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40/7, 1299-i 305 (1994)

#{149} Drug


and Toxicology

Simultaneous Measurement of Cocaine, Cocaethylene, Their Metabolites, and “Crack” Pyrolysis Products by Gas Chromatography-Mass Spectrometry
Edward J. Cone,’ Mary Hiilsgrove, and William D. Darwin era! analytical techniques, including HPLC (9-14), gas-liquid chromatography (4, 15-18), and gas chromatography-mass spectrometry (GC-MS) (19_28).2 Generally, these methods focus on the determination of cocaine, benzoylecgonine, or ecgonine methyl ester, but do not include other metabolites. Solid-phase extraction (SPE) systems have been reported for the rapid and efficient removal of numerous basic drugs and metabolites (29). The ability of these copolymeric bonded phase columns to extract both basic and aniphoteric cocaine metabolitesallows the design of an assay for the simultaneous determination of multiple metabolites of cocaine. We have developed a combined assay for cocaine, cocaethylene, anhydroecgonine methyl ester, and six additional metabolites in biological fluids. The assay is based on rapid SPE extraction and measurement by GC-MS operating in the selected-ion monitoring (SIM) mode. We used the assay to measure cocaine and its metabolites in specimens collected from human subjects after administration of cocaine by intravenous, smoking, and intranasal routes.

We developed a sensitive and specific assay for the simultaneous measurement of cocaine, cocaethylene, six of their metabolites, and anhydroecgonine methyl ester, a pyrolysis product, in biologicalfluids. The assay involves solid-phase extraction columns containing a copolymeric bonded phase for isolation of cocaine analytes, derivatization with N,O-bis(trimethylsilyl)trifluoroacetamideand 10 gIL trimethylchlorosilane, and measurement with gas chromatography-mass spectrometry operating in the selected-ion monitoring mode. Detector responses for analytes were linear over a concentration range of 3.1-1000 zg/L. The limits of detection were -1 jtgIL for cocaine, ecgonine methyl ester, and benzoylecgonine and 3-6 g/L for the remaining analytes. Hydrolysis of cocaine and artifact formation of anhydroecgonine methyl ester during extraction and assay was <1%. Cocaine and its derivatives appear in different proportions in plasma, saliva, and urine according to the biologicalfluid and time of measurement. Each biologicalfluid provides unique information on the disposition of cocaine in human subjects. IndexIngTerms: abused drugs/drug metabolism/drug monitoring

MaterIals and Methods
Cocaine continues to be a major drug of abuse in the US. Within the last decade, the preferred route of cocaine intake changed from intranasal and intravenous to smoking (1). Alcohol (ethanol) also is commonly used in combination with cocaine (2). These changes in patterns of usage and routes of administration have resulted in significant changes in the biological excretion products of cocaine. Normally, cocaine is metabolized to benzoylecgonine and ecgonine methyl ester and lesser amounts of norcocaine and benzoylnorecgonine (Fig. 1). A smoking byproduct, anhydroecgonine methyl ester, is formed as a result of thermal degradation when cocaine base (“crack”) is smoked. This material has been identified in the urine of crack smokers and is considered a marker of cocaine use (3). In addition, the combined use of cocaine and alcohol produces a new cocaine analog, cocaethylene, in vivo. This analog was identified in the blood (4-6) and urine of cocaine users (7, 8) after simultaneous intake of cocaine and alcohol; it is not normally present in cocaine alone. Cocaethylene can be metabolized further to benzoylecgonine, norcocaethylene, and ecgonine ethyl ester (Fig. 1). Identification and measurement of these analytes are important in the diagnosis and treatment of cocaine abuse. Cocaine and its metabolites can be measured by 5evP.O. Box 5180, Addiction Research Center, National Institute on Drug Abuse, Baltimore, MD 21224 ‘Author for correspondence. Fax 410-550-1645. Received December 8, 1993; accepted March 29, 1994. Materials

