,
Specimen. The metabolites reported here were identified in a single urine specimen received in conjunction with a general drug testing program. No information is available regarding the donor, the size of the cocaine dose, or the mode and time of administration. Materials. Cocaine, benzoylecgonine, and ecgonine methyl ester were obtained from Alitech Associates; ecgonine and salicylic acid were from Sigma Chemical; methyltriphenylphosphonium iodide, hexamethylphosphoric triamide (HMPA), 1,3-dicyclohexylcarbodiimide (DCC), boron trifluoride etherate ( B F J E h O ) , and m-hydroxybenzoic acid were from Aldrich Chemical; p-hydroxybenzoic acid was from Eastman Kodak; pentafluoropropionic anhydride (PFPA) was from Regis Chemical; and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was from Pierce Chemical. Hydrolysis and extraction. To a 2-mL aliquot of urine in a 25-mL screw-capped extraction tube was added 4 mL of 1M sodium acetate buffer (pH 5) containing 20,000 units of/3glucuronidase. The mixture was incubated overnight at 37~ After the pH was adjusted to 8.5 with 1N NH,OH, the incubated sample was extracted twice with 5-mL volumes of chloroformisopropanol (3:1). The combined organic fractions were then back-extracted into 5 mL of 0.1N HCI. The organic layer was discarded and the aqueous phase was adjusted to pH 8.5 with IN NH,OH and extracted twice with 5-mL volumes of chloroform-isopropanol (3:1). The organic layers were combined and evaporated to dryness at 50~ under a gentle stream of air. Derivatization. To the tube containing the urine extract, 70/zL of PFPA and 30 #L of HFIP were added. The mixture was vortexed for 10 s and then heated at 70~ for 10 min. After evaporation of the mixture to dryness under a gentle stream of air at 50~ the residue was taken up in 20 #L of ethyl acetate and analyzed by GC/MS (Figure 1). Synthetic procedures
Ecgonidine methyl ester. Ecgonidine methyl ester was synthesized from ecgonine methyl ester by modification of a published procedure for dehydration of alicyclic alcohols (11). A mixture of 0.5 mg methyltriphenylphosphonium iodide in 1 mL of hexamethylphosphoric triamide was added to 400 ttg of ecgonine methyl ester in a 5-mL microvial. The microvial was wrapped in black paper to protect it from light, and the mixture
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Journal of Analytical Toxicology, Vol. 14, July/August 1990
was then stirred for 4 h at 50~ and cooled to room temperature. Water (2 mL) was added and the solution was made acidic by addition of 1N HCI. The mixture was then washed with 2 mL of chloroform. After separation of the phases, the aqueous layer was adjusted to a pH of 8.5 with IN N H , O H and extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The combined organic layers were evaporated to dryness and the unpurified product was taken up in ethyl acetate and analyzed by GC/MS. The major chromatographic peak gave a mass spectrum consistent with ecgonidine methyl ester. Ecgonidine. Ecgonidinr was obtained by hydrolysis of ecgonidine methyl ester. Ecgonidine methyl ester (about 200/zg) was refluxed with 1 mL of 2N NaOH for 2 h. After cooling to room temperature, the solution was adjusted to pH 8 with 2N HCI and extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The organic layer was evaporated to dryness and the residue was derivatized by treatment with 30 ~L of H F I P and 70/~L of P F P A at 70~ for 10 min. After evaporation to dryness, the product was dissolved in ethyl acetate and analyzed by G C / M S . Norecgonine methyl ester. Norecgonine methyl ester was prepared from purified norcocaine via norecgonine. Norcocaine (1 rag) was refluxed with 2 mL o f 2 N NaOH in a 50-mL roundbottom flask for 4 h. After cooling, the solution was made acidic by addition of 2N HCI and extracted with 2 mL of chloroform to remove the liberated benzoic acid. The aqueous layer was evaporated to dryness on a hot plate under a stream of air. The residue was taken up in 2 mL of methanol containing 0.1 mL of boron trifluoride-etherate (BF~-Et20). The solution was refluxed for 2 h, cooled to room temperature, and poured into 5 mL of water. After its pH was adjusted to 8.5 with IN NH,OH, the solution was extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The combined organic extracts were then evaporated to dryness. The residue was derivatized by treatment with 70/~L of pentafluoropropionic anhydride at 70~ for 10 min and the product was analyzed by G C / M S . Norecgonidine methyl ester. Norecgonidine methyl ester was synthesized from norecgonine methyl ester by the same procedure described above for the synthesis of ecgonidine methyl ester from ecgonine methyl ester. Hydroxybenzoylecgonines and hydroxycocaines. The ortho-, meta-, and para-hydroxybenzoylecgonine isomers were prepared by coupling ecgonine to o-, m-, and p-hydroxybenzoic acids, respectively, as illustrated in the following example.
