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The purpose of this study was to compare results from five commercial hair testing laboratories conducting workplace drug testing with regard to bias, precision, selectivity and decontamination efficiency. Nine blind hair specimens, including cocaine-positive drug user specimens (some contaminated with methamphetamine) and negative specimens contaminated with cocaine, were submitted in up to five replicates to five different laboratories. All laboratories correctly identified cocaine in all specimens from drug users. For an undamaged hair specimen from a cocaine user, within-laboratory Coefficients of Variation (CVs) of 5-22% (median 8%) were reported, showing that it is possible to produce a homogenous proficiency testing sample from drug user hair. Larger CVs were reported for specimens composed of blended hair (up to 29%) and curly/damaged hair (19-67%). Quantitative results appeared to be method-dependent, and the reported cocaine concentrations varied up to 5-fold between the laboratories, making interlaboratory comparisons difficult. All laboratories reported at least one positive result in specimens contaminated with cocaine powder, followed by sweat and shampoo treatments. Benzoylecgonine, norcocaine, cocaethylene and hydroxylated cocaine metabolites were all detected in cocaine powder-contaminated specimens. This indicates that current industry standards for analyzing and reporting positive cocaine results are not completely effective at identifying external contamination. Metabolite ratios between meta- or para-hydroxy-cocaine and cocaine were 6- and 10-fold lower in contaminated specimens compared to those observed in cocaine user specimens, supporting their potential use in distinguishing samples positive due to contamination and drug use., (Published by Oxford University Press 2022. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

A highly discussed step in hair sample preparation for forensic analytics is the applied decontamination. The here presented investigations aim to gain insight and give recommendations on how to conduct this decontamination for the analysis of cocaine consumption in hair. Key insights were gained from the investigation of cocaine consumer hair, which was artificially contaminated in a humid atmosphere with 13C6 labelled cocaine and from cocaine powder contaminated hair. Several decontamination protocols were investigated, whereby the usage of a decontamination protocol consisting of multiple short repetitive washes allowed to visualize the wash out of (13C6-) cocaine. Multiple methanol washes proved to be an efficient and simple decontamination approach. Our findings showed that decontamination protocols can successfully wash out recent cocaine contaminations. They were observed to be rather quickly washed out, whereas cocaine from consumption or "older" cocaine contaminations were shown to eliminate both at a constant rate (from inner hair compartments). Thus, the usage of decontamination protocols to differentiate between consumption and contamination was shown to be limited. As contamination can happen any time at any level, only the application of elaborated decision trees, based on cocaine metabolite ratios and thresholds, can provide the distinction between consumption and contamination. Thus, the authors highly recommend the usage of such tools on all hair samples analyzed for cocaine consumption., (© The Author(s) 2020. Published by Oxford University Press.)

Given that external contamination must be considered in hair analysis, there is still a demand for reliable tools to differentiate between incorporation of drugs into the hair as a result of drug consumption and of the hair shaft by external contamination. With the aim of establishing alternative discrimination parameters, some of the hydroxy metabolites of cocaine i.e., para- and meta-hydroxycocaine and para- and meta-hydroxybenzoylecgonine were measured together with cocaine, benzoylecgonine, cocaethylene, and norcocaine in five seized street cocaine samples and in hair samples from different cohorts: cohort 1 (in vivo external contamination study, n = 28), cohort 2 (individuals with self-reported cocaine use, n = 92), and cohort 3 (individuals with suspected cocaine use or contamination, n = 198). Statistical evaluation of the data of cohort 1 and 2 using ROC curves yielded metabolic ratios indicating cocaine use. Based on these results, a decision workflow was established for the discrimination between cocaine use and external contamination. The power of this approach was finally statistically validated across the different cohorts., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

A reliable method based on high-performance liquid chromatography-tandem mass spectrometry has been developed for the assessment of coca alkaloids/metabolites [cocaine (COC), benzoylecgonine (BE), cocaethylene (CE), ecgonine methyl ester (EME), anhydroecgonine methyl ester (AEME), tropococaine (TRO), transcinnamoylcocaine (trCIN), cuscohygrine (CUS) and hygrine (HYG)] in oral fluid samples from cocaine abusers and from coca leaves consumers (coca leaves chewers and coca tea drinkers). Oral fluid samples were collected by the passive drool technique (spitting), and after centrifugation the supernatant was treated for protein removal by adding acidified acetonitrile. The developed method was fully validated according to the international criteria and good results have been obtained (intraday and inter-day precisions were lower than ±20%, intraday and inter day accuracy was within the 75-116% range, and LODs/LOQs was lower and close to cut-off values for COC and BE). The proposed method has been successfully applied to oral fluid samples from cocaine abusers, and also from coca leave chewers and coca tea drinkers. CUS and HYG were only found in oral fluid from people who chewed coca leaves and drank coca tea and were not detected in cocaine abusers. Both CUS and HYG could be good markers in oral fluid for distinguishing people who consume coca leaves legally (coca leave chewers and coca tea drinkers) from those who consume illegal cocaine., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In this study, a quantitative polarity switching liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS) method was developed to detect and quantify cocaine and metabolites (cocaethylene, benzoylecgonine and meta-hydroxybenzoylecgonine), phencyclidine (PCP) and barbiturates (phenobarbital and butalbital) in meconium. Accuracy and precision samples at 0.0125% and 75% of the upper limit of quantitation (ULOQ) were analyzed in triplicate over 5 days with accuracy above 84% and average %CV values below 11%. Within-run (n = 15) and between-run (n = 15) %CV values were ≤5%. Analytical measurement ranges were reproducible and linear (R ≥ 0.995) for cocaine and metabolites (20-2,000 ng/g), PCP (10-1,000 ng/g) and barbiturates (50-5,000 ng/g). Accuracy of 100 ± 20% was observed at (the limits of detection) 10 ng/g for cocaine and metabolites, 2.5 ng/g for PCP and 25 ng/g for barbiturates. No carryover was observed at 2X ULOQ and no interfering substances were identified. Sample preparation recoveries were 53-83%. Fifty-one authentic patient samples previously characterized correlated with the newly developed test having R2 values ≥0.996. This combined method allows accurate quantitation of the targeted drugs in a complex matrix while decreasing sample preparation and analysis time with reduced sample volume. Clinical data and positivity rates were similar to previously published positivity rates. Validation data and positivity rate agreement signifies a reliable and robust assay.

