Ecgonine Methyl Ester Research Articles

Cocaine and Certain of Its Long-Acting Metabolites Stimulate Endothelial Cell Von Willebrand Factor Secretion

Abstract 1148Cocaine ingestion is associated with increased risk of myocardial infarction, stroke, and deep vein thrombosis. Elevated risk for these thrombotic events persists for 7–10 days after cocaine ingestion, despite the fact that cocaine is rapidly cleared from the blood (with a half-life in circulation of less than 1 hour) being rapidly converted into numerous metabolites that circulate for 1–2 weeks. Thus, it is likely that these longer-lived cocaine metabolites account for the long-term thrombotic risk with cocaine use. Cocaine ingestion has been associated with increased plasma VWF concentration, early atherosclerosis, and platelet-rich arterial thrombi. VWF is the major adhesive ligand that attaches platelets to the vessel wall, either to the subendothelium at sites of injury where the endothelium has been denuded or, during inflammation, to intact endothelium, from which it is secreted. VWF is secreted by endothelial cells from intracellular storage granules (Weibel-Palade bodies) in a large, hyperadhesive multimeric form (ultra-large VWF, or ULVWF) that either remains tethered to the endothelial surface or is released into bulk flow. Platelet-VWF adhesion, and subsequent thrombus formation, may be augmented further by cocaine-induced vaso-constriction, increasing shear stress. Thus, we hypothesized that cocaine and/or its metabolites would stimulate endothelial VWF secretion as a mechanism of thrombotic risk. To test this possibility, we exposed cultured human endothelial cells from umbilical vein (HUVEC), brain microvasculature (BMVEC), or coronary artery (CAEC) to cocaine or one of its four major metabolites at concentration ranges reported to occur in plasma following cocaine use. The cocaine metabolites we tested were benzoylecgonine (BE), cocaethylene (CE), norcocaine (NC), and ecgonine methyl ester (EME). We assayed VWF release by platelet-VWF string formation in a parallel-plate flow chamber (2.5 dyne/cm2) and by measuring the concentration of VWF released into the supernatant. Cocaine concentrations as low as 0.1 μg/ml induced VWF release from HUVEC; 1–2 μg/ml cocaine was as effective in releasing VWF as 25 μM histamine or 4 mg/ml dDAVP. Of the cocaine metabolites, only BE and CE induced VWF release from endothelial cells. BMVEC were 10-fold more sensitive to cocaine and metabolites BE and CE than HUVEC in both platelet-string formation and VWF antigen assays. In CAEC, VWF release was slightly reduced compared to HUVEC in response to cocaine, BE, or CE. Consistent with this pattern, staining for intracellular VWF revealed the following hierarchy of intracellular VWF: BMVEC > HUVEC >> CAEC. In BMVEC and HUVEC, VWF staining was restricted to Weibel-Palade bodies, with CAEC also demonstrating a diffuse cytoplasmic pattern. We tested whether intracellular cAMP levels increased after cocaine or cocaine metabolite exposure to explore whether VWF release was dependent on Protein Kinase A, as is the case with dDAVP. Intracellular cAMP did not increase following exposure to cocaine or its metabolites in any of the endothelial cell lines. These results suggest that cocaine contributes to thrombosis by activating endothelial cells to secrete ULVWF via a mechanism that does not involve increased intracellular cAMP. The prolonged thrombotic risk after cocaine ingestion likely relates to the continued action of cocaine metabolites BE and/or CE on endothelial ULVWF secretion. VWF secretion is likely to vary between vascular beds, with brain endothelial cells being particularly sensitive. Furthermore, these results suggest that clinical management of cocaine-induced ischemia or vaso-occlusion may benefit from therapies aimed at disrupting the ULVWF–platelet interaction. Disclosures:No relevant conflicts of interest to declare.