9
4
11
To a 5-mL microvial were added 0.1 mg of ecgonine, 0.5 mg of o-hydroxybenzoic acid (salicylic acid), 1 mg of 1,3-dicyclohexylcarbodiimide (DCC), and 2 drops o f pyridine in 1 mL of methylene chloride. The solution was stirred at 50~ for 2 h. After cooling, the reaction mixture was washed with 2 mL of water and extracted with 2 mL of 2N HCI. The acidic solution was extracted with methylene chloride to remove nonbasic components. The aqueous solution was then adjusted to a pH of 8.5 with IN N H , O H and extracted with two 2-mL volumes of chloroform-isopropanol (3:1), and the combined organic extracts were evaporated to dryness. The unpurified o-hydroxybenzoylecgonine was characterized by G C / M S analysis after conversion to its H F I P / P F P derivative by treatment with 70 ~L of pentafluoropropionic anhydride and 30 ~L of hexafluoroisopropanol at 70~ for 10 min. The excess derivatizing agents were removed by evaporation before addition of 20/~L of ethyl acetate and injection of a I-/~L aliquot into the G C / M S . The ortho-, meta-, and para-isomers o f hydroxycocaine were synthesized in a similar manner by substituting ecgonine methyl ester for ecgonine in the above procedure. Instrumentation
All G C / M S analyses were performed on a Finnigan-MAT Model 800 ion trap detector (ITD) coupled to a Hewlett-Packard Model 5890 gas chromatograph and an IBM AT microcomputer. The ion trap manifold was maintained at 225~ All electron ionization mass spectra were acquired using the automatic gain control (AGC) program, while chemical ionization mass spectra were acquired using the automatic reaction control (ARC) program. The ion trap detector was autotuned according to manufacturer's recommendations. Electron ionization mass spectra (EIMS) were acquired by repetitively scanning the mass range m/z 70 to 650 at a rate of one scan per second. The electron multiplier voltage was set at the voltage required to give a 10' gain. The filament emission current was set at 5 #A. Chemical ionization mass spectra (CIMS) were acquired under the following operating conditions: reagent gas, methanol; ionization RF level, 8 amu; reaction RF level, 20 amu; ionization time, 120 ms; filament emission, 10 #A; and the electron multiplier set at the voltage required to give a 10' gain. The methanol pressure within the ion trap was adjusted in the following manner. With the ARC function turned off and the B value (sensitivity) set to a minimum, the reagent gas metering valve was gradually opened until the area of the m/z 33 ion reached 4000 counts. The G C / M S analyses employed at 15-m x 0.25-mm i.d. J&W Scientific DB-I capillary column with a film thickness of 0.25 gin. The carrier gas (hydrogen) linear velocity was 60 cm/s measured at an oven temperature of 100~ After a splitless injection, the oven temperature was maintained at 100~ for 0.5 min with the split flow turned off, and then the oven temperature was programmed to 250~ at 10~
T0I 1~12 2! 3 '
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1. The total ion current profile from the GC/MS analysis of the derivatized urine extract.
Figure
202
and
Discussion
Cocaine and 11 of its metabolites were detected and identified in the urine specimen from a cocaine user. The total ion current profile from G C / E I M S analysis of the derivatized urine extract is shown in Figure 1. The mass spectrum recorded for chromatographic peak 9
Journal of Analytical Toxicology, Vol, 14, July/August 1990
(Figure 2a) is consistent with mass spectra of cocaine that have been reported and discussed previously (7,9,12,13). The most abundant ion (rn/z 82) is formed by fragmentation of the bicyclic ring system to form a protonated methylpyrrole structure. Additional abundant ions are present at m / z 105, corresponding to the benzoyl ion, and at m / z 182, corresponding to the loss of the benzoate radical from the molecular ion. There is also a structurally useful ion observed at m/z 272 which is formed by the loss of the methoxyl radical from the molecular ion. Analogous fragmentation processes are observed in the mass spectra of the derivatized metabolites of cocaine. For example, the mass spectrum corresponding to chromatographic peak 4 (Figure 2b) shows the same abundant ions at m / z 82 and 182 that are in the cocaine spectrum, indicating that the cocaine bicyclic structure and the methyl ester are present. However, the absence of the benzoyl ion (m/z. 105) and the shift of the molecular ion to m / z 345 identify the compound as the pentafluoropropionyl derivative of ecgonine methyl ester. In the mass spectrum recorded for chromatographic peak 7 (Figure 2c), the m / z 82, 105, and 272 ions are present, but the molecular ion and the M-C,H,CO, ion have shifted to rn/z 439 and 318, respectively. These features clearly indicate that the compound corresponds to the hexafluoroisopropyl derivative of benzoylecgonine. The mass spectrum from chromatographic peak 2 (Figure 2d) is similar to the mass spectrum of the benzoylecgonine derivative in that it shows an abundant ion peak at m / z 318. However, because in this case the m / z 318 ion is formed by loss of the pentafluoropropionate radical from the molecular ion at ra/z 481, the spectrum identifies the compound as the hexafluoroisopropyl/pentafluoropropionyi derivative of ecgonine.
Chromatographic peak 11 was identified as the N-pentafluoropropionyl derivative of norcocaine. The m/z. 82 ion is absent in its spectrum (Figure 3) and is replaced by prominent ions at m / z 213 and 194, which are observed in the mass spectra of all N-desmethyl (nor-) metabolites. Curiously, the molecular ion of the norcocaine derivative undergoes loss of benzoic acid, rather than loss of the benzoate radical, to give the prominent ion at m / z 313. Chromatographic peak 3 was identified as ecgonidine methyl ester based on comparison of its mass spectrum (Figure 4) with that reported by Lowry et al. (7) for what they referred to as "methylecgonidine." Ecgonidine methyl ester may not be a true metabolite of cocaine, because it can be formed as a pyrolysis product when cocaine is smoked (14). However, it has been identified in rat urine following interperitoneal administration of cocaine (15) and in bile from a young man who died from an overdose of cocaine after apparent intravenous administration (7). Also, we know that the ecgonidine methyl ester detected in our study was not formed during the extraction and derivatization steps, because no ecgonidine methyl ester was detected when drug-free urine specimens were fortified with cocaine and subjected to the same extraction and derivatization conditions. The most abundant ion in the mass spectrum of ecgonidine methyl ester occurs at m / z 152, which is 29 atomic mass units below the molecular ion (m/z 181). A possible mechanism for this fragmentation is loss of the ethylene bridge along with a hydrogen atom to form the relatively stable N-methyl pyridinium ion shown in the following equation (15): CH3
CH3 N§
O
+ N ~
OCH3 -- CHi--CH2 +H ~--- 0 C-OCH3 mlz 152 0
m/z 1111
The mass spectra recorded for the chromatographic peaks I and 5 also show prominent M-29 ions, The short retention time of peak I) an apparent molecular ion at m / z 317, and the presence of a prominent m / z 82 ion suggest that the compound
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Figure 3. The El-MS of the N-pentafluoropropionyl derivative of norcocaine (chromatographic peak 11 in Figure 1).