Samples from a self-proclaimed cocaine (COC) user, from 19 drug users (postmortem) and from 27 drug chemists were extensively washed and analyzed for COC, benzoylecgonine, norcocaine (NC), cocaethylene (CE) and aryl hydroxycocaines by liquid chromatography-tandem mass spectrometry. Published wash criteria and cutoffs were applied to the results. Additionally, the data were used to formulate new reporting criteria and interpretation guidelines for forensic casework. Applying the wash and reporting criteria, hair that was externally contaminated with COC was distinguished from hair collected from individuals known to have consumed COC. In addition, CE, NC and hydroxycocaine metabolites were only present in COC users' hair and not in drug chemists' hair. When properly applied, the use of an extended wash, along with the reporting criteria defined here, will exclude false-positive results from environmental contact with COC., (Published by Oxford University Press 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

The United States Public Health Service Substance Abuse and Mental Health Services Administration is alerting medical professionals that a substantial percentage of cocaine imported into the United States is adulterated with levamisole, a veterinary pharmaceutical that can cause blood cell disorders such as severe neutropenia and agranulocytosis. Levamisole HCl is the active ingredient in a number of veterinary drugs approved to treat worm infestations in animals. Levamisole HCl was also the active ingredient in a human drug for oral administration approved on June 18, 1990, as adjuvant treatment in combination with fluorouracil after surgical resection in patients with Duke's stage C colon cancer. This drug was withdrawn from the U.S. market around 2000, and it has not been marketed in the U.S. since then. The objective of this study was to develop a method to determine the amount of levamisole in urine samples. The procedure will be provided to state health laboratories as needed to be used in the evaluation of patients that have developed neutropenia or agranulocytosis in the setting of recent cocaine use. A gas chromatography-mass spectrometry method was validated and tested at two different laboratories, and the method limit of detection for levamisole is 1 ng/mL in urine when using a 5-mL sample. Confirmation of the stereoisomer of levamisole was done by high-performance liquid chromatography using a chiral column.

Opiates, cocaine, and metabolites were quantified by liquid chromatography-mass spectrometry (LC-MS) in 284 urine specimens, collected thrice weekly, to monitor possible drug relapse in 15 pregnant heroin-dependent women. Opiates were detected in 149 urine specimens (52%) with limits of quantification (LOQ) of 10-50 microg/L. Morphine, morphine-3-glucuronide, and/or morphine-6-glucuronide were positive in 121 specimens; 6-acetylmorphine, a biomarker of heroin ingestion, was quantifiable in only 7. No heroin, 6-acetylcodeine, papaverine, or noscapine were detected. One hundred and sixty-five urine specimens (58%) from all 15 participants were positive for one or more cocaine analytes (LOQ 10-100 microg/L). Ecgonine methylester (EME) and/or benzoylecgonine were the major cocaine biomarkers in 142. Anhydroecgonine methylester, a biomarker of smoked cocaine, was positive in six; cocaethylene and/or ecgonine ethylester, biomarkers of cocaine and ethanol co-ingestion, were found in 25. At the current Substance Abuse Mental Health Services Administration cutoffs for total morphine (2000 microg/L), codeine (2000 microg/L), 6-acetylmorphine (10 microg/L), and benzoylecgonine (100 microg/L), 16 opiate- and 29 cocaine-positive specimens were identified. Considering 100 microg/L EME as an additional urinary cocaine biomarker would identify 51 more positive cocaine specimens. Of interest is the differential pattern of opiate and cocaine biomarkers observed after LC-MS as compared to gas chromatography-mass spectrometry analysis.

Cocaine (COC) is a potent CNS stimulant that is metabolized to benzoylecgonine (BE) and further metabolized to minor metabolites such as m-hydroxybenzoylecgonine (m-HOBE). COC is also metabolized to norcocaine (NC). Cocaethylene (CE) is formed when cocaine and ethyl alcohol are used simultaneously. Anhydroecgonine methyl ester (AEME) is a unique marker following smoked cocaine, and anhydroecgonine ethyl ester (AEEE) is found in cocaine smokers who also use ethyl alcohol. We developed a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection and quantitation of COC, BE, NC, CE, m-HOBE, AEME, and AEEE in urine. Two hundred samples previously analyzed by gas chromatography (GC) coupled with MS were extracted using solid-phase extraction. Chromatographic separation was achieved using a gradient consisting of mobile phase A [20 mM ammonium formate (pH 2.7)] and mobile phase B (methanol/acetonitrile, 50:50), an XDB-C(8) (50 x 2.1 mm, 1.8 microm) column and a flow rate of 270 microL/min. Concentrations were calculated by comparing the peak-area with the internal standard and plotted against a standard curve. The assay displayed linearity from 1.0 to 100 ng/mL. Within- and between-run coefficients of variation were < 10% throughout the linear range. A method comparison between GC-MS and LC-MS-MS showed good correlation for COC (r(2) = 0.982) and BE (r(2) = 0.955). We report here on a sensitive method to identify clinically and forensically relevant cocaine and associated analytes at concentrations as low as 1.0 ng/mL.

