William D. Darwin

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography-mass spectrometry (GC-MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester;. ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40 degrees C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC-MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.

Cocaine metabolism and urinary excretion after different routes of administration.

Cocaine abusers frequently self-administer cocaine by different routes of administration. A controlled-dosing study was performed to assess the effect of different routes of administration on the excretion profile of cocaine and metabolites in urine. Single bioequivalent doses of cocaine were administered by the intravenous, intranasal, and smoked routes to six human subjects. Urine specimens were collected for 3 days after drug administration and were analyzed for cocaine, metabolites, and anhydroecgonine methyl ester, the thermal degradation product of cocaine, by gas chromatography-mass spectrometry. Cocaine was rapidly absorbed, metabolized, and excreted in urine. Peak cocaine concentrations were generally present in the first specimen collected; thereafter, concentrations declined quickly and were usually below the limit of detection (approximately 1 ng/ml) within 24 hours. The metabolite benzoylecgonine was present in the highest concentration and represented approximately 39%, 30%, and 16%, of the administered dose by the intravenous, intranasal, and smoked routes, respectively. Combined amounts of ecgonine methyl ester and six minor metabolites (norcocaine, benzoylnorecgonine, m-hydroxycocaine, p-hydroxycocaine, m-hydroxybenzoylecgonine, and p-hydroxybenzoylecgonine) accounted for approximately 18%, 15%, and 8% of the administered dose by the intravenous, intranasal, and smoked routes, respectively. Anhydroecgonine methyl ester was present in trace amounts (0.02% dose) in specimens collected after smoked cocaine administration. Because many of these metabolites exhibit pharmacologic activity, their presence in urine may indicate that they play complex biologic roles in the overall activity of cocaine.

The occurrence of cocaine, heroin and metabolites in hair of drug abusers

The analysis of hair for drugs of abuse reveals information regarding past drug exposure. We developed methods for washing, extraction and analysis of hair samples for cocaine, heroin and metabolites. Twenty paired head- and arm-hair samples, collected from known heroin/cocaine abusers, were analyzed with a new comprehensive GC/MS assay for cocaine, heroin and metabolites. Cocaine and 6-acetylmorphine (6-AM) were the major analytes present in both head- and arm-hair samples. Cocaine was detected in all head- and 17 arm-hair samples. The concentration of cocaine found was 4-760 ng/10 mg in head hair and 0-1090 ng/10 mg in arm hair. Less benzoylecgonine was present in a concentration range of 0-158 ng/10 mg of head hair and 0-125 ng/10 mg of arm hair. Heroin was found in only 2 head-hair samples, whereas 6-AM was present in 14 head and 6 arm-hair samples. The concentration of 6-AM was 0-8 ng/10 mg in head hair and 0-31 ng/10 mg in arm hair. Morphine was present in 3 head-hair samples in a range of 2-9 ng/10 mg and was not detected in arm-hair samples. When results were compared by groups (head hair versus arm hair, Caucasoid versus Africoid), only two significant differences were found. Cocaine concentrations in both head and arm hair were significantly (P < 0.05) higher in the Africoid group than in the Caucasoid group. The reasons for these differences were not readily apparent, but could have been due to differences in the level of cocaine use or to ethnic differences in the deposition of drug in hair.

Extended urinary Δ9-tetrahydrocannabinol excretion in chronic cannabis users precludes use as a biomarker of new drug exposure

BACKGROUNDnGenerally, urinary 11-nor-9-carboxy-Delta9-tetrahydrocannabinol (THCCOOH) after alkaline hydrolysis is monitored to detect cannabis exposure, although last use may have been weeks prior in chronic cannabis users. Delta9-Tetrahydrocannabinol (THC) and 11-hydroxy-THC (11-OH-THC) concentrations in urine following Escherichia coli beta-glucuronidase hydrolysis were proposed as biomarkers of recent (within 8h) cannabis use.nnnOBJECTIVEnTo test the validity of THC and 11-OH-THC in urine as indicators of recent cannabis use.nnnMETHODSnMonitor urinary cannabinoid excretion in 33 chronic cannabis smokers who resided on a secure research unit under 24h continuous medical surveillance. All urine specimens were collected individually ad libidum for up to 30 days, were hydrolyzed with a tandem E. coli beta-glucuronidase/base procedure, and analyzed for THC, 11-OH-THC and THCCOOH by one- and two-dimensional-cryotrap gas chromatography mass spectrometry (2D-GCMS) with limits of quantification of 2.5 ng/mL.nnnRESULTSnExtended excretion of THC and 11-OH-THC in chronic cannabis users urine was observed during monitored abstinence; 14 of 33 participants had measurable THC in specimens collected at least 24h after abstinence initiation. Seven subjects had measurable THC in urine for 3, 3, 4, 7, 7, 12, and 24 days after cannabis cessation. 11-OH-THC and THCCOOH were detectable in urine specimens from one heavy, chronic cannabis user for at least 24 days.nnnCONCLUSIONnFor the first time, extended urinary excretion of THC and 11-OH-THC is documented for at least 24 days, negating their effectiveness as biomarkers of recent cannabis exposure, and substantiating long terminal elimination times for urinary cannabinoids following chronic cannabis smoking.