Cocaine-induced kidney toxicity: an in vitro study using primary cultured human proximal tubular epithelial cells

Renal failure resulting from cocaine abuse has been well documented, although the underlying mechanisms remain to be investigated. In the present study, primary cultured human proximal tubular epithelial cells (HPTECs) of the kidney were used to investigate its ability to metabolize cocaine, as well as the cytotoxicity induced by cocaine and its metabolites benzoylecgonine (BE), ecgonine methyl ester (EME) and norcocaine (NCOC). Gas chromatography/ion trap-mass spectrometry (GC/IT-MS) analysis of HPTECs exposed to cocaine (1mM) for 72h confirmed its metabolism into EME and NCOC, but not BE. EME levels increased along the exposure time to cocaine, while NCOC concentration diminished after reaching a maximum at 6h, indicating a possible secondary metabolism for this metabolite. Cocaine promoted a concentration-dependent loss of cell viability, whereas BE and EME were found to be non-toxic to HPTECs at the tested conditions. In contrast, NCOC revealed to have higher intrinsic nephrotoxicity than the parent compound. Moreover, cocaine-induced cell death was partially reversed in the presence of ketoconazole (KTZ), a potent CYP3A inhibitor, supporting the hypothesis that NCOC may play a role in cocaine-induced nephrotoxicity. Cocaine-induced cytotoxicity was found to involve intracellular glutathione depletion at low concentrations and to induce mitochondrial damage at higher concentrations. Under the present experimental conditions, HPTECs death pathway followed an apoptotic pattern, which was evident for concentrations as low as 0.1mM.

The Ability of Bacterial Cocaine Esterase to Hydrolyze Cocaine Metabolites and Their Simultaneous Quantification Using High-Performance Liquid Chromatography-Tandem Mass Spectrometry

Cocaine toxicity is a widespread problem in the United States, responsible for more than 500,000 emergency department visits a year. There is currently no U.S. Food and Drug Administration-approved pharmacotherapy to directly treat cocaine toxicity. To this end, we have developed a mutant bacterial cocaine esterase (DM-CocE), which has been previously shown to rapidly hydrolyze cocaine into inert metabolites, preventing and reversing toxicity with limited immunogenic potential. Herein we describe the ability of DM-CocE to hydrolyze the active cocaine metabolites norcocaine and cocaethylene and its inability to hydrolyze benzoylecgonine. DM-CocE hydrolyzes norcocaine and cocaethylene with 58 and 45% of its catalytic efficiency for cocaine in vitro as measured by a spectrophotometric assay. We have developed a mass spectrometry method to simultaneously detect cocaine, benzoylecgonine, norcocaine, and ecgonine methyl ester to quantify the effect of DM-CocE on normal cocaine metabolism in vivo. DM-CocE administered to rats 10 min after a convulsant dose of cocaine alters the normal metabolism of cocaine, rapidly decreasing circulating levels of cocaine and norcocaine while increasing ecgonine methyl ester formation. Benzoylecgonine was not hydrolyzed in vivo, but circulating concentrations were reduced, suggesting that DM-CocE may bind and sequester this metabolite. These findings suggest that DM-CocE may reduce cocaine toxicity by eliminating active and toxic metabolites along with the parent cocaine molecule.

Toxicokinetics of cocaine and metabolites in a body-packer becoming symptomatic

Life-threatening complications may occur in body-packers and the rupture of a single packet containing cocaine may lead to fatality. We report the case of a 35-year-old body-packer who developed at the airport clinical signs of cocaine toxicity. There was evidence of bowel obstruction. The plasma concentration of cocaine, benzoylecgonine (BZE) and ecgonine methyl ester (EME) was determined 1 h after symptoms onset, during surgery and postoperative period. The measured peak value at 1 h was 594 ng/ml for cocaine, 9423 ng/ml for BZE and 3261 ng/ml for EME. We confirm the following order BZE > EME > cocaine for peak plasma concentrations. A rebound in plasma levels was found during surgery, together with electrocardiographic changes. A total of 107 packets were eliminated, and the patient survived.