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Figure 2. The El-MS of cocaine and three derivatized metabolites identified in a urine specimen. The spectra correspond as follows: (2a) cocaine, (2b) the pentafluoropmpionyl derivative of ecoonine methyl ester, (2c) the hexafluoroisopropyl derivative of benzoylecgonine, (2d) the hexafluoroisopropyl-pentafluoropropionyl derivative of ecoonine.
7
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. .. ..
ill
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Figure 4. The El-MS of ecgonidine methyl ester (chromatographic peak 3 in Figure 1).
203
Journal of Analytical Toxicology, Vol, 14, July/August 1990
was the hexafluoroisopropyl derivative of ecgonidine. An abundant m/z 194 ion and an apparent molecular weight of 313 indicated that peak 5 corresponds to the N-pentafluoropropionyl derivative of a previously unreported cocaine metabolite, norecgonidine methyl ester. These tentative identifications were subsequently confirmed by comparison of the retention times and the mass spectra of each metabolite with the retention times and mass spectra (Figure 5a and 5b) of synthetic standards (Table I). The mass spectrum from chromatographic peak 6 (Figure 6) shows prominent ions at m/z 194 and 213 and the absence of an abundant ion at m/z 82, suggesting that the compound was also a N-desmethyl (nor-) metabolite. Further evidence for this compound's structure was provided by the apparent molecular ion at m/z 477 and a fragment ion at m/z 313 corresponding to loss of a pentafluoropropionic acid from the molecular ion. Confirmation that the compound corresponded to the N,O-bis(pentaftuoropropionyl) derivative of norecgonine methyl ester was provided by comparison of its retention time and mass spectrum with those of a synthetic standard. Chromatographic peaks 10 and 12 gave very similar mass
-
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spectra, each showing an apparent molecular ion at m / z 465, a M-OCH~ ion at m/z 434, and prominent fragment ions at m/z 82 and 182. Both mass spectra also show a prominent ion at m/z 267 which is consistent with a pentafluoropropionatesubstituted benzoyl ion. These features indicate that the two cocaine metabolites were hydroxylated on the aromatic ring. In order to determine the exact location of the hydroxyl group in each metabolite, the three positional isomers of arylhydroxylated cocaine were synthesized. The mass spectra and retention times of chromatographic peaks 10 and 12 are in good agreement wih the mass spectra (Figure 7b and 7c) and retention times (Table I) of the pentafluoropropionyl derivatives of meta- and para-hydroxycocaine, respectively. The mass spectrum (Figure 8) of chromatographic peak 8 also contains a prominent m/z 267 ion, indicating the presence of a pentafluoropropionate-substituted benzoyl ion. However, a shift of the m/z 182 ion to m/z 318 indicates that the metabolite had been converted to a hexafluoroisopropyl ester in the derivatization process. The apparent molecular ion at m/z 601 further indicates that the compound was a hexafluoroisopropyl-pentafluoropropionyl derivative of an aryl-hydroxylated benzoylecgonine. Again, it was necessary to synthesize each of the three possible positional isomers in order to determine the exact location of the metabolite's hydroxyl group. Not surprisingly, the E1 mass spectra of the three derivatized hydroxy benzoyl-
=~=. . . . 6,i . . . . . . ~ki ....... ;,,ki ...... id,i ,,,,='
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Figure 6. The El-MS of the N,O-bis-(pentafluoropropionyl)derivativeof
norecgonine methyl ester (chromatographicpeak 6 in Figure 1). Figure 5. The El-MS of the hexafluoroisopropylderivative of synthetic ecgonidine(5a) and the N-pentafluoropropionylderivative of synthetic norecgonidine methyl ester (5b), 434
Table I. Gas Chromatographic Relative Retention Times for Cocaine, Its Metabolites and Related C o m p o u n d s Analyzed as their HFIP/PFP Derivatives
,p:= ,= '~
Relative retention times 9
Compounds Ecgonidine Ecgonine Ecgonidine methyl ester Ecgonine methyl ester Norecgonidinemethyl ester Norecgonine methyl ester Benzoylecgonine o-Hydroxybenzoylecoonine m-Hydroxybenzoylecgonine p-Hydroxybenzoylecgonine Cocaine o-Hydroxycocaine m-Hydroxycocaine Norcocaine p-Hydroxycocaine Cinnamoylecgonine Cinnamoylcocaine
204
Peak no. 1 2 3... 4 5 6 7 ND 8 ND 9 ND 10 11 12 13 14
Urine specimen 0,176 0,266 0,332 0.360 0.415 0.445 0.845 0,915
Synthetic standard 0,176 0,329
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Figure 7. The El-MSof the pentafluoropropionylderivativesof synthetic ortho-, meta., and para.hydroxycocaine (Figures 7a, 7b and 7c, respectively).
Journal of Analytical Toxicology, Vol. 14, July/August 1990
ecgonine isomers are too similar to permit a conclusive identification of the urinary metaholite based solely on comparison of their mass spectra. The derivatized metabolite's mass spectrum and GC retention time do however agree best with the synthetic standard of the meta-isomer. The aryi-hydroxylated metabolites were apparently present in the urine specimen as glucuronide or sulfate conjugates, since they were not detected when the urine specimen was extracted and analyzed without enzyme hydrolysis. It is interesting that cinnamoylcocaine (peak 14) and its metabolite cinnamoylecgonine (peak 12) were also found in the urine sample. Cinnamoylcocaine is present in most illicit cocaine samples (16). Derivatization of the metabolites by treatment with hexafluoroisopropanol and pentafluoropropionic anhydride proved highly advantageous: The resulting ion current profile was less complex and the mass spectra were easier to interpret than when the urine extract was derivatized by trimethylsilylation. The identifications were also greatly facilitated by the extreme sensitivity of the ion trap detector and its ability to provide both EI and C[ mass spectra. Although the C[ mass spectra are not shown here, they provided valuable supporting information, such as firmly establishing the molecular weight of each metabolitc. It is possible that some of the identified compounds are not true metabolites of cocaine but were formed during sample storage and processing. However, none of the previously unreported cocaine metabolites were detected when drug-free urine was fortified with cocaine, benzoylecgonine, and ecgonine methyl ester and analyzed as described.