Cocaine hydrochloride was obtained from Mallinckrodt, St. Louis, MO. Benzoylecgonine tetrahydrate, norcocaine, benzoylnorecgonine, and ecgonine methyl ester hydrochloride were obtained from the National Institute on Drug Abuse (NIDA), Rockville, MD. Cocaethylene and norcocaethylene fumarate were obtained from Research Triangle Institute, Research Triangle Park, NC (courtesy of F.I. Carroll). Anhydroecgonine methyl ester oxalate and ecgonine ethyl ester were obtained from the Addiction Research Center, NIDA, Baltimore, MD (courtesyof Andrew Allen).Trideuterated analogs of cocaine, benzoylecgonine, and ecgornne methyl ester were obtained from Sigma Chemical Co., St. Louis, MO. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 10 g/L trimethylchlorosilane (TMCS) was obtained from Pierce Chemical Co., Rockford, IL. All organic solvents were HPLC-grade and chemicals were reagentgrade. Acetate buffer (pH 4.0 ± 0.1) was prepared with 2.0 moIJL sodium acetate and 2.0 mol/L acetic acid. The elution solvent (methylene chloride:isopropanol:concentrated aqueous amnionium hydroxide, 80:20:2 by vol) was prepared daily. Extraction columns, Clean Screen#{174} (ZSDAUO2O), 4-mL reservoirs (RFVO2F4P), and the 2Nonstandard abbreviations: GC-MS, gas chromatographymass spectrometiy; SPE, solid-phase extraction; SIM, selected-ion monitoring, NIDA, National Institute on Drug Abuse; BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMCS, trimethylchlorosilane; and LOD, limit of detection.
CLINICAL CHEMISTRY, Vol. 40, No. 7, 1994

360. The mass-ion defect for the quantification ion for each analyte was 1300 Sample collection. 364. and programmed for to 260#{176}C The GC-MS was tuned daily with perfluorotributylat 3. ol.40. anhyProcedures droecgonine methyl ester. tively. m/z 140. Sample preparation. 184. [2H3]ecgonine methyl ester.Hs H’ H Bsnzoylecgonlne LH. programmed for to 250#{176}C 10#{176}C/mm at and maintained at 250#{176}C 15 s. (196). the citric acid in the candy effectively inhibits the hydrolysis of cocaine (30). At least seven concentrations per detector. determined daily. Packard (Wilmington. m/z (152). 1. ecgonine ethyl ester. (96). Metabolic and pyrolysis products of cocaine.CHs N -OcOC. (96). All samples were refrigerated at 2-4#{176}C until processed. Calibration curves were pre- CUNICALCHEMISTRY.) silanized borosilicate and relative abundance of confirming ions with the corlinerwith a silanized glass-woolplug. The initial oven temperature was 70#{176}C. 271. mlz (155). (185).. compound in the following elution order: [2H3]anhydroecgonine methyl ester. 166. cocaethylene. 298. mlz 82. vacuum manifold system (VMFO24GL) were purchased from United Chemical Technologies. m/z 140. V 1994 . Urine specimens were collected without preservatives. 346. Norcocalne Ecgonlne Ethyl Ester Anhydraecgonlne Methyl Ester Coceethylene Norcoceethylen. 181. m/z 85. Injections were responding values for authentic calibrators assayed made in the splitless mode with a purge time of 0. cocaine. and injector seal. for amine and the electron multiplier was operated at 200 eV relative to the tuned value. cocaethylene. 240. it was programmed to 220#{176}C 35#{176}C/min maintained at and at Drug-freesamples collected from staff were used as controls. The injection port and transfer line temperatures were 250 and 280#{176}C. Saliva specimens were collected by stimulation of saliva flow with sour candy. 274. .8 mldmin. Horshain. 306. mm. (182). [2H3]benzoylecgonine. monitored ions at a dwell time of 10 ms for each GC-MS included clipping the GC column and replacing the septum. 