P.1.c.060 (±)3,4-Methylenedioxyamphetamine inhibits a TEA-sensitive potassium current in the hippocampus

The detection and quantitation of drugs of abuse in blood is of growing interest in forensic and clinical toxicology. With the development of highly sensitive chromatographic methods, such as high-performance liquid chromatography (HPLC) with sensitive detectors and gas chromatography–mass spectrometry (GC–MS), more and more substances can be determined in blood. This review includes methods for the determination of the most commonly occurring illicit drugs and their metabolites, which are important for the assessment of drug abuse: Methamphetamine, amphetamine, 3,4-methylenedioxymethamphetamine (MDMA), N-ethyl-3,4-methylenedioxyamphetamine (MDEA), 3,4-methylenedioxy-amphetamine (MDA), cannabinoids (delta-9-tetrahydrocannabinol, 11-hydroxy-delta-9-tetrahydrocannabinol, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol), cocaine, benzoylecgonine, ecgonine methyl ester, cocaethylene and the opiates (heroin, 6-monoacetylmorphine, morphine, codeine and dihydrocodeine). A number of drugs/drug metabolites that are structurally close to these substances are included in the tables. Basic information about the biosample assayed, work-up, GC column or LC column and mobile phase, detection mode, reference data and validation data of each procedure is summarized in the tables. Examples of typical applications are presented.

Identification of cocaine and its metabolites in urban wastewater and comparison with the human excretion profile in urine

The most relevant human urinary metabolites of cocaine (nine metabolites) were measured in urban wastewater in Italy and USA. A novel analytical method based on liquid chromatography tandem mass spectrometry allowed the identification of ecgonine, ecgonine methyl ester and the pyrolytic derivatives of cocaine in untreated wastewater. The aim of this study was to verify whether the pattern of cocaine metabolites in wastewater reflected the human excretion profile in urine. The performance of the method was good, with recoveries higher than 60% and limits of quantifications in the low ng/L range. The stability in untreated wastewater was assessed for all metabolites and the best storage condition resulted freezing samples immediately after collection and keep them frozen until analysis. All the selected compounds were measured in wastewater at concentrations up to 1.5 μg/L and their weekly loads were calculated during a five weeks monitoring campaign in Milan (Italy). The profiles of cocaine metabolites in wastewater matched with those in human urine reported in the literature, suggesting that measures in wastewater reflect the real human excretion and that wastewater analysis is suitable for assessing drug consumption. Benzoylecgonine was confirmed as the best target for estimating cocaine use by wastewater analysis, while cocaine itself should not be considered because its amount in wastewater is affected by other environmental sources such as transport, handling and consumption. Results suggested that the measurement of other metabolites in combination with benzoylecgonine might reflect 60% of an administered dose of cocaine providing also information on different patterns of use.

Evaluation of the hydrolytic activity of a long-acting mutant bacterial cocaine in the presence of commonly co-administered drugs

Background Cocaine toxicity is a prevalent problem in the Unites States for which there is currently no FDA-approved pharmacotherapy. We have developed a bacterial cocaine esterase (CocE) towards this indication. A thermostabilized mutant of CocE (DM-CocE) retains the hydrolytic activity of the wild-type esterase, rapidly hydrolyzing cocaine into the inactive metabolites ecgonine methyl ester and benzoic acid, and can prevent cocaine toxicities in rodent and non-human primate models. To advance DM-CocE towards clinical use, we examine here how the hydrolytic activity of DM-CocE is altered by some drugs commonly co-administered with cocaine. Methods We employed a spectrophotometric cocaine hydrolysis assay to evaluate whether pharmacologically relevant doses of alcohol, nicotine, morphine, phencyclidine, ketamine, methamphetamine, naltrexone, naloxone, or midazolam would alter the Michaelis–Menten kinetics of DM-CocE for cocaine. Mass spectrometry was used to evaluate interaction with diazepam as this drug interferes with the absorbance spectra of cocaine critical for the spectrophotometric assay. Results Alcohol, nicotine, morphine, phencyclidine, ketamine, methamphetamine, naltrexone, naloxone, and midazolam did not alter cocaine hydrolysis by DM-CocE. However, diazepam significantly slowed DM-CocE cocaine hydrolysis at very high concentrations, most likely through interaction of the phenyl ring of the benzodiazepine with the active site of DM-CocE. Conclusions DM-CocE does not display significant drug interactions, with the exception of diazepam, which may warrant further study as DM-CocE progresses towards a clinically used pharmacotherapy for cocaine toxicity. Alternate benzodiazepines, e.g., midazolam could be used to avoid this potential interaction.