Conclusions Cocaine and 11 of its metabolites have been identified in this qualitative examination of a urine specimen from a cocaine user. Four of the metabolites are reported for the first time. The relative sizes of the chromatographic peaks in Figure I give a rough indication of the relative concentrations of the metabolites in the urine specimen, but the ability to make quantitative conclusions is severely limited by the absence of information regarding the dose of cocaine, the mode and time of administration, and the efficiency with which each of the metabolites was extracted and derivatized. These data suggest that neither the metabolism nor the subsequent urinary excretion products of cocaine in man are as simple or predictable as previously reported.
FIgurs 8. The El-MS of chromatographicpeak 8 (Figure 1), tentatively identified as the hexafiuoroisopropyl-pentafluoropropionylderivativeof meta.hydroxy benzoylecgonine.
Acknowledgment The authors gratefully acknowledge support for this work from the Finnigan Foundation.
References 1. R.T. Jones in The Pharmacology of Cocaine. U.S. Government Printing Office, Washington, D.C., 1984, pp. 34-53. 2. F. Fish and W.D.C. Wilson. Excretion of cocaine and its metaboiites in man. J. Pharm. Phermacol. 21:135-38 (1969). 3. J.J. Ambre, M. Fischman, and T.-I. Ruo. Urinary excretion of ecgonine methyl ester, a major metabolite of cocaine in humans. J. Anal. Toxicol. 8:23-25 (1984). 4. J. Ambre, T.I. Ruo, J. Nelson, and S. Belknap. Urinary excretion of cocaine, benzoylecgonine, and ecgonine methyl ester in humans. J. Anal. Toxicot. 12:301-306 (1988). 5. R.L. Hawks, I.J. Kopin, R.W. Colburn, and N.B. Thoa. Norcocaine: A pharmacologically active metabolite of cocaine found in the brain. Life Sci. 15:2189-95 (1974). 6. A.L. Misra, RK. Nayak, R. Block, and S.J. Mul~. Estimation and disposition of ZH-benzoylecgonine and pharmacological activity of some cocaine metabolites. J. Pharm. Pharmacol. 27:784-86 (1~r5). 7. W.T. Lowry, J.N. Lomonte, D. Hatchett, and J.C. Garriott. Identification of two novel cocaine metabolites in bile by gas chromatography and gas chromatography/mass spectrometry in a case of acute intravenous cocaine overdose. J. Anal. Toxicol. 3:91-95 (1979). 8. R.M. Smith. Arylhydroxy metabolites of cocaine in the urine of cocaine users. J. Anal. Toxicol. 8:35-37 (1984). 9. R.M. Smith, M.A. Poquette, and P.J. Smith. Hydroxymethoxybenzoylmethylecgonine: New metabotites of cocaine from human urine. J. Anal, Toxico/. 8:29-34 (1984). 10. S.J. Mul6 and G.A. Casella. Confirmation and quantitation of cocaine, benzoylecgonine, ecgonine methyl ester in human urine by GCIMS. J. Anal. Toxicol. 12:153-55 (1988). 11. C.W. Spangler and T.W. Hartford. Dehydration of 2-cyclohexen-l-ols and hexadienols with methyltriphenoxyphosphonium iodide in hexamethylphosphoric triamide: A simple route to conjugated dienes and trtenes. Synthesis pp. 108-10 (1976). 12. S.R Jindal, T. Lutz, and P. Vestergaard. Mass spectrometric determination of cocaine and its biologically active metaboUte, norcocaine, in human urine. Blomed. Mass Spectrom. 5:658-63 (1978). 13 S.P. Jindal and R Vestergaard. Quantitation of cocaine and its principal metabolite, benzoylecgonine, by GLC-mass spectrometry using stable isotope labeled analogs as internal standards. J. Pharm. Sci. 67:811-14 (1978). 14. B,R. Martin, L.R Lue, and J.R Boni. Pyrolysis and volatilization of cocaine. J. Anat. Toxico/. 13:158-62 (1989). 15. S.R Jindal and T. Lutz. Ion cluster technique in drug metabolism: Use of a mixture of labeled and unlabeled cocaine to facilitate metabolite identification. J. Anal. Toxicol. 10:150-55 (1986). 16. J.M. Moore. Identification of cis- and trans-cinnamoylcocaine in illicit cocaine seizures. J. Assoc. Off. Anal. Chem. 56:1199-1205 (1973). Manuscript received August 14, 1989; revision received January 3, 1990.
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Cocaine Metabolism in Man! Identification of Four Previously Unreported Cocatne Metabolites in Human Urine* Ji Y u e Z h a n g and R o d g e r L. F o l t z * *
Center for Human Toxicology, University of Utah, Salt Lake City, Utah 84108
I Abstract
Experimental
I
Cocaine and 11 of Its metabolites were Identified In a urine specimen from e cocaine user. Four of the metabolltes are reported for the first time: ecgonidine, norecgonidlne methyl ester, norecgonine methyl ester, and m-hydroxybenzoylecgonine. The structures of the newly identified metabolitea were confirmed by comparison of their gas chromatographic retention times and their electron Ionization and chemical Ionization mass spectra with the corresponding data obtained on synthesized standards. Other metabolitea present were benzoylecgonine, ecgonlne methyl ester, ecgonine, ecgonidine methyl ester, norcocaine, p~ cocaine, and m-hydroxycoceine.
