Cocaine and Metabolite Excretion in Saliva under Stimulated and ...

Journal of Analytical Toxicology, Vol. 17, October 1993

Cocaine and Metabolite Excretion in Saliva under Stimulated and NonstimulatedConditions Kenichi Kato, Mary Hillsgrove, Linda Weinhold, David A. Gorelick, William D. Darwin, and E d w a r d J. C o n e *

Addiction Research Center, National Institute on Drug Abuse, Baltimore, MD 21224

Abstract The accessibility of saliva for rapid, nonlnveelve sampling makes It an attractlve blologlcel fluld for detectlng drug use. However, llttle Is known about sallvary excratlon patterns of the major cocalne metabolltes, benzoylecgonlne (BE) and ecgonlne methyl ester (EME). Addltlonally, there Is a general lack of Informstlon on the effects of sallvary collecUon condltlons on cocelne excretion In sallva. Thls study documents the profile of cocaine and metabolltee In human sallva under stlmulated and nonstlmulated sallva flow condltlons. Saliva samples were obtained perlodlcally from six healthy volunteers who were administered three, equally spaced, slngle Intravenous doses of 25 mg of cocelne durlng a 6-h test seeslon. On dlfferent days, whole sallva was obtained either under nonstlmulated or stlmulated (sour candy) conditions. The samples were analyzed for cocalne and metabolites by GCIMS. Cocaine, BE, and EME were detected and quantitated In the saliva of all subjects. Cocaine was the predominant analyte identified in all samples. Nonstimulated saliva contalned substantially more drug than stimulated samples. The ratio of the area under the curve (AUC) of cocalne in nonstlmulated saliva to that of stimulated saliva was varlable and ranged from 3.0 to 9.5. The AUC ratlos of BE and EME were similar to those observed for cocalne. The lowering of cocalne concentratlon In saliva In the stimulated flow condltlon was likely due to an increase In sallva pH assoclated with Increased sallva flow rate; it Is known that an increase in saliva pH retards cocalne partitioning Into this blologlcal fluid. Generally, the results of this study indicated that cocaine is the predomlnant analyte In saliva and that concentratlons of cocaine and metabolltes can be influenced substantially by the method of collectlon. These factors should be taken Into account In the deslgn of sallva tests for detection of cocaine exposure.

testing, but it has certain disadvantages. With advance notice, individuals can attempt to negate test results by drinking large amounts of water. In addition, urine can be adulterated during collection, sometimes leading to invalid results. Collection and testing of blood specimens also has drawbacks. Blood can be difficult to obtain from drug abusers and carries some risk of spreading infection. In contrast, saliva is a biological fluid easily obtainable through noninvasive measures, which can be useful for chemical validation of drug exposure. Numerous drugs of abuse have been reported to occur in saliva, and the usefulness of saliva in forensic detection has been reviewed (1,2). Cocaine has been detected in saliva after oral (3) and intravenous administration (4,5). In recent studies, saliva cocaine levels have been shown to correlate significantly with blood levels and with pharmacologic effects following intravenous administration (5). However, there is little information on the effect of collection conditions on the excretion of cocaine in saliva. Schramm et al. (2) also commented upon the lack of information on the occurrence of cocaine metabolites in saliva. They reported, in preliminary investigations, the presence of benzoylecgonine (BE), ecgonine methyl ester(EME), and ecgonine in the saliva of a single cocaine user (2). This study was designed to determine the pattern of excretion of cocaine and its metabolites in saliva of human subjects after intravenous administration of cocaine. Collection procedures were controlled to determine if cocaine and metabolite levels differed under different saliva flow conditions. Saliva samples were collected following cocaine administration, extracted by solid-phase extraction (SPE), and assayed by gas chromatography/mass spectrometry (GC/MS).

Materials and Methods Subjects Introduction

With the high incidence of drug abuse in the United States, there is continuing interest in the development of new chemical methods for drug detection. Urine is used most frequently for *Address reprint requests to Dr. EdwardJ. Cone, ARC, NIDA, P.O. Box 5180, Baltimore, MD 21224.

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Six healthy, male, volunteer subjects with a history of intravenous cocaine abuse participated in the study. Informed consent was obtained, and the study was conducted under the guidelines for the protection of human subjects. All subjects resided in a closed research unit of the Addiction Research Center, National Institute on Drug Abuse, during the study. Their ages ranged from 30 to 42 years, and their weights ranged from 57.7 to 85.5 kg.