Excessive fluid intake can substantially dilute urinary drug concentrations and result in false-negative reports for drug users. Methods for correction ("normalization") of drug/metabolite concentrations in urine have been utilized by anti-doping laboratories, pain monitoring programs, and in environmental monitoring programs to compensate for excessive hydration, but such procedures have not been used routinely in workplace, legal, and treatment settings. We evaluated two drug normalization procedures based on specific gravity and creatinine. These corrections were applied to urine specimens collected from three distinct groups (pain patients, heroin users, and marijuana/ cocaine users). Each group was unique in characteristics, study design, and dosing conditions. The results of the two normalization procedures were highly correlated (r=0.94; range, 0.78-0.99). Increases in percent positives by specific gravity and creatinine normalization were small (0.3% and -1.0%, respectively) for heroin users (normally hydrated subjects), modest (4.2-9.8%) for pain patients (unknown hydration state), and substantial (2- to 38-fold increases) for marijuana/cocaine users (excessively hydrated subjects). Despite some limitations, these normalization procedures provide alternative means of dealing with highly dilute, dilute, and concentrated urine specimens. Drug/metabolite concentration normalization by these procedures is recommended for urine testing programs, especially as a means of coping with dilute specimens.

A novel fast gas chromatography-mass spectrometry (FGC-MS) analytical method for benzoylecgonine (BZE) has been developed to improve the efficiency of specimen analysis without diminishing the reliability of metabolite identification and quantification. Urine specimens were spiked with deuterated internal standard (BZE-d8), subjected to solid-phase extraction, and derivatized with pentafluoropropionic anhydride (PFPA) and pentafluoropropanol (PFPOH). The pentafluoropropyl ester derivative of BZE was identified and quantified using both a standard GC-MS method and the newly developed FGC-MS method. Shorter GC analyte retention times were made possible in the FGC-MS method by employing a 220-volt GC oven controller, which allowed an increased temperature ramp rate. The FGC-MS method was linear between 25 and 10,000 ng/mL of BZE yielding a correlation coefficient of 0.9994. The intra-assay precision of a 100 ng/mL BZE standard (n=15) yielded an average concentration of 99.7 ng/mL and a coefficient of variation of 1.2%. The interassay precision of 21 sets of 50, 100, and 125 ng/mL BZE controls was found to be acceptable, with coefficients of variation less than 2.4%. No interference was observed when the FGC-MS method was challenged with cocaine, ecgonine, ecgonine methyl ester, and nine other drugs of abuse. Analysis of presumptively positive specimens (n=146) by both analytical methods yielded comparable results with a correlation coefficient of 0.996. The FGC-MS method, when compared with a standard GC-MS method, reduces total assay time by approximately 50% while demonstrating comparable reliability.

The National Laboratory Certification Program undertook an evaluation of the dynamics of external contamination of hair with cocaine (COC) while developing performance testing materials for Federal Drug-Free Workplace Programs. This characterization was necessary to develop performance materials that could evaluate the efficacy of hair testing industry's decontamination procedures. Hair locks (blonde to dark brown/black) from five different individuals were contaminated with cocaine HCl. Hair locks were then treated with a synthetic sweat solution and hygienic treatments to model real-life conditions. Hair locks were shampooed daily (Monday through Friday) for 10 weeks, and samples of the hair locks were analyzed for COC, benzoylecgonine (BE), cocaethylene (CE), and norcocaine (NCOC). Three commercial analytical laboratories analyzed samples under three protocols: no decontamination procedure, individual laboratory decontamination, or decontamination by an extended buffer procedure at RTI International. Results indicated substantial and persistent association of all four compounds with all hair types. Hair that was not decontaminated had significantly greater quantities of COC and BE than did hair that was decontaminated. The only hair samples below detection limits for all four compounds were those decontaminated 1 h after contamination. Additionally, BE/COC ratios increased significantly over the 10-week study (regardless of decontamination treatment). From 21 days postcontamination until the end of the study, the mean BE/COC ratio for all hair types exceeded 0.05, the proposed Federal Mandatory Guidelines requirement. The largest variability in results was observed for samples decontaminated by participant laboratories. This suggests that current laboratory decontamination strategies will increase variability of performance testing sample results. None of the decontamination strategies used in the study were effective at removing all contamination, and some of the contaminated hair in this study would have been reported as positive for cocaine use based on the proposed Federal Mandatory Guidelines.