Prolonged Toxic Effects After Cocaine Challenge in Butyrylcholinesterase/Plasma Carboxylesterase Double Knockout Mice: A Model for Butyrylcholinesterase-Deficient Humans

Death and toxicity after cocaine use do not correlate with cocaine blood levels. One explanation for this observation is that cocaine abusers may posses one or more of the 58 possible known mutations in the butyrylcholinesterase gene (BCHE). Butyrylcholinesterase (BChE) serves as the primary cocaine hydrolase producing a nontoxic product ecgonine methyl ester. A reduction in endogenous levels of BChE may result in increased metabolism by hepatic carboxylesterase to produce norcocaine, a toxic product. Humans have carboxylesterase in tissues but not in plasma, whereas wild-type mice have significant amounts of carboxylesterase in tissues and plasma. Knockout mice with no plasma carboxylesterase were created to eliminate the contribution of plasma carboxylesterase in cocaine hydrolysis, thereby simulating human enzyme levels. This study tested the hypothesis that reductions in BChE such as those in humans with BChE mutations contribute to increased toxicity after cocaine use. Carboxylesterase and BChE double knockout mice, models for humans with BChE deficiency, were challenged with a nonlethal dose of 100 mg/kg (-)-cocaine. Carboxylesterase/BChE double knockout mice demonstrated toxic signs significantly longer than did wild-type and carboxylesterase knockout mice. The carboxylesterase/BChE-deficient mice took approximately 2.5 times as long to recover from cocaine toxicities, including the following: hypothermia, hyperactivity, stereotypical behavior, ocular effects, and dorsiflexion of the tail. The carboxylesterase/BChE double knockout mouse model demonstrates the importance of endogenous BChE for protection against cocaine toxicity and provides an in vivo system for studying drug sensitivity of humans who carry a BChE mutation.

Cocaine surface contamination and the medico-legal implications of its transfer

The question posed by this research involves how frequently one can expect to contact cocaine in day-to-day living experiences where drug use may not normally be suspected. Issues concerning contamination are germane to medico-legal investigators who evaluate the significance of drug test results in (1) questioned deaths, (2) public health concerns, (3) drugs crimes, and (4) drug use toxicological tests potentially caused by external contamination, such as hair, sweat, and skin swabs. Previous surface studies focus mainly on currency drug contamination; few have addressed other common surfaces.Public surfaces handled by a large number of people, such as building entrance door handles, bank currency dispensing machines, food store shopping carts, and service station fuel pumps within the New Haven, Connecticut metropolitan area were tested. Socio-economically, the distribution of items tested ranged from working-class to upper-middle class areas. Results were obtained using the Securetec Drugwipe II® which is an immunological, on-site test device. Precautions were taken to avoid carry-over and other potential contamination from handling including single-use latex gloves which were pre-tested as a negative control immediately before swabbing. Analysis was performed according to the manufacturer’s instructions and in the laboratory to standardize conditions. Drugwipe II® limit of detection (LOD) is ∼50ng.Test results revealed 78% positive for cocaine substances as follows: fuel pump buttons for credit card authorization, 100% positive (n=10 individual locations); ATM machines for currency withdrawal, 100% positive (n=10 individual locations); grocery store shopping carts, 70% positive; academic building entrance doors, 30% positive (n=10); and shopping mall entrance/exit doors, 100% positive (n=5).Forensic scientists and medico-legal investigators responsible for interpreting surface test results are cautioned to consider contamination before ascribing drug activity to a specific individual. For legal proceedings these results are indicative but not conclusive because immunological tests are considered presumptive and may react with the parent substance (cocaine), related compounds (benzoylecgonine, ecgonine methyl ester, ecgonine, nor-ecgonine, and cocaethylene) and immunologically similar materials.