361. norcocaethylene. cocaine. blood specimens were stabilized by addition of sodium fluoride and acetic acid. (99).. m/z 83. Fig. 303. 285. (254). ecgonine methyl ester. benzoylecgonine. 220#{176}C 15 s. cocaethylene. DE) 5890 gas chromatograph norcocaine./*“ Benzoylnorecgonlne CocaIne LH.d. Daily maintenance of the The mass selective detector.) x 0. operated in the SIM mode. norcocaethylene. AnhydroecgoGC-MS analyses were performed with a Hewlettnine methyl ester. (404). 7. [2H3]cocaine. PA..d.zoylnorecgonine were quantified by ratios of peak areas for each analyte equipped with a 7673A automated liquid sampler and to those of the nearest structurally related deuterated interfaced with a Hewlett-Packard 5970 mass selective internal standard. The ions noted in parentheses were used for quantification. (243). ecgornne ethyl ester. liner.2 mm (i. 169.5#{176}C/mm maintained and at 260#{176}C 15 s. mlz (140). mlz 85. and cross-linked methyl silicone gum capillary column [HPanalytes were identified by comparison of retention time 1. (240). mlz 82. respeccontrol samples collectedfrom volunteers residing on the clinical ward of the Addiction Research Center. and benzoylecgonine were quantified by ratios of peak areas for each analyte to those of the correspondGC-MS ing deuterated internal standard analogs. 12 m x 0. m/z 82.75 daily. and benzoylnorecgonine. Ultrapure-grade helium was used as the carrier Background interferences were assessed for each biological fluid by including in each analytical run predrug gas at a flow rate of 0. m/z 85. and ben. then stored frozen at -30#{176}C. No. The gas chromatograph was equipped with a analyte were used to construct calibration curves. mlz 82. 317. norcocaine. Because cocaine is hydrolyzed in the presence of esterases. Ecgonine methyl ester.33-jrni film thickness (Hewlett-Packard)] and a 4-mm (i.

9% to 88. benzoylecgornne. Subjects resided on the clinical ward of the Addiction Research Center. and acetate buffer (pH 4. depending on the quality of the column and injector. typically. and saliva samples were diluted with 3 mL of acetate buffer(pH 4.1-1000 g/L for cocaine.0 pg/L (minimal signal-to-noise ratio of 5). and benzoylnorecgonine-ranged from 36% to 78%. Methanol was added (2 x 1 mL) and the cartridges were again aspirated to dryness for 2 min. SPE extraction. intranasal.3%.L of acetic acid (31). and anhydroecgonine methyl ester were extracted from biological fluids in a one-step SPE process. The remaining analytes were quantified with use of the nearest structurally related deuterated analog as the internal standard. and saliva were prepared by adding 0. cocaethylene.1 mol/L HC1 (1. For stabilization of cocaine. for 1 pL of derivative was injected for GC-MS analysis. The cartridges were aspirated to dryness for 2 mm. and the vial was sealed and heated at 60#{176}C 30 mm. CUNICAL CHEMISTRY. and benzoylecgonine. required -10 min. and norcocaethylene were >95%. An entire run.5 mL). The copolymeric phase of the extractioncolumns allowed rapid and efficient isolation of the analytes. At concentrations 1000 pg/L. which ranged from 78. temperature This allowed efficient volatilization of all analytes. The study was conducted under the guidelines for protection of human subjects. ecgonine methyl ester. two contained all analytes at a concentration of 250 or 500 pgfL each. Samples (0. ecgenine methyl ester. washing. blood samples (5 mL) were collected into 125 L of sodium fluoride (saturated solution) plus 125 j. The limits of detection (LODs) for cocaine.10 mm. then filtered through a 4-mL fitted reservoir. methanol (2 x 2 mL). The injector port of the GC-MS was maintained at 250#{176}C. with minimal thermal decomposition of cocaine to anhydroecgonmne methyl ester. whereas recoveries of the more polar compounds-anhydroecgonmne methyl ester. Within-run and betweenrun CVs were <10% for all analytes except for the between-run CV for benzoylhorecgonmne (16. LODs