It has been proposed that issues of external contamination of hair can be resolved by the presence of metabolites. This is true for some drugs in some circumstances. However, even if some metabolites such as cocaethylene are sufficiently definitive to indicate certainty of use, others such as benzoylecgonine can occur as contaminants. In addition, for phencyclidine, only the parent compound is determined. Therefore, effective washing procedures are needed to rule out contamination as the source of the parent or metabolite. The protection provided by cutoffs is also dependent on washing to remove surface contamination, which can range from very little to greater than 20 times the amount in the hair. In cocaine-positive head hair samples from 67 subjects with cocaine-positive urine tests, contamination of positive hair samples ranged from 0.7 to 8009 ng/10 mg hair, demonstrating that analyses of such samples to determine use is meaningless without removal of contamination. Wash procedures have been a focus of this laboratory for many years. In this paper, the effectiveness of our wash procedure was further challenged by application to negative hair (blonde, auburn, brown, and black) contaminated by soaking for 1 h in 1000, 10,000, and 50,000 ng cocaine/mL water. The uptake of the cocaine was approximately linear with increasing concentrations of cocaine in the soaking solutions. By application of the wash criterion, all samples were clearly identified as contaminated (i.e., negative). The effects of hair porosity on uptake of cocaine were also studied with 10 hair samples of all colors. Permed and unpermed samples were soaked in 10,000 ng cocaine/mL for 1 hour. All hair responded to the perm with increased uptake, and all were decontaminated or identified as contaminated (i.e., negative for cocaine use). Porosity, not hair color, determined the rate of penetration of hair by cocaine in solution, and, likewise, washing characteristics were unrelated to hair color.

A quantitative analysis was developed for the determination of cocaine, benzoylecgonine, and cocaethylene in oral fluid using liquid chromatography-tandem mass spectrometry. After internal standardization and solid-phase extraction, chromatographic separation was achieved on a reversed-phase column by gradient elution. The reconstructed mass chromatograms of the collision-induced dissociation transitions of m/z 290 --> m/z 168 (benzoylecgonine), m/z 304 --> m/z 168+119 (2'-methylbenzoylecgonine), m/z 304 --> m/z 182 (cocaine), m/z 318 --> m/z 196 (cocaethylene), and m/z 318 --> m/z 182+119 (2'-methylcocaine) were used for quantitation. The developed method was adequately validated. Good linearity was obtained from 10 to 1000 microg/L. Extraction recoveries exceeded 85% for all compounds. Excellent total and within-run reproducibilities (CV% < 20%) and accuracy figures were obtained. The limit of detection (signal-to-noise ratio >/= 3) was 1 microg/L for all three compounds. As such, a method for drug abuse confirmation analysis in oral fluid, compatible with the present day saliva collecting devices, is obtained. The method was applied to real samples (n = 15) obtained from suspected drug users, of which seven proved positive. The concentrations found in the positive samples were between 10.2 and 200.6 microg/L for cocaine, < limit of quantification (LOQ) and 10.5 microg/L for cocaethylene, and < LOQ and 59.2 microg/L for benzoylecgonine.

The purpose of these studies was to determine the performance characteristics of the Cozart microplate EIA for the detection of cocaine and cocaine metabolites in oral fluid. A total of 217 samples were collected using the Cozart RapiScan oral fluid Collection System from donors who were receiving treatment and being monitored for their addiction. The samples were screened in the laboratory using the Cozart microplate EIA for cocaine and metabolites and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. Of the 217 samples tested, 116 were positive for cocaine and/or cocaine metabolites. The Cozart microplate EIA for cocaine, using a cutoff of 30 ng/mL benzoylecgonine equivalents in neat oral fluid, had a sensitivity of 95.7% +/- 2.0% and specificity of 100% +/- 0.7%. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Sweat collection patches are used for drug abuse monitoring. We investigated the effect of sweat patch location (back and shoulder) on cocaine levels after controlled intravenous cocaine exposure (210 mg/70 kg) in 12 subjects (Experiment 1). Gas chromatographic-mass spectrometric analyses show cocaine and metabolites levels in Pharmchek trade mark patches were eightfold higher on the back than those on the shoulders. To assess the mechanisms for possible loss of cocaine from patches during wear, 48 sweat patches with a small amount of cocaine-d(5) (100 ng as base/patch) were placed on the backs of eight cocaine-naive volunteers for up to 72 h (Experiment 2). Drug-free patches were applied over eight of the cocaine-d(5) (100 ng) containing patches to measure loss through the patch. Cocaine levels in spiked patches declined over time (p = 0.002), with levels at 48 h postapplication 30% less than control, consistent with possible drug reabsorption. Cocaine was detectable (> 2 ng/patch, LOQ) in four of eight initially cocaine-free patches placed on top of the cocaine-containing patches, indicating transfer through the patch outer membrane. Conversion to benzoylecgonine was detectable but at low levels (< 2%). Reabsorption (back transfer), degradation or hydrolysis, and loss of cocaine to the environment may account for substantial loss of cocaine from skin sweat collection patches during patch wear.

This study reports the results of the detection and quantitation of cocaine and its metabolites in liver tissues fixed in formalin and in the formalin solutions in which the same tissues were fixed. Toxicological analyses were performed on formalin-fixed liver samples from four cases of death of cocaine abusers and on formalin solutions (10% buffered, pH 7) in which the samples were preserved. Analyses carried out at the time of autopsy on body fluids and tissues allowed identification of cocaine and the metabolite benzoylecgonine. Liver tissue samples were preserved in formalin solutions for four weeks before analysis. Results only showed the presence of benzoylecgonine in the studied materials. The mean levels of recovery of benzoylecgonine in fixed tissues were 12.31% in liver and 84.47% in formalin from liver. Results indicated that benzoylecgonine has good stability, even in biological specimens subjected to chemical fixation.