Illicit drugs in alternative biological specimens: A case report

Postmortem tissues (e.g. liver, kidney) have been long used in forensic applications especially in those cases where blood is unavailable. The aim of this paper is to demonstrate the importance of the information provided to the forensic toxicologist at the time of carrying out the toxicological analysis, especially in cases where the samples commonly used in forensic toxicology are unavailable. This work describes the toxicological findings in a violent death resulting from a man who was hit by a train. Vitreous humor, liver and kidney were sent for toxicological analysis, once it was not possible to obtain blood and urine. The validated procedures used in the routine casework of Forensic Toxicology Laboratory of the Centre Branch of the National Institute of Legal Medicine, were applied in the analysis of liver, kidney and vitreous humor, using gas chromatography–mass spectrometry after solid-phase extraction and gas chromatography-flame ionization detector for the analysis of drugs of abuse and ethanol, respectively. Morphine, codeine, cocaine, benzoylecgonine and ecgonine methyl ester were found in the liver and in the kidney and no ethanol was found in the vitreous humor. The method validation included the study of specificity, selectivity, limits of detection, recovery and carryover. Although blood and urine are the most common and preferred matrices used for toxicological studies involving drugs of abuse, sometimes the choice of specimen is determined by the case under investigation. The forensic pathologist must be aware that relevant information must be provided so that the toxicological analysis can be conducted in accordance with case history, particularly when the only samples available for analysis are these “unconventional” specimens, since the interpretation of the obtained results is more difficult.

A one year investigation of the occurrence of illicit drugs in wastewater from Brussels, Belgium

Daily 24 hour composite influent wastewater samples were collected from the wastewater treatment plant of Brussels-North (Belgium) for eight months to study variations in the concentrations and mass loads of nine illicit drugs and metabolites: cocaine (COC) and its metabolites benzoylecgonine (BE) and ecgonine methyl ester (EME), amphetamine (AMP), methylenedioxymethamphetamine (MDMA), methamphetamine (METH), methadone (MTD) and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and the specific metabolite of heroin, 6-monoacetylmorphine (6-MAM). Samples were analyzed using a validated analytical method based on solid-phase extraction and hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry. The selected compounds could be detected and quantified in all samples. The measured concentrations were in agreement with earlier published concentrations, when available. All illicit drugs showed significant differences in mass loads between months. Daily variations were observed for MDMA, AMP and COC and metabolites, with higher mass loads observed during the weekend. This is probably related to their recreational consumption pattern and was further confirmed when holiday periods (New Year's Eve, Belgian national holiday) were in detail investigated. For METH, 6-MAM, MTD and EDDP, stable and consistent amounts were observed during the week. In all samples, the ratio of the concentration of the parent compound to its metabolite(s) was calculated to evaluate whether measured concentrations reflect human excretion of the illicit drugs. For MTD, the ratio of parent compound/metabolite was in agreement with the excretion pattern, while for COC some deviations were observed, resulting from excretion pattern uncertainties, and stability and discharge issues.

Simultaneous analysis of buprenorphine, methadone, cocaine, opiates and nicotine metabolites in sweat by liquid chromatography tandem mass spectrometry

A liquid chromatography tandem mass spectrometry method for buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine, ecgonine methyl ester (EME), morphine, codeine, 6-acetylmorphine, heroin, 6-acetylcodeine, cotinine, and trans-3'-hydroxycotinine quantification in sweat was developed and comprehensively validated. Sweat patches were mixed with 6 mL acetate buffer at pH 4.5, and supernatant extracted with Strata-XC-cartridges. Reverse-phase separation was achieved with a gradient mobile phase of 0.1% formic acid and acetonitrile in 15 min. Quantification was achieved by multiple reaction monitoring of two transitions per compound. The assay was a linear 1-1,000 ng/patch, except EME 5-1,000 ng/patch. Intra-, inter-day and total imprecision were <10.1%CV, analytical recovery 87.2-107.7%, extraction efficiency 35.3-160.9%, and process efficiency 25.5-91.7%. Ion suppression was detected for EME (-63.3%) and EDDP (-60.4%), and enhancement for NBUP (42.6%). Deuterated internal standards compensated for these effects. No carryover was detected, and all analytes were stable for 24 h at 22 °C, 72 h at 4 °C, and after three freeze/thaw cycles. The method was applied to weekly sweat patches from an opioid-dependent BUP-maintained pregnant woman; 75.0% of sweat patches were positive for BUP, 93.8% for cocaine, 37.5% for opiates, 6.3% for methadone and all for tobacco biomarkers. This method permits a fast and simultaneous quantification of 14 drugs and metabolites in sweat patches, with good selectivity and sensitivity.