for the remaining analytes varied from 3 to 6 pg/L. and benzoylecgonine were -1.1 to 1000 zgfL. Derivatization. Derivatizing reagent (20 L of BSTFA with 10 g/L TMCS) was added. cocaine. two contained 500 LgIL cocaine. the solutions were stored at room temperature for 15-20 min. hydrolysis to benzoylecgonine or ecgonine methyl ester during extraction was consistently <1%. Whole blood required greater dilution (1:10) to facilitate passage through the extraction column. The cartridge was not permitted to dry between conditioning and applica- Results Sample extraction.u. ecgonine methyl ester. r was >0.pared in the appropriate matrix in a concentration range of 3. ClinicalStudies Male volunteers with a history of cocaine abuse participated in the study. and elution. and urine were linear with concentrations from 3. ecgonine methyl ester. ecgonine methyl ester. Subjects were administered cocaine or placebo by the intravenous. 7. The analytes were collected in a clean tube after addition of the elution solvent (6 x 1 mL). including recycle time. Cocaine. During cartridge conditioning. Thermal conversion of [2H3]cocaine produces [2H3]anhydroecgonine methyl ester. All analytes were resolved with a separation of at least 0. Relative accuracy and precision of the analysis for cocaine and cocaine metabolites are shown in Table 1. plasma.1-mL autosampler vials. Cocaine and cocaine analytes were analyzed simultaneously by GC-MS with SIM. and all subjects provided informed consent. Vol. with use of a stock solution containing 20 mgfL cocaine. The three most abundant ions for each analyte were preselected from electron-impact spectra. and benzoylecgonine and 10 mgfL for each remaining analyte. Detector response for the m/z 155 ion was consistently <1% of the response obtained with deuterated cocaine (m/z 185). the pressure was adjusted to maintain a flow rate of 1-2 mldmin.5-1 mL) of whole blood. No. benzoylecgonine.g/L for anhydroecgonine methyl ester. The responsesfor cocaine and analytes extracted from plasma. norcocaine.2%). benzoylecgonine. or smoking route under supervised medical conditions.1 mL) each of deuterated analogs of ecgunine methyl ester. followed by deionized water (2 mL) and 0. Extracts were free of interferences at the appropriate retention times of each analyte. 1994 tion of samples.1 pg (0. GC-MS. The stability of cocaine throughout the extraction process was routinely monitored. ecgonine ethyl ester. Recoveries of cocaine. deionized water (2 x 2 mL). Filtered samples were added to the cartridges. ecgonine ethyl ester. Each sample batch included eight controls. 2 mL). dilutions of sample to less than this concentration provided greater quantitative accuracy. norcocaine. 40. Deuterated analogs were available for cocaine. responses were not always linear. urine. urine. two contained all analytes at a concentration of 50 or 100 gfL each. norcocaine. Plasma. Plasma was separated and frozenimmediately. norcocaethylene. 1301 . Within-run and between-run relative accuracies for all analytes were >90%. Urine and saliva samples were collected without preservatives and frozenfor later analysis. The extraction cartridges were conditioned with elution solvent (1 mL). norcocaethylene. and ecgonine methyl ester and were used for quantifying these analytes. benzoylnorecgonmne. The potential formation of anhydroecgonmne methyl ester as an artifact was monitored via the m/z 155 ion. and two were prepared as blank matrix samples. After mixing. cocaethylene. cocaethylene.6-500 .0).995. Ionization of [2H3]anhydroecgonine methyl ester produces the m/z 155 ion. saliva. except for benzoylecgornne and the nor-analytes within-run determinations. and benzoylecgonine and 1. Extracts were evaporated to dryness at ambient temperature under nitrogen and reconstituted in 20 tL of acetonitrile. and benzoylnorecgonine. The samples were transferred to 0.