The object of this study was to evaluate the suitability of the Neogen Corporation microtiter plate enzyme-linked immunoassay (ELISA) for cocaine and metabolites for screening of postmortem blood. Sixty-five postmortem whole blood specimens were obtained from drug-involved deaths that had been screened and confirmed positive for cocaine and/or benzoylecgonine (BE). Fifty-eight negative specimens were obtained from noncocaine-involved deaths. Specimens were tested using the Neogen Cocaine/BE microtiter plate ELISA assay. No matrix effects were found for whole blood in this assay. The effect of dilutions of the whole blood specimens of 1:5 through 1:50 was studied. A dilution of 1:5 was chosen to correspond to that used for other Neogen microtiter plate assays for drugs in whole blood. True positives, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), GC-nitrogen-phosphorus detection, and case histories. From these graphs and the receiver operating characteristic curves, the optimal cutoff for the Neogen Cocaine/BE ELISA was found to be 5 ng/mL BE equivalents at a 1:5 dilution. The optimum cutoff for a 1:50 dilution was 50 ng/mL BE equivalents. The Neogen Cocaine/BE ELISA had a sensitivity of 93.8% +/- 2.9% and a specificity of 96.6% +/- 2.4% versus GC-MS at a cutoff of 5 ng/mL BE equivalents (1:5 dilution) and a sensitivity of 100% +/- 0.5% and specificity of 98.3% +/- 1.7% versus GC-MS at a 50 ng/mL BE equivalents cutoff (1:50 dilution).

Oral fluid has become a widely accepted alternative matrix for drugs of abuse detection. Immunoassays have been developed for on-site testing of cocaine and metabolites in oral fluid. The performance of the Cozart RapiScan Oral Fluid Drug Testing System (CRS) was evaluated in comparison with Cozart Microplate Enzyme Immunoassay Cocaine Oral Fluid Kit (COC ELISA) and gas chromatography-mass spectrometry (GC-MS) at several screening and confirmation cutoffs, including those proposed by SAMHSA and those currently in use in the U.K. Oral fluid samples (n = 1271) were collected prior to and following controlled clinical cocaine administration. CRS provides a qualitative screen at a preset cutoff of 30 microg/L. Sensitivity, specificity, and efficiency for CRS (30 microg/L) as compared with COC ELISA with a cutoff of 30 microg/L were 92.1%, 91.8%, and 92.0%. The comparison of CRS (30 microg/L) with the 8-mg/L proposed SAMHSA confirmation cutoffs for cocaine and/or benzoylecgonine exhibited a sensitivity of 82.7%, a specificity of 94.5%, and an efficiency of 87.6%. For this study, an alternative CRS cutoff of 20 microg/L was also evaluated. Performance characteristics of CRS (20 microg/L) at the proposed SAMHSA confirmation cutoffs were 89.9%, 89.7%, and 89.8%, respectively. At cutoffs in use in the U.K., 30- micro g/L CRS screen and 15- microg/L GC-MS cutoffs for cocaine, benzoylecgonine, and/or ecgonine methyl ester sensitivity, specificity, and efficiency were 89.4%, 92.2%, and 90.7%, respectively. Cozart RapiScan had performance similar to the COC ELISA assay for the detection of cocaine exposure and suitable sensitivity and specificity at the proposed SAMHSA cutoffs.

In order to evaluate the effectiveness of a wash procedure using isopropanol followed by multiple extended phosphate buffer washes as compared with a methanol wash procedure previously reported, a contamination experiment was designed involving the soaking of human head hair in cocaine-contaminated aqueous solutions. Fourteen negative human head hair samples were soaked in a solution of cocaine HCl (1000 ng/mL) at room temperature for 1 h, then rinsed with distilled water and dried at room temperature. Using the extensive wash procedures (15-min isopropanol wash, followed by three 30-min phosphate buffer washes and then two 60-min washes), in no case would any of the samples be reported out as positive at a cut-off of 5 ng cocaine/ 10 mg hair. With the methanol procedure, 8 of the 14 methanol-washed samples exceeded a cut-off of 5 ng/10 mg hair. Extensive washing was shown to be far more effective for removal of external contamination than the methanol wash procedure reported. In all cases, the extensive aqueous wash protocol would allow the differentiation of ingestion versus external contamination, as defined by this soaking experiment. All samples underwent solid-phase extraction and derivatization followed by liquid chromatographic-tandem mass spectrometric analysis. Analysis was performed on a triple quadrupole API 2000 PerkinElmer Sciex mass spectrometer (MS) equipped with an atmospheric pressure ionization source via an ion spray interface. The MS operated in the positive Cl multiple reaction mode.

A novel extraction and derivatization procedure for the cocaine metabolite benzoylecgonine (BZE) was developed and evaluated for use in a high-volume forensic urine analysis laboratory. Extractions utilized a Speedisk 48 positive pressure extraction manifold and polymer-based cation-exchange extraction columns. Samples were derivatized by the addition of pentafluoropropionic anhydride and pentafluoropropanol. All analyses were performed in selected ion monitoring mode; ions included m/z 421, 300, 272, 429, and 303 with m/z 421 to 429 ratio used for quantitation. The average extraction efficiency was 80%. Seventy-five common over-the-counter products, including prescription drugs, drug metabolites, and other drugs of abuse, demonstrated no significant interference with respect to chromatography or quantitation. The limit of detection and limit of quantitation were calculated at 12.5 ng/mL, and the assay was linear from 12.5 to 20,000 ng/mL with an r2 of 0.99932. A series of 20 precision samples (100 ng/mL) produced an average response of 97.8 ng/mL and a percent coefficient of variation of 4.1%. A set of 79 archived human urine samples that had previously been found to contain BZE were analyzed by 3 separate laboratories. The results did not differ significantly from prior quantitation or between laboratories. The Speedisk has proven viable for a high-volume production facility reducing overall cost of analysis by decreasing analysis time and minimizing waste production while meeting strict forensic requirements.