Δ9-Tetrahydrocannabinol (THC), 11-Hydroxy-THC, and 11-Nor-9-carboxy-THC Plasma Pharmacokinetics during and after Continuous High-Dose Oral THC

BACKGROUNDnDelta(9)-tetrahydrocannabinol (THC) is the primary psychoactive constituent of cannabis and an active cannabinoid pharmacotherapy component. No plasma pharmacokinetic data after repeated oral THC administration are available.nnnMETHODSnSix adult male daily cannabis smokers resided on a closed clinical research unit. Oral THC capsules (20 mg) were administered every 4-8 h in escalating total daily doses (40-120 mg) for 7 days. Free and glucuronidated plasma THC, 11-hydroxy-THC (11-OH-THC), and 11-nor-9-carboxy-THC (THCCOOH) were quantified by 2-dimensional GC-MS during and after dosing.nnnRESULTSnFree plasma THC, 11-OH-THC, and THCCOOH concentrations 19.5 h after admission (before controlled oral THC dosing) were mean 4.3 (SE 1.1), 1.3 (0.5), and 34.0 (8.4) microg/L, respectively. During oral dosing, free 11-OH-THC and THCCOOH increased steadily, whereas THC did not. Mean peak plasma free THC, 11-OH-THC, and THCCOOH concentrations were 3.8 (0.5), 3.0 (0.7), and 196.9 (39.9) mug/L, respectively, 22.5 h after the last dose. Escherichia coli beta-glucuronidase hydrolysis of 264 cannabinoid specimens yielded statistically significant increases in THC, 11-OH-THC, and THCCOOH concentrations (P < 0.001), but conjugated concentrations were underestimated owing to incomplete enzymatic hydrolysis.nnnCONCLUSIONSnPlasma THC concentrations remained >1 mug/L for at least 1 day after daily cannabis smoking and also after cessation of multiple oral THC doses. We report for the first time free plasma THC concentrations after multiple high-dose oral THC throughout the day and night, and after Escherichia coli beta-glucuronidase hydrolysis. These data will aid in the interpretation of plasma THC concentrations after multiple oral doses.

Subjective and Physiological Effects After Controlled Sativex and Oral THC Administration

Sativex is a cannabis‐plant extract delivering nearly 1:1 Δ9‐tetrahydrocannabinol (THC) and cannabidiol (CBD) by oromucosal spray. It has been suggested that CBD attenuates THC‐induced tachycardia, anxiety, and euphoria. In this study, pharmacodynamic effects were compared over 10.5 h in nine cannabis smokers randomly assigned to receive placebo, 5 and 15 mg oral synthetic THC, and low (5.4 mg THC, 5.0 mg CBD) and high (16.2 mg THC, 15.0 mg CBD) doses of Sativex. At therapeutic doses, no substantial CBD‐induced modulation of THCs effects was evident. Oral THC and Sativex produced similar, clinically insignificant increases in heart rate, anxiety, and “good drug effects” with no serious adverse events. Oral and oromucosal THC have slower absorption, lower rate of THC delivery to the brain, and fewer associated adverse events as compared with smoked cannabis. These results indicate that Sativex has a pharmacodynamic safety profile comparable to that of oral THC at low, therapeutic doses.

Diazepam and methadone blood levels following concurrent administration of diazepam and methadone

Results of a previous study indicated that the opioid effects of methadone were enhanced by the concurrent administration of diazepam in methadone-maintained subjects. To determine whether a pharmacokinetic interaction might account for this methadone-diazepam interaction, the plasma levels of methadone, diazepam and diazepam metabolites were determined in blood samples collected during that study. Five adult male patients on methadone maintenance (50-60 mg/day) were administrated single doses of placebo, diazepam (20 and 40 mg), methadone (100%, 150% and 200% of the maintenance dose), and four diazepam-methadone dose combinations (20 and 40 mg diazepam in combination with 100% and 150% of the maintenance dose). The results showed that the concurrent administration of methadone and diazepam did not significantly change the time-course or areas under the plasma concentration-time curve of methadone, diazepam or N-desmethyl-diazepam compared to the levels following the administration of either drug alone. Thus, plasma drug level analysis does not indicate a pharmacokinetic interaction between diazepam and methadone.