1) 97.5 (2.6 3. one-step isolation procedure. 3D). saliva.0 (2.6 3.8) 52. ecgonine methyl ester. 3. Figure 3C illustrates cocaine plasma concentrations in a subject after three routes of cocaine administration.6 (2. Use of the copolymeric bonded-phase columns allowed extraction of the analytes in a rapid.8 96.3 96. Ecgonine methyl ester concentrations remained low throughout the 3-h sampling period.5) 88. benzoylecgonine. 1994 V . cocaine. by smoking. Urinary excretion of anhydroecgonine methyl ester. The two minor metabolites. 33).0 9.8 96.4 97. and ecgonine methyl ester were present in the urine sample collected from the crack smoker. were present in low concentrations (5-27 and 3-16 pg/L.2) 78. but reached a higher concentration that was sustained over time. 7.5) 111. Substantial amounts of cocaine.5 (4. and recovery of cocaine metabolites.1 6. Plasma concentrations increased immediately after the intravenous and smoking routes.4 40.5 (3.5) 39.3 (3.8 95. plasma Discussion The use of multimodal copolymeric bonded phases in SPE provides a convenient means of isolating drugs from biological specimens (32). a zero urine control sample. and benzoylnorecgonine were evident.8 (5.3 (2. respectively) and were detectable for 28 h.1) (2.0) (5. ecgonine methyl ester did not peak until 24 h after. Concentration vs time curves for cocaine. Cocaine concentrations diminished rapidly over time and became undetectable by 40 h.3) (1. and intranasally. dose.3 98.6) 48.7 (4.4 Figure 2 illustrates characteristic SIM recordings of an extracted cocaine urine standard.4 (3. benzoylecgonine. Cocaine and metabolite concentrations were measured in plasma. was detectable at low concentrations (5-27 pgfL) immediately after smoking and remained detectablefor 28 h. Benzoylecgonine excretion rose to peak concentrations in urine 12 h after drug administration.0) 41.% 98.5 5.5) 98. and urine of human volunteers who had taken cocaine intravenously. Clinical studies. The appearance of benzoylecgonine in was slower by the intranasal route than by the intravenous and smoking route (Fig.9 16. Neutral and acidic compounds are absorbed onto the columns through hydrophobic interactions. The pyrolysis product. The intermediate methan- 1302 CLINICAL CHEMISTRY.9) 49. norcocaine. 3A.3) 51.6) 102.1) 101.8 92. whereas only traces of anhydroecgonine methyl ester. saliva. while basic drugs are bound through both hydrophobic and ionic interactions.4 (3. ol.9 97. % 5.1 95. benzoylecgonine and ecgonine methyl ester declined in similar fashion and were not detectable beyond 72 h.25 h after a subject smoked crack (42 mg). 3B) were greater than plasma concentrations.7(5.9 (6.6 Recovery.2 (5.4 (6. 4.3 98.6 Norcocaethylene Benzoylnorecgonine 9. Peak cocaine plasma concentrations were reached immediately after administration by the intravenous route (Fig. and urine.4 5. and the minor metabolites norcocaine and benzoylnorecgonine after smoking crack (42 mg) is illustrated in Fig. and a urine specimen obtained 1.Table 1. anhydroecgnnine methyl ester.1 83.5 6. precision.9) 102. We used SPE for the simultaneous isolation of cocaine and eight cocaine metabolite derivatives from blood.2 7. Subsequently.1 94. following an erratic but similar time course of disappearance. Benzoylecgonine and ecgonine methyl ester saliva concentrations remained low.2) Cocaine Benzoylecgonine Ecgonine methyl ester Cocaethylene Norcocaine 98.1 78.1) 36. benzoylecgonine.9 (1.9 83.5) 98.4) 6.4 50. Benzoylecgonine concentrations rose quickly and exceeded cocaine concentrations during the first hour.8 48. L An&yte Within-run Target 100 100 100 50 50 50 Measureda Relative accuracy. and ecgonine methyl ester in plasma and saliva are shown for two volunteers in Fig.7 (5. whereas concentrations rose gradually by the intranasal route and reached a peak at 60 mm.2) 47. 