Distribution of Concentrations of Cocaine and its Metabolites in Hair Collected Postmortem from Cases with Diverse Causes/Circumstances of Death

The concentrations and ratios of cocaine, benzoylecgonine (BE), and ecgoninemethylester (EME) in 360 head hair segments and 34 pubic hair samples collected at coroner's postmortem examinations were reviewed. The cases included diverse histories and causes/circumstances of death. The hair was analyzed using a validated method published previously; hair was shampoo washed, solvent washed, followed by extraction using 0.1 M hydrochloric acid and SPE clean-up, and quantitative analysis by gas chromatography-mass spectrometry in selected ion monitoring mode. A statistical evaluation demonstrated that, in head hair, the respective lower, middle, and upper concentration (ng/mg) ranges were < 10 ng total-0.8, > 0.8-18.9, and > 18.9-384.7 cocaine; < 10 ng total-0.6, > 0.6-7.9, and > 7.9-142.2 BE; and < 10 ng total-0.3, > 0.3-0.9, and > 0.9-39.5 EME. In pubic hair, the concentrations (ng/mg) detected were 0.2-236.2 cocaine, < 10 ng total-74.0 BE, and < 10 ng total-3.2 EME. The BE/cocaine ratio range in head hair was 0.01-43.00 (mean 1.39, median 0.28), and in pubic hair it was 0.31-2.67 (mean 0.59, median 0.31). The EME/cocaine ratio in head hair ranged from < 0.01 to 0.46 (mean 0.04, median 0.02), and in pubic hair, it ranged from < 0.01 to 0.32 (mean 0.07, median 0.04). Results reported as < 10 ng total were above the limit of detection and below the limit of quantitation (LOQ) (LOQ = 0.2 ng/mg for 50 mg of hair).

HPLC/MS Analysis of Historical Pharmaceutical Preparations of Heroin and Cocaine

Pharmaceutical preparations of heroin and cocaine more than seventy years old were analyzed using RP-HPLC. The composition of mobile phase was optimized. The components were identified by MS2 or MS3, and the APPI fragmentation mechanisms of compounds found were proposed. The sample of heroin hydrochloride injection solution consists of 96.1% morphine and 3.9% of codeine. The sample of cocaine hydrochloride injection solution consists of 26.9% cocaine, 31.5% benzoylecgonine, 17.4% ecgonine, and 24.2% ecgonine methyl ester.

Development and validation of a gas chromatography/ion trap-mass spectrometry method for simultaneous quantification of cocaine and its metabolites benzoylecgonine and norcocaine: Application to the study of cocaine metabolism in human primary cultured renal cells

Acute renal failure is a common finding in cocaine abusers. While cocaine metabolism may contribute to its nephrotoxic mechanisms, its pharmacokinetics in kidney cells is hitherto to be clarified. Primary cultures of human proximal tubular cells (HPTCs) provide a well-characterized in vitro model, phenotypically representative of HPTCs in vivo. Thus, the present work describes the first sensitive gas chromatography/ion trap-mass spectrometry (GC/IT-MS) method for measurement of cocaine and its metabolites benzoylecgonine (BE) and norcocaine (NCOC) using a primary culture of HPTCs as cellular matrix, following solid phase extraction (SPE) and derivatization with N-methyl- N-(trimethylsilyl)trifluoroacetamide (MSTFA). The application of this methodology also enables the identification of two other cocaine metabolites: ecgonine methyl ester (EME) and anhydroecgonine methyl ester (AEME). The validation of the method was performed through the evaluation of selectivity, linearity, precision and accuracy, limit of detection (LOD), and limit of quantification (LOQ). Its applicability was demonstrated through the quantification of cocaine, BE and NCOC in primary cultured HPTCs after incubation, at physiological conditions, with 1 mM cocaine for 72 h. The developed GC/IT-MS method was found to be linear ( r 2 > 0.99). The intra-day precision varied between 3.6% and 13.5% and the values of accuracy between 92.7% and 111.9%. The LOD values for cocaine, BE and NCOC were 0.97 ± 0.09, 0.40 ± 0.04 and 20.89 ± 1.81 ng/mL, respectively, and 3.24 ± 0.30, 1.34 ± 0.14 and 69.62 ± 6.05 ng/mL as LOQ values.