The testing for drugs of abuse in hair is increasingly used to detect illicit substances. Laboratories have implemented various decontamination, or washing, procedures in order to eliminate concerns regarding potential contamination of the hair with drug from the environment. However, the effect of these decontamination procedures on drug incorporated into the hair shaft via systemic exposure is unknown. This study evaluated the effect of four simple laboratory wash procedures on the quantitative measurement of cocaine and its metabolites in hair from rats administered cocaine by intraperitoneal injection. Washes included (1) methanol only; (2) 0.1 M phosphate buffer, pH 6.0; (3) 0.1 M phosphate buffer, pH 8.0; and (4) isopropanol and phosphate buffer, pH 5.5. Cocaine and its major metabolites, benzoylecgonine, norcocaine, ecgonine methyl ester, and cocaethylene, were analyzed using high-performance liquid chromatography coupled to atmospheric pressure electrospray ionization mass spectrometry. All four washes resulted in significant differences from unwashed hair controls (p < or = 0.05) for some or all of the detectable analytes. Because different wash procedures lead to significant differences in the measured concentrations of analytes in hair known to contain drug, quantitative data must be interpreted cautiously based on the wash procedures employed.

The usual specimens submitted by a medical examiner for toxicological analysis include blood, urine, bile, vitreous humor, stomach contents, and solid-organ tissue. The detection of drugs in these specimens typically involves a combination of techniques including colorimetry, immunoassay, and gas chromatography. Although many laboratories rely principally on urine for the detection of drugs of abuse by immunoassay, these assays may be applied to other specimen types. An evaluation of Microgenics Corporation's cloned enzyme donor immunoassay (CEDIA) was conducted in order to evaluate its use in the detection of cocaine/cocaine metabolites in vitreous humor specimens. During a 14-month period, 392 vitreous humor specimens were analyzed by the CEDIA DAU Cocaine assay. Instrument parameters were set according to published manufacturer's guidelines. All presumptive positive immunoassay results prompted confirmatory testing and quantitation by gas chromatography-mass spectrometry (GC-MS) of other specimens including blood. Vitreous humor specimens were not tested by GC-MS. Using a approximately 100-ng/mL cutoff, the CEDIA assay produced 23 presumptive positive results, 22 of which were confirmed by GC-MS. The only specimen which could not be confirmed, elicited an immunoassay screen value near the cutoff limit. Routine analysis of blood, urine, bile, and/or bladder wash specimens by gas chromatography-nitrogen phosphorus detection revealed the presence of cocaine/cocaine metabolites in only 7 (31.8%) of the 22 confirmed cases. The concentration ranges of cocaine and benzoylecgonine in the blood specimens were none detected to 337 ng/mL and 17 to 8598 ng/mL, respectively. Cocaethylene was not detected in these cases. Analysis of vitreous humor specimens by CEDIA improved the detection rate of cocaine/cocaine metabolites by 0.7% in the cases submitted to our laboratory during the 14-month period.

In order to support studies on various medication protocols for the treatment of cocaine abuse, an accurate, precise, and sensitive (2.5 to 750 ng/mL) liquid chromatography-tandem mass spectrometry assay was developed to determine cocaine and benzoylecgonine in human plasma. Cocaine-d3 and benzoylecgonine-d3 were added as internal standards and samples were subjected to solid-phase extraction. Cocaine recovery was 94.4% and benzoylecgonine was 80.3% at 2.5 ng/mL. The selected reaction monitoring of parent ions at m/z 304 and 290 resulted in strong fragments at m/z 182 and 168 for cocaine and benzoylecgonine, respectively. The method was fully validated. The mean measured concentration at the 2.5 ng/mL, the lower limit of quantitation, was within 10.8% of the target and the precision determined at the low (5 ng/mL), medium (50 ng/mL), and high (650 ng/mL) quality controls ranged from 0.9 to 6.2 %CV. Cocaine and benzoylecgonine concentrations in plasma treated with 1% NaF showed changes of less than 10% when maintained at room temperature for up to 7 h and no significant changes when subjected to three freeze-thaw cycles. The concentrations of cocaine and benzoylecgonine remained stable in plasma samples stored at -20 degrees C for up to 11 months. Methanolic stock solutions of both analytes are stable, staying within 2% of the freshly prepared stock solutions, when stored at -20 degrees C for up to 235 days. Both extracted analytes reconstituted in methanolic solutions are stable for up to seven days whether stored at -20 degrees C or at room temperature on the autosampler. The method is rugged, rapid, and robust and has been applied to the batch analysis of more than 700 samples during pharmacokinetic profiling to assess potential interactions between intravenous (i.v.) cocaine challenge and treament medications. Results from three of these subjects receiving 40 mg (i.v.) cocaine demonstrate the utility of the method.