Introduction In the body, cocaine is rapidly converted to metabolites by enzymatic and chemical processes, so very little cocaine is usually excreted in the urine (1). The two major urinary metabolites of cocaine are benzoylecgonine (2) and ecgonine methyl ester (3). In a recent study, Ambre et ai. reported that 14-17% of a cocaine dose was excreted in the urine as benzoylecgonine, and 12-21% as ecgonine methyl ester (4). Other cocaine metabolites reported in human urine specimens include norcocaine (5), ecgonine (2), benzoylnorecgonine (6), ecgonidine methyl ester (7), cocaine hydroxylated on the benzoyl ring (8), and hydroxymethoxy-substituted benzoylecgonine (9)~, Here we report the results of an extensive GC/MS analysis of a urine specimen received as part of a drug testing program and initially found to be positive for benzoylecgonine. A total of 11 metabolites of cocaine were identified, including four that had not been previously reported. Identifications were based on comparison of the GC retention times and electron ionization (EI) and chemical ionization (C1) mass spectra of the derivatized metabolites with the corresponding data obtained on purchased or synthesized standards. The metabolites were derivatized by treatment with hexafluoroisopropanol and pentafluoropropionic anhydride (10). 9Presented In part at the 37th ASMS Conference on Mass Spectrometryand AlUedTopics, Mtamt Beach, Flonda, May 22-26, 1989. "* Author to whom correspondence should be addressed,
Specimen. The metabolites reported here were identified in a single urine specimen received in conjunction with a general drug testing program. No information is available regarding the donor, the size of the cocaine dose, or the mode and time of administration. Materials. Cocaine, benzoylecgonine, and ecgonine methyl ester were obtained from Alitech Associates; ecgonine and salicylic acid were from Sigma Chemical; methyltriphenylphosphonium iodide, hexamethylphosphoric triamide (HMPA), 1,3-dicyclohexylcarbodiimide (DCC), boron trifluoride etherate ( B F J E h O ) , and m-hydroxybenzoic acid were from Aldrich Chemical; p-hydroxybenzoic acid was from Eastman Kodak; pentafluoropropionic anhydride (PFPA) was from Regis Chemical; and 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) was from Pierce Chemical. Hydrolysis and extraction. To a 2-mL aliquot of urine in a 25-mL screw-capped extraction tube was added 4 mL of 1M sodium acetate buffer (pH 5) containing 20,000 units of/3glucuronidase. The mixture was incubated overnight at 37~ After the pH was adjusted to 8.5 with 1N NH,OH, the incubated sample was extracted twice with 5-mL volumes of chloroformisopropanol (3:1). The combined organic fractions were then back-extracted into 5 mL of 0.1N HCI. The organic layer was discarded and the aqueous phase was adjusted to pH 8.5 with IN NH,OH and extracted twice with 5-mL volumes of chloroform-isopropanol (3:1). The organic layers were combined and evaporated to dryness at 50~ under a gentle stream of air. Derivatization. To the tube containing the urine extract, 70/zL of PFPA and 30 #L of HFIP were added. The mixture was vortexed for 10 s and then heated at 70~ for 10 min. After evaporation of the mixture to dryness under a gentle stream of air at 50~ the residue was taken up in 20 #L of ethyl acetate and analyzed by GC/MS (Figure 1). Synthetic procedures
Ecgonidine methyl ester. Ecgonidine methyl ester was synthesized from ecgonine methyl ester by modification of a published procedure for dehydration of alicyclic alcohols (11). A mixture of 0.5 mg methyltriphenylphosphonium iodide in 1 mL of hexamethylphosphoric triamide was added to 400 ttg of ecgonine methyl ester in a 5-mL microvial. The microvial was wrapped in black paper to protect it from light, and the mixture
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201
Journal of Analytical Toxicology, Vol. 14, July/August 1990
was then stirred for 4 h at 50~ and cooled to room temperature. Water (2 mL) was added and the solution was made acidic by addition of 1N HCI. The mixture was then washed with 2 mL of chloroform. After separation of the phases, the aqueous layer was adjusted to a pH of 8.5 with IN N H , O H and extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The combined organic layers were evaporated to dryness and the unpurified product was taken up in ethyl acetate and analyzed by GC/MS. The major chromatographic peak gave a mass spectrum consistent with ecgonidine methyl ester. Ecgonidine. Ecgonidinr was obtained by hydrolysis of ecgonidine methyl ester. Ecgonidine methyl ester (about 200/zg) was refluxed with 1 mL of 2N NaOH for 2 h. After cooling to room temperature, the solution was adjusted to pH 8 with 2N HCI and extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The organic layer was evaporated to dryness and the residue was derivatized by treatment with 30 ~L of H F I P and 70/~L of P F P A at 70~ for 10 min. After evaporation to dryness, the product was dissolved in ethyl acetate and analyzed by G C / M S . Norecgonine methyl ester. Norecgonine methyl ester was prepared from purified norcocaine via norecgonine. Norcocaine (1 rag) was refluxed with 2 mL o f 2 N NaOH in a 50-mL roundbottom flask for 4 h. After cooling, the solution was made acidic by addition of 2N HCI and extracted with 2 mL of chloroform to remove the liberated benzoic acid. The aqueous layer was evaporated to dryness on a hot plate under a stream of air. The residue was taken up in 2 mL of methanol containing 0.1 mL of boron trifluoride-etherate (BF~-Et20). The solution was refluxed for 2 h, cooled to room temperature, and poured into 5 mL of water. After its pH was adjusted to 8.5 with IN NH,OH, the solution was extracted twice with 2-mL volumes of chloroform-isopropanol (3:1). The combined organic extracts were then evaporated to dryness. The residue was derivatized by treatment with 70/~L of pentafluoropropionic anhydride at 70~ for 10 min and the product was analyzed by G C / M S . Norecgonidine methyl ester. Norecgonidine methyl ester was synthesized from norecgonine methyl ester by the same procedure described above for the synthesis of ecgonidine methyl ester from ecgonine methyl ester. Hydroxybenzoylecgonines and hydroxycocaines. The ortho-, meta-, and para-hydroxybenzoylecgonine isomers were prepared by coupling ecgonine to o-, m-, and p-hydroxybenzoic acids, respectively, as illustrated in the following example.