Pharmacokinetics of Cocaine and Metabolites in Human Oral Fluid and Correlation With Plasma Concentrations After Controlled Administration

Oral fluid is an attractive alternative matrix for drug testing with a noninvasive and directly observed collection, but there are few controlled cocaine administration studies to guide interpretation. While residing on a closed research unit for up to 10 weeks under constant medical supervision, 19 participants were administered 75 mg/70 kg subcutaneous cocaine and 14 received 150 mg/70 kg. The disposition of cocaine, benzoylecgonine (BE), and ecgonine methyl ester (EME) into oral fluid was determined by gas chromatography-mass spectrometry for 0.08 to 48 hours after administration. In oral fluid collected by citric acid candy-stimulated expectoration, cocaine first appeared in oral fluid 0.08 to 0.32 hours after dosing and was rapidly eliminated with half-lives of 1.1 to 3.8 hours. BE and EME were first detected 0.08 to 1.0 hours after dosing with longer half-lives of 3.4 to 13.8 (BE) and 2.4 to 15.5 hours (EME) (P < 0.05). Oral fluid and plasma concentrations were significantly correlated for cocaine, BE, and EME (P < 0.0001). There were no significant differences (P > 0.05) in first and last detection times with the 8-μg/L cutoff proposed by the Substance Abuse and Mental Health Services Administration or the 10-μg/L cutoff from the European initiative, Driving Under the Influence of Drugs, Alcohol and Medicines. Metabolite:cocaine ratios increased after cocaine administration, potentially helpful for interpreting time of last use. Comparison of oral fluid collection through citric acid candy-stimulated expectoration, citric acid-treated Salivette, and neutral cotton Salivette devices did not reveal significant differences between devices for areas under the curve for cocaine, BE, or EME (P > 0.05). These results provide additional evidence for interpreting cocaine and metabolite concentrations in oral fluid and oral fluid's usefulness as an alternative matrix for drug testing.

Determination of 17 illicit drugs in oral fluid using isotope dilution ultra-high performance liquid chromatography/tandem mass spectrometry with three atmospheric pressure ionizations

The collection of oral fluid for drug testing is easy and non-invasive. This study developed a drug testing method using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC–MS/MS) in selected-reaction monitoring (SRM) mode. We tested the method on the analysis of four opiates and their metabolites, five amphetamines, flunitrazepam and its two metabolites, and cocaine and its four metabolites in oral fluid. 100-μL samples of oral fluid were diluted with twice the amount of water then spiked with isotope-labeled internal standards. After the samples had undergone high-speed centrifugation for 20 min, we analyzed the supernatant. The recovery of the sample preparation ranged from 81 to 108%. We compared the performance of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). The ion suppression of most analytes on ESI (28–78%) was lower than that of APCI and APPI. A post-column flow split (5:1) did not reduce the matrix effect on ESI. Direct APPI performed better than dopant-assisted APPI using toluene. ESI, APCI and APPI limits of quantitation mostly ranged from 0.11 to 1.9 ng/mL, 0.02 to 2.2 ng/mL and 0.02 to 2.1 ng/mL, respectively, but were much higher on amphetamine and ecgonine methyl ester (about 2.7–4.7 ng/mL, 8.7–14 ng/mL, and 10–19 ng/mL, respectively). Most of the bias percentages (accuracy) and relative standard deviations (precision) on spiked samples were below 15%. This method greatly simplifies the process of sample preparation and shortens the chromatographic time to only 7.5 min per run and is able to detect analytes at sub-ppb levels.

Quantification of drugs of abuse in municipal wastewater via SPE and direct injection liquid chromatography mass spectrometry

We present an isotopic-dilution direct injection reversed-phase liquid chromatography-tandem mass spectrometry method for the simultaneous determination of 23 drugs of abuse, drug metabolites, and human-use markers in municipal wastewater. The method places particular emphasis on cocaine; it includes 11 of its metabolites to facilitate assessment of routes of administration and to enhance the accuracy of estimates of cocaine consumption. Four opioids (6-acetylmorphine, morphine, hydrocodone, and oxycodone) are also included, along with five phenylamine drugs (amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine, methylbenzodioxolyl-butanamine, and 3,4-methylenedioxy-N-ethylamphetamine) and two human-use markers (cotinine and creatinine). The method is sufficiently sensitive to directly quantify (without preconcentration) 18 analytes in wastewater at concentrations less than 50 ng/L. We also present a modified version of this method that incorporates solid-phase extraction to further enhance sensitivity. The method includes a confirmatory LC separation (selected by evaluating 13 unique chromatographic phases) that has been evaluated using National Institute of Standards and Technology Standard Reference Material 1511 Multi-Drugs of Abuse in Freeze-Dried Urine. Seven analytes (ecgonine methyl ester, ecgonine ethyl ester, anhydroecgonine methyl ester, m-hydroxybenzoylecgonine, p-hydroxybenzoyl-ecgonine, ecgonine, and anhydroecgonine) were detected for the first time in a wastewater sample.