Benzoylecgonine (BZE) extraction from urine was explored using Cerex Polycrom Clin II solid-phase extraction (SPE) columns and the Speedisk 48 Pressure Processor as an alternative to the Prep1 automated sample processor and XTRX Type RP/W columns. Linearity for urine standards extracted using the Cerex-Speedisk method ranged from 20 to 3000 ng/mL. The mean recovery at the 100-ng/mL cutoff for three lots of columns was 92%. The mean of the within-run means for three batches, which had coefficients of variations of 1.8% or less, was 101.3 ng/mL at the 100-ng/mL cutoff level. Forty-six specimens known to contain BZE were analyzed by both the Prep1-Type RP/W and Cerex-Speedisk methods. The correlation for specimen BZE concentrations between the two methods gave an r2 of 0.9999 and a slope of 1.03. The Cerex-Speedisk system is an inexpensive alternative to the Prep1-Type RP/W system. It is less costly, requires little maintenance, has a small footprint, is hood compatible, and can process four times the number of specimens in a given time.

Heroin, with a plasma half-life of approximately 5 min, is rapidly metabolized to 6-acetylmorphine (6-AM). 6-AM, a specific marker for heroin use, which also has a short half-life of only 0.6 h, is detected in urine for only a few hours after heroin exposure. Ingestion of poppy seeds and/or licit opiate analgesics can produce positive urine opiate tests. This has complicated the interpretation of positive opiate results and contributed to the decision to raise opiate cutoff concentrations and to require 6-AM confirmation in federally mandated workplace drug-testing programs. Microgenics Corp. has developed the CEDIA 6-AM assay, a homogeneous enzyme immunoassay for semiquantitative determination of 6-AM in human urine, in addition to its CEDIA DAU opiate assay. Urine specimens were collected 3 times per week from 27 participants enrolled in a clinical research trial evaluating a contingency management treatment program for heroin and cocaine abuse. Of the 1377 urine specimens screened, 261 (18.9%) were positive for opiates at > or = 300 ng/mL, 153 (11.1%) were positive for opiates at > or = 2000 ng/mL, and 55 (4.0%) were positive for 6-AM at > or = 10 ng/mL. For opiate-positive screens > or = 300 and > or = 2000 ng/mL, 91.3% and 80.8% confirmed positive for morphine or codeine at the respective gas chromatography-mass spectrometry (GC-MS) cutoffs. All specimens screening positive for 6-AM also confirmed positive by GC-MS at > or = 10 ng/mL. Increasing the opiate screening and confirmation cutoffs for the federal workplace drug-testing program resulted in 8% fewer opiate-positive tests; however, recent heroin use was not affected by this change.

In this retrospective study, we examined the levels of cocaine and its major metabolites in plasma and urine from 29 randomly selected emergency department patients (19 males and 10 females, aged 19 to 55) whose urine screened positive for benzoylecgonine using fluorescence polarization immunoassay. Levels of cocaine along with benzoylecgonine, ecgonine methyl ester, and norcocaine were quantitated in EDTA plasma and urine from each patient using gas chromatography-mass spectrometry with selected ion monitoring. Admission diagnosis and history were also obtained for each patient. In plasma, the levels were 16-130 ng/mL for cocaine (n = 3), 27-96 ng/mL for ecgonine methyl ester (n = 9), and 18-1390 ng/mL for benzoylecgonine (n = 22). Norcocaine was not detected in any of the plasma samples. In urine, the concentration ranges were 4-40,130 ng/mL for cocaine (n = 23), 36-660,500 ng/mL for ecgonine methyl ester (n = 27), and 9-2520 ng/mL for norcocaine (n = 9). All urine samples were positive for benzoylecgonine (106-3,361,000 ng/mL), and benzoylecgonine was the only metabolite present in two urine samples (at concentrations of 407 and 435 ng/mL). Two patients had plasma and urine samples positive for all analytes (except norcocaine in plasma). The patient with the highest urinary concentrations of cocaine (40,130 ng/mL), ecgonine methyl ester (660,500 ng/mL), benzoylecgonine (3,361,000 ng/mL), and norcocaine (2520 ng/mL) had a small quantity of benzoylecgonine (465 ng/mL) in plasma. No correlation was noted with patient history, admitting diagnosis or symptomatology, or plasma/urine levels of cocaine or any of its metabolites.