9
4
11
To a 5-mL microvial were added 0.1 mg of ecgonine, 0.5 mg of o-hydroxybenzoic acid (salicylic acid), 1 mg of 1,3-dicyclohexylcarbodiimide (DCC), and 2 drops o f pyridine in 1 mL of methylene chloride. The solution was stirred at 50~ for 2 h. After cooling, the reaction mixture was washed with 2 mL of water and extracted with 2 mL of 2N HCI. The acidic solution was extracted with methylene chloride to remove nonbasic components. The aqueous solution was then adjusted to a pH of 8.5 with IN N H , O H and extracted with two 2-mL volumes of chloroform-isopropanol (3:1), and the combined organic extracts were evaporated to dryness. The unpurified o-hydroxybenzoylecgonine was characterized by G C / M S analysis after conversion to its H F I P / P F P derivative by treatment with 70 ~L of pentafluoropropionic anhydride and 30 ~L of hexafluoroisopropanol at 70~ for 10 min. The excess derivatizing agents were removed by evaporation before addition of 20/~L of ethyl acetate and injection of a I-/~L aliquot into the G C / M S . The ortho-, meta-, and para-isomers o f hydroxycocaine were synthesized in a similar manner by substituting ecgonine methyl ester for ecgonine in the above procedure. Instrumentation
All G C / M S analyses were performed on a Finnigan-MAT Model 800 ion trap detector (ITD) coupled to a Hewlett-Packard Model 5890 gas chromatograph and an IBM AT microcomputer. The ion trap manifold was maintained at 225~ All electron ionization mass spectra were acquired using the automatic gain control (AGC) program, while chemical ionization mass spectra were acquired using the automatic reaction control (ARC) program. The ion trap detector was autotuned according to manufacturer's recommendations. Electron ionization mass spectra (EIMS) were acquired by repetitively scanning the mass range m/z 70 to 650 at a rate of one scan per second. The electron multiplier voltage was set at the voltage required to give a 10' gain. The filament emission current was set at 5 #A. Chemical ionization mass spectra (CIMS) were acquired under the following operating conditions: reagent gas, methanol; ionization RF level, 8 amu; reaction RF level, 20 amu; ionization time, 120 ms; filament emission, 10 #A; and the electron multiplier set at the voltage required to give a 10' gain. The methanol pressure within the ion trap was adjusted in the following manner. With the ARC function turned off and the B value (sensitivity) set to a minimum, the reagent gas metering valve was gradually opened until the area of the m/z 33 ion reached 4000 counts. The G C / M S analyses employed at 15-m x 0.25-mm i.d. J&W Scientific DB-I capillary column with a film thickness of 0.25 gin. The carrier gas (hydrogen) linear velocity was 60 cm/s measured at an oven temperature of 100~ After a splitless injection, the oven temperature was maintained at 100~ for 0.5 min with the split flow turned off, and then the oven temperature was programmed to 250~ at 10~
T0I 1~12 2! 3 '
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Results T , I $CAH NO
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1. The total ion current profile from the GC/MS analysis of the derivatized urine extract.
Figure
202
and
Discussion
Cocaine and 11 of its metabolites were detected and identified in the urine specimen from a cocaine user. The total ion current profile from G C / E I M S analysis of the derivatized urine extract is shown in Figure 1. The mass spectrum recorded for chromatographic peak 9
Journal of Analytical Toxicology, Vol, 14, July/August 1990
(Figure 2a) is consistent with mass spectra of cocaine that have been reported and discussed previously (7,9,12,13). The most abundant ion (rn/z 82) is formed by fragmentation of the bicyclic ring system to form a protonated methylpyrrole structure. Additional abundant ions are present at m / z 105, corresponding to the benzoyl ion, and at m / z 182, corresponding to the loss of the benzoate radical from the molecular ion. There is also a structurally useful ion observed at m/z 272 which is formed by the loss of the methoxyl radical from the molecular ion. Analogous fragmentation processes are observed in the mass spectra of the derivatized metabolites of cocaine. For example, the mass spectrum corresponding to chromatographic peak 4 (Figure 2b) shows the same abundant ions at m / z 82 and 182 that are in the cocaine spectrum, indicating that the cocaine bicyclic structure and the methyl ester are present. However, the absence of the benzoyl ion (m/z. 105) and the shift of the molecular ion to m / z 345 identify the compound as the pentafluoropropionyl derivative of ecgonine methyl ester. In the mass spectrum recorded for chromatographic peak 7 (Figure 2c), the m / z 82, 105, and 272 ions are present, but the molecular ion and the M-C,H,CO, ion have shifted to rn/z 439 and 318, respectively. These features clearly indicate that the compound corresponds to the hexafluoroisopropyl derivative of benzoylecgonine. The mass spectrum from chromatographic peak 2 (Figure 2d) is similar to the mass spectrum of the benzoylecgonine derivative in that it shows an abundant ion peak at m / z 318. However, because in this case the m / z 318 ion is formed by loss of the pentafluoropropionate radical from the molecular ion at ra/z 481, the spectrum identifies the compound as the hexafluoroisopropyl/pentafluoropropionyi derivative of ecgonine.