Differentiating new cannabis use from residual urinary cannabinoid excretion in chronic, daily cannabis users

AIMSnTo develop and validate empirically a mathematical model for identifying new cannabis use in chronic, daily cannabis smokers.nnnDESIGNnModels were based on urinary creatinine-normalized (CN) cannabinoid excretion in chronic cannabis smokers.nnnSETTINGnFor model development, participants resided on a secure research unit for 30 days. For model validation, participants were abstinent with daily observed urine specimens for 28 days.nnnPARTICIPANTSnA total of 48 (model development) and 67 (model validation) daily cannabis smokers were recruited.nnnMEASUREMENTSnAll voided urine was collected and analyzed for 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) by gas chromatography-mass spectrometry (GCMS; limit of quantification 2.5 ng/ml) and creatinine (mg/ml). Urine THCCOOH was normalized to creatinine, yielding ng/mg CN-THCCOOH concentrations. Urine concentration ratios were determined from 123,513 specimen pairs collected 2-30 days apart.nnnFINDINGSnA mono-exponential model (with two parameters, initial urine specimen CN-THCCOOH concentration and time between specimens), based on the Marquardt-Levenberg algorithm, provided a reasonable data fit. Prediction intervals with varying probability levels (80, 90, 95, 99%) provide upper ratio limits for each urine specimen pair. Ratios above these limits suggest cannabis re-use. Disproportionate numbers of ratios were higher than expected for some participants, prompting development of two additional rules that avoid misidentification of re-use in participants with unusual CN-THCCOOH excretion patterns.nnnCONCLUSIONSnFor the first time, a validated model is available to aid in the differentiation of new cannabis use from residual creatinine-normalized 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (CN-THCCOOH) excretion in chronic, daily cannabis users. These models are valuable for clinicians, toxicologists and drug treatment staff and work-place, military and criminal justice drug-testing programs.

63Ni electron-capture gas chromatographic assay for buprenorphine and metabolites in human urine and feces

A 63Ni electron-capture gas chromatographic assay is described for buprenorphine, a potent narcotic agonist--antagonist. In addition, the assay is useful for the measurement of the metabolite norbuprenorphine and demethoxybuprenorphine, a rearrangement product resulting when buprenorphine is exposed to acid and heat. An extraction procedure was developed which optimized recovery of buprenorphine from biological samples and produced minimal background interferences and emulsion problems. Extract residues were derivatized with pentafluoropropionic anhydride and assayed by gas chromatography. Samples were analyzed with and without enzyme hydrolysis, thus providing a selective and sensitive assay for both free and conjugated buprenorphine, norbuprenorphine and demethoxybuprenorphine. The lower limits of detection following extraction of a 1-ml sample were ca. 10 ng/ml for buprenorphine and demethoxybuprenorphine and 5 ng/ml for norbuprenorphine. Application of the assay to human samples following a 40-mg oral dose of buprenorphine produced no evidence for the presence of demethoxybuprenorphine in urine or feces. Norbuprenorphine (free and conjugated) was present in urinary and fecal samples; buprenorphine (free and conjugated) was found in high amounts only in feces and in trace amounts in urine as conjugated buprenorphine. The urinary and fecal excretion pattern observed for a human subject following oral dosing of buprenorphine suggests enterohepatic circulation of buprenorphine.

Disposition of Cannabinoids in Oral Fluid after Controlled Around-the-Clock Oral THC Administration

BACKGROUNDnOral fluid, a promising alternative matrix for drug monitoring in clinical and forensic investigations, offers noninvasive sample collection under direct observation. Cannabinoid distribution into oral fluid is complex and incompletely characterized due to the lack of controlled drug administration studies.nnnMETHODSnTo characterize cannabinoid disposition in oral fluid, we administered around-the-clock oral Delta(9)-tetrahydrocannabinol (THC) (Marinol) doses to 10 participants with current daily cannabis use. We obtained oral fluid samples (n=440) by use of Quantisal collection devices before, during, and after 37 20-mg THC doses over 9 days. Samples were extracted with multiple elution solvents from a single SPE column and analyzed by 2-dimensional GC-MS with electron-impact ionization for THC, 11-hydroxy-THC (11-OH-THC), cannabidiol, and cannabinol and negative chemical ionization for 11-nor-9-carboxy-THC (THCCOOH). Linear ranges were 0.5-50 microg/L, with the exception of cannabinol (1-50 microg/L) and THCCOOH (7.5-500 ng/L).nnnRESULTSnTHCCOOH was the most prevalent analyte in 432 samples (98.2%), with concentrations up to 1117.9 ng/L. In contrast, 11-OH-THC was not identified in any sample; cannabidiol and cannabinol were quantified in 3 and 8 samples, respectively, with maximum concentrations of 2.1 and 13 microg/L. THC was present in only 20.7% of samples, with highest concentrations near admission (median 4.2 microg/L, range 0.6-481.9) from previously self-administered smoked cannabis.nnnCONCLUSIONSnMeasurement of THCCOOH in OF not only identifies cannabis exposure, but also minimizes the possibility of passive inhalation. THCCOOH may be a better analyte for detection of cannabis use.