A total of 30 human head-hair samples were analyzed for cocaine (COC), cocaethylene (CE), benzoylecgonine (BE), methylecgonine (EME), and norcocaine (NCOC) using a sensitive positive ion chemical ionization gas chromatography-tandem mass spectrometry (GC-MS-MS) method. All 30 hair samples had been previously submitted to the laboratory and had confirmed positive for cocaine. Hair samples (20 mg each) were cut into small segments (2-5 mm) and incubated overnight at 45 degrees C in 0.1 N HCl after the addition of 50 microL of an internal standard mix of COC-d3 (1.0 ng/mg), BE-d3 (0.5 ng/mg), EME-d3 (0.25 ng/mg), and NCOC-d3 (0.25 ng/mg). The samples were then extracted with Clean Screen extraction columns from United Chemical Technologies, Inc. The final extract was evaporated to dryness and derivatized with 50 microL of 1,1,1,3,3,3-hexafluoro-2-propanol and 50 microL of trifluoroacetic anhydride at 90 degrees C for 15 min. The derivatized samples were allowed to cool to room temperature, evaporated to dryness, and then reconstituted in 50 microL of ethyl acetate. Parent set masses (unbolded ions) and product ions were m/z 304 and m/z 182 and 82 (COC), m/z 307 and m/z 185 and 85 (COC-d3), m/z 318 and m/z 196 and 82 (CE), m/z 440 and m/z 318 and 105 (BE), m/z 443 and m/z 321 and 105 (BE-d3), m/z 296 and m/z 182, and 82 (EME), m/z 299 and m/z 185 and 85 (EME-d3), m/z 403 and m/z 386 and 105 (NCOC), m/z 406 and m/z 389 and 105 (NCOC-d3). Quantitation was accomplished by calculating the area ratio of the higher mass product ion (underlined ions) of analyte to the respective internal standard based on multilevel calibrations from 0.01 to 10.0 ng/mg. The GC-MS-MS method had a limit of detection of 0.01 ng/mg and a limit of quantitation of 0.05 ng/mg for all five analytes. COC, BE, and EME were detected in all 30 samples, and CE and NCOC were found in 19 and 29 samples, respectively. The average relative percentages of each metabolite normalized to the cocaine concentrations were 12.8%, 15.4%, 1.8%, and 2.5% for BE, CE, EME, and NCOC, respectively.

Hair pigmentation is a critical factor in the interpretation of the concentration of certain compounds and their metabolites incorporated into hair. Melanin is responsible for the pigmentation. The color and the melanin content of human hair samples differs over a wide range. Once deposited into hair, drug may remain detectable for a period of months to years. However, if drug disposition into hair is influenced by those properties attributed to hair color, then certain persons may test positive more frequently than other persons. Removal of the melanin from hair digests prior to drug analysis may reduce the effect of melanin on the total drug concentration by excluding the drug bound to the pigment. In this study, the effect of melanin removal by centrifugation of hair digests on cocaine concentrations was investigated. Two sets of hair samples from five cocaine users were analyzed for cocaine and metabolites. A solution consisting of 10 mL of 0.5M Tris buffer (pH 6.4) to which is added 60 mg D,L-dithiothreitol, 200 mg SDS, and 200 U Proteinase K, was used to digest the hair. Two milliliters of this solution was added to 20 mg of hair and incubated at 37 degrees in a shaking water bath (90 oscillations/min) overnight. The samples were removed from the water bath and mixed. One set was centrifuged at 2000 rpm and divided into supernatant and melanin pellet. The other set was not centrifuged. Internal standards were added to all tubes. The samples were further extracted, derivatized, and analyzed by gas chromatography-mass spectrometry. A mean of 8.8% (standard deviation [SD] 7.0%) of the total cocaine concentration (supernatant and pellet) was left behind in the pellet. The same experiment was repeated except that the melanin pellet was redigested with 0.1 N HCl. After redigestion of the melanin pellet, the mean cocaine concentration in the pellet was 3.8% +/- 4.0% (mean +/- SD) of the total cocaine concentration in hair. These data demonstrate that removal of melanin from hair digests by centrifugation does not eliminate hair color bias when interpreting cocaine concentrations.

Drug users can be highly motivated to obtain negative results on urine drug tests and may attempt to subvert the process by in vivo adulteration. The use of herbal products for "flushing" and "detoxification" is frequently advertised as an effective means of passing drug tests. Accordingly, a study was designed to determine the effects of ingestion of two herbal products, Naturally Klean Herbal Tea and Golden Seal root, and a diuretic medication, hydrochlorothiazide. The herbal tea was prepared in 1 gal of water as specified by the manufacturer. All other products were consumed with 1 gal of water. Two control conditions in which the subject consumed only water (1 gal; 12 oz) were included. The 1-gal liquid treatments were divided into 4-qt aliquots, and 1-qt was consumed each hour for 4 h. All treatments were begun approximately 22 h after smoking of a marijuana cigarette (3.58% THC) and 22 h after intranasal administration of cocaine hydrochloride. Following all treatments with excess fluid, creatinine and specific gravity dropped in 1.5-2.0 h to levels indicative of diluted specimens (<20 mg/dL creatinine, <1.003 specific gravity). Marijuana and cocaine metabolite concentrations by immunoassay (EMIT and TDx) also dropped rapidly, and the results frequently switched from positive to negative. By the time subjects had consumed 2 qt of any fluid, they were generally producing false-negative results. For example, ingestion of excess water produced dilute specimens (<20 mg/dL creatinine; <1.003 specific gravity) in an average time plus or minus the standard error of the mean of 1.47 +/- 0.17 h (N = 5) and 1.45 +/- 0.2 h (N = 5) following smoked marijuana and intranasal cocaine, respectively. In comparison, ingestion of Klean Tea produced dilute specimens in 1.36 +/- 0.07 h (N = 4) and 1.39 +/- 0.11 h (N = 4) following marijuana and cocaine administration. Recovery of urine test measures to pre-treatment levels occurred over a period of 8-10 h. Average detection times for marijuana metabolite appeared to be slightly shorter following ingestion of 1 gal of fluids compared with ingestion of 12 oz of water as a result of the time of testing being near the end of the cannabinoid metabolite excretion phase. Consequently, negative cannabinoid results induced by fluid ingestion rarely returned to positive after excess water was eliminated. In contrast, negative cocaine results reverted to positive quickly after the dilution effects disappeared. It was concluded that excess water ingestion can produce false-negative test results, but the claims of herbal products to be an aid in passing a urine test appear to be unfounded.