Chromatographic peak 11 was identified as the N-pentafluoropropionyl derivative of norcocaine. The m/z. 82 ion is absent in its spectrum (Figure 3) and is replaced by prominent ions at m / z 213 and 194, which are observed in the mass spectra of all N-desmethyl (nor-) metabolites. Curiously, the molecular ion of the norcocaine derivative undergoes loss of benzoic acid, rather than loss of the benzoate radical, to give the prominent ion at m / z 313. Chromatographic peak 3 was identified as ecgonidine methyl ester based on comparison of its mass spectrum (Figure 4) with that reported by Lowry et al. (7) for what they referred to as "methylecgonidine." Ecgonidine methyl ester may not be a true metabolite of cocaine, because it can be formed as a pyrolysis product when cocaine is smoked (14). However, it has been identified in rat urine following interperitoneal administration of cocaine (15) and in bile from a young man who died from an overdose of cocaine after apparent intravenous administration (7). Also, we know that the ecgonidine methyl ester detected in our study was not formed during the extraction and derivatization steps, because no ecgonidine methyl ester was detected when drug-free urine specimens were fortified with cocaine and subjected to the same extraction and derivatization conditions. The most abundant ion in the mass spectrum of ecgonidine methyl ester occurs at m / z 152, which is 29 atomic mass units below the molecular ion (m/z 181). A possible mechanism for this fragmentation is loss of the ethylene bridge along with a hydrogen atom to form the relatively stable N-methyl pyridinium ion shown in the following equation (15): CH3
CH3 N§
O
+ N ~
OCH3 -- CHi--CH2 +H ~--- 0 C-OCH3 mlz 152 0
m/z 1111
The mass spectra recorded for the chromatographic peaks I and 5 also show prominent M-29 ions, The short retention time of peak I) an apparent molecular ion at m / z 317, and the presence of a prominent m / z 82 ion suggest that the compound
M9
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Figure 3. The El-MS of the N-pentafluoropropionyl derivative of norcocaine (chromatographic peak 11 in Figure 1).
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Figure 2. The El-MS of cocaine and three derivatized metabolites identified in a urine specimen. The spectra correspond as follows: (2a) cocaine, (2b) the pentafluoropmpionyl derivative of ecoonine methyl ester, (2c) the hexafluoroisopropyl derivative of benzoylecgonine, (2d) the hexafluoroisopropyl-pentafluoropropionyl derivative of ecoonine.
7
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ill
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Figure 4. The El-MS of ecgonidine methyl ester (chromatographic peak 3 in Figure 1).
203
Journal of Analytical Toxicology, Vol, 14, July/August 1990
was the hexafluoroisopropyl derivative of ecgonidine. An abundant m/z 194 ion and an apparent molecular weight of 313 indicated that peak 5 corresponds to the N-pentafluoropropionyl derivative of a previously unreported cocaine metabolite, norecgonidine methyl ester. These tentative identifications were subsequently confirmed by comparison of the retention times and the mass spectra of each metabolite with the retention times and mass spectra (Figure 5a and 5b) of synthetic standards (Table I). The mass spectrum from chromatographic peak 6 (Figure 6) shows prominent ions at m/z 194 and 213 and the absence of an abundant ion at m/z 82, suggesting that the compound was also a N-desmethyl (nor-) metabolite. Further evidence for this compound's structure was provided by the apparent molecular ion at m/z 477 and a fragment ion at m/z 313 corresponding to loss of a pentafluoropropionic acid from the molecular ion. Confirmation that the compound corresponded to the N,O-bis(pentaftuoropropionyl) derivative of norecgonine methyl ester was provided by comparison of its retention time and mass spectrum with those of a synthetic standard. Chromatographic peaks 10 and 12 gave very similar mass
-
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spectra, each showing an apparent molecular ion at m / z 465, a M-OCH~ ion at m/z 434, and prominent fragment ions at m/z 82 and 182. Both mass spectra also show a prominent ion at m/z 267 which is consistent with a pentafluoropropionatesubstituted benzoyl ion. These features indicate that the two cocaine metabolites were hydroxylated on the aromatic ring. In order to determine the exact location of the hydroxyl group in each metabolite, the three positional isomers of arylhydroxylated cocaine were synthesized. The mass spectra and retention times of chromatographic peaks 10 and 12 are in good agreement wih the mass spectra (Figure 7b and 7c) and retention times (Table I) of the pentafluoropropionyl derivatives of meta- and para-hydroxycocaine, respectively. The mass spectrum (Figure 8) of chromatographic peak 8 also contains a prominent m/z 267 ion, indicating the presence of a pentafluoropropionate-substituted benzoyl ion. However, a shift of the m/z 182 ion to m/z 318 indicates that the metabolite had been converted to a hexafluoroisopropyl ester in the derivatization process. The apparent molecular ion at m/z 601 further indicates that the compound was a hexafluoroisopropyl-pentafluoropropionyl derivative of an aryl-hydroxylated benzoylecgonine. Again, it was necessary to synthesize each of the three possible positional isomers in order to determine the exact location of the metabolite's hydroxyl group. Not surprisingly, the E1 mass spectra of the three derivatized hydroxy benzoyl-
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norecgonine methyl ester (chromatographicpeak 6 in Figure 1). Figure 5. The El-MS of the hexafluoroisopropylderivative of synthetic ecgonidine(5a) and the N-pentafluoropropionylderivative of synthetic norecgonidine methyl ester (5b), 434
Table I. Gas Chromatographic Relative Retention Times for Cocaine, Its Metabolites and Related C o m p o u n d s Analyzed as their HFIP/PFP Derivatives
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Relative retention times 9
Compounds Ecgonidine Ecgonine Ecgonidine methyl ester Ecgonine methyl ester Norecgonidinemethyl ester Norecgonine methyl ester Benzoylecgonine o-Hydroxybenzoylecoonine m-Hydroxybenzoylecgonine p-Hydroxybenzoylecgonine Cocaine o-Hydroxycocaine m-Hydroxycocaine Norcocaine p-Hydroxycocaine Cinnamoylecgonine Cinnamoylcocaine
204
Peak no. 1 2 3... 4 5 6 7 ND 8 ND 9 ND 10 11 12 13 14
Urine specimen 0,176 0,266 0,332 0.360 0.415 0.445 0.845 0,915
Synthetic standard 0,176 0,329
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Figure 7. The El-MSof the pentafluoropropionylderivativesof synthetic ortho-, meta., and para.hydroxycocaine (Figures 7a, 7b and 7c, respectively).