25 mg of cocaine HC1). norcocaine and benzoylnorecgonine. The study was performed in a crossover design in which each subject was administered cocaine by all three routes.6 6. After the 25-mg intravenous cocaine dose.2 (10. 107.7 CV. % 98.4 98.6 4. Conc. No.5) 41. RelatIve accuracy. There have been several reports of application of SPE techniques to a wide variety of drugs and matrices (30.6 (8. in contrast.7) 50 Anhydroecgonine methyl ester 50 Between runs 100 Cocaine 100 Benzoylecgonine 100 Ecgonine methyl ester 50 Cocaethylene 50 Norcocaine 50 Norcocaethylene Benzoylnorecgonine 50 Anhydroecgonine methyl ester 50 a Mean (SD) of n = 15 determinations for each analyte.4 6. No interferences were present in the zero urine control sample.6 (3.5 (3. cocaine salivaconcentrations (Fig.40.8 9.

ecgonlne ethyl ester. BE. when saliva has a pH of 6. 3 positive controls. 14 calibrators (seven-point calibration curve in duplicate).. tion. 03c 6. If specimens were suspected of being more concentrated than the highest point on the calibration curve. AEME. norcocaethylene. SIM GC-MS of cocaine calibrator(A). Signs of peak broadening by these analytes served as a warning sign that chromatographic conditions were deteriorating and maintenance should be performed. Ecgonine methyl ester appears in plasma in very low amounts compared with that of benzoylecgonine and often goes undetected. After evaporation and derivatization. peaks in 1-3 h. norcocaine.5 6. whereas saliva ecgonine methyl ester concentrations were higher. samples were analyzed by GC-MS with an autosampler. D3BE.4 (35). Samples were analyzed in the following sequence: 1 zero control. This sequence order allowed evaluation for sample deterioration over the entire analysis period. H3Icocaine. D3EME. Instrument run time was -12 h for a batch of 50 specimens with accompanying calibrators and controls. 15 specimens. attesting to the stability of the derivatized sample over the period of analysis. solvent injections were added between specimens to prevent carryover.- COCBE EEE D3BE / AEME. Assay Characteristics SPE combined with automated GC-MS analyses allowed the measurement of cocaine metabolites in plasma.3% within run) and the nor-metabolites (78. the composition depending on both type of specimen and time of sampling. which exhibited a 16. NCE. 12H3]ecgonine methyl ester. No. 3C). Benzoylecgonine. and 8 control samples could be extracted in -6 h. 2.8 and plasma has a pH of 7.9% within run).0 4. in-plasma-soon after.5 4.5 7. tabolites indicated that each biological specimen contained different amounts of analytes.cocaineadministra-. 1994 1303 V . 7 calibrators. with CVs <10% for all analytes except benzoylnorecgonine. ol.. EEE. benzoylnorecgonine. Batches of 50 specimens. AEME/I D3EM D3COC.8 for the cocaine saliva/plasma ratio. The peak cocaine concentration in saliva was about threefold that measured in plasma. Deterioration was not noticeable within 48 h after derivatization if the sealed autosampler vial had not been punctured. Thereafter. 20 specimens. cocaine concentrations rose slowly over the first hour to peak concentrations. The extracts could be conveniently stored in methanol at -30#{176}C overnight before GC-MS analysis. calibration curves constructed from the two sets were superimposable. and urine sample (C) from a subject 1. The pattern of cocaine dispositionin saliva was substantially different from that observed in plasma. olic wash procedure removed matrix provided an extract free of interferences. and urine relatively efficiently. 1 zero control.c25 - CocEME BE x50 5 I II NCOC NBE and 7 calibrators. The precision of the assay was acceptable. Responses from the first set of calibrators were compared with responses from the second set of calibrators (at the end of the run) to determine if deterioration had occurred. 