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Journalof AnalyticalToxicology,Vol.17,October1993 Procedure Test days were at least 48 hours apart. On the morning of each test day, a heparinized intravenous catheter was placed in the subject's arm vein at approximately 8:15 a.m. Injections of 25 mg of cocaine hydrochloride were made through the catheter at approximately 9:40 a.m., 11:40 a.m., and 1:40 p.m. Pre-drug control saliva was collected 10 min before the first injection. Saliva specimens were collected at various time intervals after each drug administration. Saliva was collected under either stimulated (using citric-acid-type sour candy) or nonstimulated conditions. The same collection conditions were maintained throughout the day. Collection conditions alternated from day to day throughout the subject's participation in the study. The time needed for collection of stimulated saliva (10 mL) ranged from 3 to 5 min. Smaller volumes of nonstimulated saliva samples were collected over time periods equivalent to the time of collection of stimulated saliva. Mixed saliva was collected by expectoration into 50-mL screwcapped polypropylene tubes. After collection, the saliva samples were frozen until the time of analysis. Chemicals Cocaine hydrochloride was obtained from Mallinckrodt, Inc. Ecgonine methyl ester (EME) HC1 and benzoylecgonine (BE) tetrahydrate were obtained from the Research Technology Branch, National Institute on Drug Abuse. The internal standards, 8-(21-13methyl)-cocaine, 8-(2H3-methyl)-benzoylecgonine tetrahydrate, and 8-(2H3-methyl)-ecgonine methyl ester HC1 hydrate, were purchased from Sigma Chemical Co. Methanol, methylene chloride, 2-propanol, and acetonitrile (J.T. Baker) were HPLC grade solvents. N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) was purchased from Pierce Chemical Co. All other chemicals were reagent grade. SPE columns (Clean Screen | DAU, 200 mg-10 mL) were purchased from United Chemical Technologies, Inc. GC/MS autosampler microvials were purchased from Sun Brokers TM, Inc.

i.d.) with a 0.33-1am film thickness was linked to the MSD through a direct capillary interface. The multimode inlet was operated in the splitless mode with a 4-mm i.d. glass insert containing a silanized glass wool plug. The carrier gas was helium with a flow rate of 1 mL/min. The injector and transfer line temperatures were 250~ and 280~ respectively. The oven temperature was maintained at 70~ for 1 min, programmed to 220~ at 35~ maintained at 220~ for 15 s, programmed to 250~ at 10~ maintained at 250~ for 15 s, and then programmed to 260~ at 3.5~ The MSD was operated in the selected ion monitoring (S1M) mode. The following ions were monitored for each compound at their respective retention times (Rt) (ions used for quantitation are shown in italics): EME, m/z 82, 96, 271, Rt = 4.970 rain; D 3EME, m/z 85, 99, 274, R t = 4.964 min; cocaine, m/z 82, 182, 303, R t = 7.599 min; D3-cocaine, m/z 85, 185, 306, R t = 7.590 min; BE, m/z 82, 240, 361, R t = 8.014 min; and D3-BE m/z 85, 243, 3 6 4 , R t =8.005 min. Analytes were identified based on comparison of retention times and relative abundance of the two confirming ions to the corresponding values of authentic standards run on a daily basis. A dwell time of 10 ms was used for ion acquisition. Standard curves (12.5-1000 ng) were constructed based on ion peak-height ratios of analyte to their respective deuterated analogs. Control samples containing target concentrations of 500 ng/mL of cocaine, BE, and EME were assayed in duplicate with each GC/MS run. The mean concentration and coefficients of variation (CV, N = 5) were as follows: cocaine, 485.2 ng/mL, 3.9% CV; BE, 505.7 ng/mL, 4.4% CV; and EME, 520.3 ng/mL, 8.8% CV.

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Extraction and derivatizatlon procedures Aliquots of saliva (1 mL) and the internal standard (100 IlL, containing 1 ng/I.tL each of D3-cocaine, D3-BE, and D3-EME) were pipetted into culture tubes and briefly mixed. Each sample was adjusted to an approximate pH of 4 by addition of 3 mL of sodium acetate buffer (2M, pH 4.0). SPE columns were mounted on an extraction vacuum manifold and conditioned with 1 mL of elution solvent (xl), followed by 2 mL of methanol (x2) and 2 mL of deionized water (x2), respectively. Sodium acetate buffer (1.5 mL) was added to the column followed by addition of the sample. Vacuum was applied to produce a flow of 1-2 mL/min. The columns were washed with 2.0 mL of deionized water, 1.5 mL of 0.1M hydrochloric acid, and 1 mL of methanol (• and then aspirated to dryness. Analytes were eluted from the column with 6 mL of methylene chloride-2-propanol-ammonium hy&oxide (40:10:1, v/v/v). The eluates were evaporated to dryness under nitrogen and reconstituted with 0.02 mL of acetonitrile. The samples were transferred to 0.1-mL autosampler vials, and 0.02 mL of BSTFA (with 1% TMCS) was added. Sample vials were sealed and heated at 60~ for 30 rain. Immediately after derivatization, the samples were analyzed by GC/MS. GC/MS a s s a y

Derivatized extracts were analyzed on a Hewlett-Packard 5890A gas chromatograph coupled to a 5970B mass selective detector equipped with a 7673A automatic liquid sampler. An HP1 cross-linked fused-silica capillary column (12 m • 0.20-mm

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