Simultaneous determination of cocaine and its metabolite ecgonine methyl ester in human blood using microwave extraction-gas chromatography

A method was developed for the simultaneous determination of cocaine (COC) and its metabolite ecgonine methyl ester (EME) in human blood using gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID). The blood sample was prepared by microwave extraction (MWE). The optimal parameters of MWE were as follows: 6 mL of chloroform-isopropanol (9: 1, v/v) mixture as extraction solvent, the pH value of the sample was adjusted at 10.0 with 0.05 mol/L Na2CO3-NaHCO3 buffer, the extraction was performed at 40 degrees C for 6 min. The COC and EME in the extract were qualified using GC-MS and quantitated using GC-FID. The average recoveries of COC and EME were from 79.91% to 99.85%, the relative standard deviations were less than 3.10%, and the limits of detection (LOD) were 60 and 40 mg/L, respectively. In the method COC and EME were detected without derivatization. The method is rapid, accurate and sensitive, and can be used for the simultaneous determination of COC and EME in blood samples.

High-throughput simultaneous analysis of buprenorphine, methadone, cocaine, opiates, nicotine, and metabolites in oral fluid by liquid chromatography tandem mass spectrometry

A method for simultaneous determination of buprenorphine (BUP), norbuprenorphine (NBUP), methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), cocaine, benzoylecgonine (BE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), morphine, codeine, 6-acetylmorphine (6AM), heroin, 6-acetylcodeine (6AC), nicotine, cotinine, and trans-3'-hydroxycotinine (OH-cotinine) by liquid chromatography tandem mass spectrometry in oral fluid (OF) was developed and extensively validated. Acetonitrile (800 μL) and OF (250 μL) were added to a 96-well Isolute-PPT+protein precipitation plate. Reverse-phase separation was achieved in 16 min and quantification was performed by multiple reaction monitoring. The assay was linear from 0.5 or 1 to 500 μg/L. Intraday, interday, and total imprecision were less than 13% (n = 20), analytical recovery was 92-114% (n = 20), extraction efficiencies were more than 77% (n = 5), and process efficiencies were more than 45% (n = 5). Although ion suppression was detected for EME, cocaine, morphine, 6AC, and heroin (less than 56%) and enhancement was detected for BE and nicotine (less than 316%), deuterated internal standards compensated for these effects. The method was sensitive (limit of detection 0.2-0.8 μg/L) and specific (no interferences) except that 3-hydroxy-4-methoxyamphetamine interfered with AEME. No carryover was detected, and all analytes were stable for 24 h at 22 °C, for 72 h at 4 °C, and after three freeze-thaw cycles, except cocaine, 6AC, and heroin (22-97% loss). The method was applied to 41 OF specimens collected throughout pregnancy with a Salivette® OF collection device from an opioid-dependent BUP-maintained pregnant woman. BUP ranged from 0 to 7,400 μg/L, NBUP from 0 to 71 μg/L, methadone from 0 to 3 μg/L, nicotine from 32 to 5,020 μg/L, cotinine from 125 to 508 μg/L, OH-cotinine from 11 to 51 μg/L, cocaine from 0 to 419 μg/L, BE from 0 to 351 μg/L, EME from 0 to 286 μg/L, AEME from 0 to 7 μg/L, morphine from 0 to 22 μg/L, codeine from 0 to 1 μg/L, 6AM from 0 to 4 μg/L, and heroin from 0 to 2 μg/L. All specimens tested negative for EDDP and 6AC. This method permits a fast and simultaneous quantification of 16 drugs and metabolites in OF, with good selectivity and sensitivity.