15 specimens. COC. Thereafter. benzoylecgonine. NCOC. D3-COC. Typically.9-83.appears. concentrations declined rapidly and were nearly superimposable (Fig. 3 positive controls.0 5.5 5.0 components and Measurementof Cocaine-RelatedAnalytes in Biological Specimens Comparison of assay results for cocaine and its me- 3. based on the HendersonHasselbalch equation.40. saliva.2% between-rmi CV. concentrations do- CLINICAL CHEMISTRY. In contrast. it is cleared rapidly from plasma (34). EME. The disposition of benzoylecgonine and ecgonine methyl ester in saliva also was different from that observed in plasma. CE.A EME. saliva benzoylecgonine concentrations were lower. (2H3jbenzoylecgonine.BE #{163} -- C . cocaethylene. The disposition of cocaine in plasma samples after different routes of administration was monitored for one subject with the GC-MS assay.0 Minutes Fig. ecgonine methyl ester. The nor-metabolites exhibited the greatest sensitivity to changes in the conditions of the injection port and the chromatographic column. B D3EME D3COC -D3BE EME -- hLu . Noteattenuationchanges(x25 and x50) in panel C. Initial cocaine concentrations were higher after smoking crack (42 mg) than after intravenous administration (25 mg). BNE. NCOC D3EME -- D3COC f NCE /NBE r - . Within-run and between-run accuracies generally were >90% for all analytes except benzoylecgonine (88. cocaine. zero control (B). This is consistent with a prediction of 3. and diminishes much more slowly than cocaine. after intranasal intake. Cocaine has a half-life in human subjects of -30 mm.anhydroecgonine methyl ester.25 h after smoking crack (42 mg). 7.

metabolites. 1. this represents the first detailed study of excretion of anhydroecgonine methyl ester and these metabolites in urine. our data indicate that it is present in substantially lower concentrations and for a shorter period than benzoylecgonine. in urine of Fig. 4. its presence has been proposed as a marker for this type of cocaine. smoking (42 mg of crack). The present assay offers substantial advantages over existing assays by providing simultaneous measurements of these metabolites in a single biological sample in a sensitive and specific manner. Investigation of the pharmacological effects of cocaine and its metabolites depends on the availability of an accurate means of measurement.37:1229-34. its metabolites. Although the presence of anhydroecgonmne methyl ester in urine has been reported previously (3). norcocaine and benzoylnorecgonine. Hierholzer R. Brower K. 7. 1994 V . and anhydroecgonine methyl ester Ina subject after smoking crack (42 mg). Benzoylecgornne concentrations plasma were -50% higher after intrain nasal administration than after the intravenous and smoking routes. However. Recent trends in cocaine abuse in a VA psychiatric population. 0 20 40 60 80 Hours Fig. The formation and excretion of norcocaine in humans continues t be of interest because of its pharmacological and toxic activity (36). We used GC-MS to monitor the excretion of anhydroecgonine methyl esterand two minor cocaine metabolites. Also. No. This indicates that only small amounts of these compounds are produced in the normal course of metabolism of cocaine. Urinaryexcretion of cocaine. As a result. Maddahian E. The higher concentration of benzoylecgonine after intranasal intake could be the result of metabolism during its passage through the nasal mucosa or of hydrolysis in the stomach and first-pass metabolism of swallowed cocaine during inhalation.40. in saliva and plasma. Abbreviationss In Fig. Cocaine. References 1. Plasma and saliva concentrations of cocaine and metabolites in human