Journal of Analytical Toxicology, Vol. 14, July/August 1990
ecgonine isomers are too similar to permit a conclusive identification of the urinary metaholite based solely on comparison of their mass spectra. The derivatized metabolite's mass spectrum and GC retention time do however agree best with the synthetic standard of the meta-isomer. The aryi-hydroxylated metabolites were apparently present in the urine specimen as glucuronide or sulfate conjugates, since they were not detected when the urine specimen was extracted and analyzed without enzyme hydrolysis. It is interesting that cinnamoylcocaine (peak 14) and its metabolite cinnamoylecgonine (peak 12) were also found in the urine sample. Cinnamoylcocaine is present in most illicit cocaine samples (16). Derivatization of the metabolites by treatment with hexafluoroisopropanol and pentafluoropropionic anhydride proved highly advantageous: The resulting ion current profile was less complex and the mass spectra were easier to interpret than when the urine extract was derivatized by trimethylsilylation. The identifications were also greatly facilitated by the extreme sensitivity of the ion trap detector and its ability to provide both EI and C[ mass spectra. Although the C[ mass spectra are not shown here, they provided valuable supporting information, such as firmly establishing the molecular weight of each metabolitc. It is possible that some of the identified compounds are not true metabolites of cocaine but were formed during sample storage and processing. However, none of the previously unreported cocaine metabolites were detected when drug-free urine was fortified with cocaine, benzoylecgonine, and ecgonine methyl ester and analyzed as described.
Conclusions Cocaine and 11 of its metabolites have been identified in this qualitative examination of a urine specimen from a cocaine user. Four of the metabolites are reported for the first time. The relative sizes of the chromatographic peaks in Figure I give a rough indication of the relative concentrations of the metabolites in the urine specimen, but the ability to make quantitative conclusions is severely limited by the absence of information regarding the dose of cocaine, the mode and time of administration, and the efficiency with which each of the metabolites was extracted and derivatized. These data suggest that neither the metabolism nor the subsequent urinary excretion products of cocaine in man are as simple or predictable as previously reported.
FIgurs 8. The El-MS of chromatographicpeak 8 (Figure 1), tentatively identified as the hexafiuoroisopropyl-pentafluoropropionylderivativeof meta.hydroxy benzoylecgonine.
Acknowledgment The authors gratefully acknowledge support for this work from the Finnigan Foundation.
References 1. R.T. Jones in The Pharmacology of Cocaine. U.S. Government Printing Office, Washington, D.C., 1984, pp. 34-53. 2. F. Fish and W.D.C. Wilson. Excretion of cocaine and its metaboiites in man. J. Pharm. Phermacol. 21:135-38 (1969). 3. J.J. Ambre, M. Fischman, and T.-I. Ruo. Urinary excretion of ecgonine methyl ester, a major metabolite of cocaine in humans. J. Anal. Toxicol. 8:23-25 (1984). 4. J. Ambre, T.I. Ruo, J. Nelson, and S. Belknap. Urinary excretion of cocaine, benzoylecgonine, and ecgonine methyl ester in humans. J. Anal. Toxicot. 12:301-306 (1988). 5. R.L. Hawks, I.J. Kopin, R.W. Colburn, and N.B. Thoa. Norcocaine: A pharmacologically active metabolite of cocaine found in the brain. Life Sci. 15:2189-95 (1974). 6. A.L. Misra, RK. Nayak, R. Block, and S.J. Mul~. Estimation and disposition of ZH-benzoylecgonine and pharmacological activity of some cocaine metabolites. J. Pharm. Pharmacol. 27:784-86 (1~r5). 7. W.T. Lowry, J.N. Lomonte, D. Hatchett, and J.C. Garriott. Identification of two novel cocaine metabolites in bile by gas chromatography and gas chromatography/mass spectrometry in a case of acute intravenous cocaine overdose. J. Anal. Toxicol. 3:91-95 (1979). 8. R.M. Smith. Arylhydroxy metabolites of cocaine in the urine of cocaine users. J. Anal. Toxicol. 8:35-37 (1984). 9. R.M. Smith, M.A. Poquette, and P.J. Smith. Hydroxymethoxybenzoylmethylecgonine: New metabotites of cocaine from human urine. J. Anal, Toxico/. 8:29-34 (1984). 10. S.J. Mul6 and G.A. Casella. Confirmation and quantitation of cocaine, benzoylecgonine, ecgonine methyl ester in human urine by GCIMS. J. Anal. Toxicol. 12:153-55 (1988). 11. C.W. Spangler and T.W. Hartford. Dehydration of 2-cyclohexen-l-ols and hexadienols with methyltriphenoxyphosphonium iodide in hexamethylphosphoric triamide: A simple route to conjugated dienes and trtenes. Synthesis pp. 108-10 (1976). 12. S.R Jindal, T. Lutz, and P. Vestergaard. Mass spectrometric determination of cocaine and its biologically active metaboUte, norcocaine, in human urine. Blomed. Mass Spectrom. 5:658-63 (1978). 13 S.P. Jindal and R Vestergaard. Quantitation of cocaine and its principal metabolite, benzoylecgonine, by GLC-mass spectrometry using stable isotope labeled analogs as internal standards. J. Pharm. Sci. 67:811-14 (1978). 14. B,R. Martin, L.R Lue, and J.R Boni. Pyrolysis and volatilization of cocaine. J. Anat. Toxico/. 13:158-62 (1989). 15. S.R Jindal and T. Lutz. Ion cluster technique in drug metabolism: Use of a mixture of labeled and unlabeled cocaine to facilitate metabolite identification. J. Anal. Toxicol. 10:150-55 (1986). 16. J.M. Moore. Identification of cis- and trans-cinnamoylcocaine in illicit cocaine seizures. J. Assoc. Off. Anal. Chem. 56:1199-1205 (1973). Manuscript received August 14, 1989; revision received January 3, 1990.
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