subjects after cocaine administration. The presence of even minor amounts of this metabolite could contribute to the toxicity of cocaine. concentrations of the minor metabolites were below the LOD of the assay. Little information is available regarding the pharmacological activity of benzoylnorecgonmne. the analyte commonly monitored for cocaine abuse. Use of this assay should aid future studies of the pharmacological role of cocaine. subjects after they smoked crack. Interestingly. its time course of excretion has not been determined. and related constituents. there may be a difference in metabolism by these routes. The slow increase in cocaine concentration over 1 h after intranasal intake accounts for the differences behavioral in effects often reported by subjects who prefer the intravenous and smoking routes over the intranasal route.benzoylacgonine (BE). 3. and intranasal(32mg ofcocaine hydrochloride)routes. To our knowledge. a 1304 CLINICAL CHEMISTRY. Subsequent concentration by the kidney allowed their detection and measurement in urine. Because this analyte should be present only in the urine of crack smokers. Hosp Community Psychiatry 1986. its usefulness appears to be limited. plasma concentrations of cocaine (C) and BE (D) in another subject after administrationof cocaine by the intravenous (25 mg of cocaine hydrochloride).200 A 600 B 400 ‘Cocaine I\ 0 80 250 AEME 200 A 60 OBE 180 300 120 180 c Intravenous ed 0 :I ix 80 40 veneus 5: _________________________ 80 10 300 A Intranasal iso so Minutes dined in a manner similar to that in other routes of administration. and ecgonine methyl ester (EME) concentrations plasma (A) and saliva (B) of one in subjectafteran Intravenous doseof 25 mg of cocainehydrochloride. ol.

Cone EJ.323:326-9.26:181-92. Determination 1980.14:353-7. Bouis P. J Chromatogr 1990. 25. Van Veen J. and plasma. 20. Isenschxnid D. Anal Biochem 1992. 19. Peat M. 5. 9. J Neurochem 1991. Mule S. benzoylecgonine. Jacob P. James RC. Cofino J. 23. Maseda C. Zhou C. and ecgonine methyl ester in blood and urine using GCIEIMS with derivatization to produce high mass molecular ions. Thompson LK. Harkey MR. 15.15:276-8. Stability of cocaine in saliva [Tech Brief]. 11. in postmortem fluids and tissues by computerized gas chromatography/mass spectrometry. 24. Cone EJ. De La Torre R.13:250-6. Confirmation of cocaine in human saliva after intravenous use. ol. Fukui Y. Ansari NA. Neugebauer M. Elias-Baker B. Morarity T. Pramanik AK. 4. Taccard G.3:59-63. Cofino J.526:447-59. Jenmson T. Caplan Y.28:894. benzoylecgonine and benzoylnorecgornne by high-performance liquid chromatography. J Anal Toxicol 1979. Determination of cocaine. Quantitation of cocaine. is in the urine of cocaine smokers. Abusada GM.25:97-104. Miller RL. Cocaethylene: a unique cocaine metabolite displays high affinity for the dopamine transporter. Forensic Sci mt 1984. J Anal Toxicol 1988. Hajar T. 36. 570:412-8. Simultaneous quantitation of cocaine and its majormetabolites in human hair by gas chromatography/chemical ionization mass spectrometry. Yousefnejad D. Stafford DT. Farre M. Correlation of saliva cocaine levels with plasma levels and with pharmacologic effects after intravenous cocaine administration in human subjects.15:223. Elias-Baker B. Benowitz N. Matsubara K. Winger G. Identification of cocaine and its metabolites in human urine in the presence of ethyl alcohol. Determination of cocaine and norcocaine in plasma and cell cultures using high-performance liquid chromatography. Tebbett IR. J Chromatogr 1991. Quantitative determination of benzoylecgonine and cocaine in human biofluids by gas-liquid chromatography.12:200-6. Taylor J. 7.33:118-9. Goenechea S. benzoylecgonine. 16. 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