Urine Mescaline Screening With a Biochip Array Immunoassay and Quantification by Gas Chromatography-Mass Spectrometry.

Mescaline, the primary psychoactive chemical in peyote cactus, has been consumed for thousands of years in ancient religious ceremonies. The US military wanted to determine if mescaline intake was a problem for personnel readiness. Twenty thousand seventeen urine specimens negative for cannabinoids, cocaine, opiates, and amphetamines were tested for mescaline with the Randox Drugs of Abuse V (DOA-V) biochip array immunoassay at the manufacturer's recommended cutoff of 6 mcg/L. A sensitive and specific method for mescaline quantification in urine was developed and fully validated. Extracted analytes were derivatized with pentafluoropropionic anhydride and pentafluoropropanol and quantified by gas chromatography-mass spectrometry (GC/MS) with electron impact ionization. Standard curves, using linear least squares regression with 1/x weighting, were linear from 1 to 250 mcg/L with coefficients of determination >0.994. Intra- and inter-assay imprecision was <4.4 coefficient of variation (%CV), with accuracies >90.4%. Mean extraction efficiencies were >92.0% across the linear range. This fully validated method was applied for the confirmation of urinary mescaline in 526 presumptive-positive specimens and 198 randomly selected presumptive-negative specimens at the manufacturer's 6 mcg/L cutoff. No specimen confirmed positive at the GC/MS limit of quantification of 1 mcg/L. Results indicated that during this time frame, there was insufficient mescaline drug use in the military to warrant routine screening in the drug testing program. However, mescaline stability, although assessed, could have contributed to lower prevalence. We also present a validated GC/MS method for mescaline quantification in urine for reliable confirmation of suspected mescaline intake.

Urinary prevalence, metabolite detection rates, temporal patterns and evaluation of suitable LC-MS/MS targets to document synthetic cannabinoid intake in US military urine specimens.

BACKGROUND: Identifying synthetic cannabinoid designer drug abuse challenges toxicologists and drug testing programs. The best analytical approach for reliably documenting intake of emerging synthetic cannabinoids is unknown. Primarily metabolites are found in urine, but optimal metabolite targets remain unknown, and definitive identification is complicated by converging metabolic pathways. METHODS: We screened 20,017 US military urine specimens collected from service members worldwide for synthetic cannabinoids between July 2011 and June 2012. We confirmed 1432 presumptive positive and 1069 presumptive negative specimens by qualitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis including 29 biomarkers for JWH-018, JWH-073, JWH-081, JWH-122, JWH-200, JWH-210, JWH-250, RCS-4, AM2201 and MAM2201. Specimen preparation included enzyme hydrolysis and acetonitrile precipitation prior to LC-MS/MS analysis. We evaluated individual synthetic cannabinoid metabolite detection rates, prevalence, temporal patterns and suitable targets for analytical procedures. RESULTS: Prevalence was 1.4% with 290 confirmed positive specimens, 92% JWH-018, 54% AM2201 and 39% JWH-122 metabolites. JWH-073, JWH-210 and JWH-250 also were identified in 37%, 4% and 8% of specimens, respectively. The United States Army Criminal Investigation Command seizure pattern for synthetic cannabinoid compounds matched our urine specimen results over the time frame of the study. Apart from one exception (AM2201), no parent compounds were observed. CONCLUSIONS: Hydroxyalkyl metabolites accounted for most confirmed positive tests, and in many cases, two metabolites were identified, increasing confidence in the results, and improving detection rates. These data also emphasize the need for new designer drug metabolism studies to provide relevant targets for synthetic cannabinoid identification.

Biochip array technology immunoassay performance and quantitative confirmation of designer piperazines for urine workplace drug testing.

Designer piperazines are emerging novel psychoactive substances (NPS) with few high-throughput screening methods for their identification. We evaluated a biochip array technology (BAT) immunoassay for phenylpiperazines (PNP) and benzylpiperazines (BZP) and analyzed 20,017 randomly collected urine workplace specimens. Immunoassay performance at recommended cutoffs was evaluated for PNPI (5 µg/L), PNPII (7.5 µg/L), and BZP (5 µg/L) antibodies. Eight hundred forty positive and 206 randomly selected presumptive negative specimens were confirmed by liquid chromatography high-resolution mass spectrometry (LC-HRMS). Assay limits of detection for PNPI, PNPII, and BZP were 2.9, 6.3, and 2.1 µg/L, respectively. Calibration curves were linear (R (2) > 0.99) with upper limits of 42 µg/L for PNPI/PNII and 100 µg/L for BZP. Quality control samples demonstrated imprecision <19.3 %CV and accuracies 86.0-94.5 % of target. There were no interferences from 106 non-piperazine substances. Seventy-eight of 840 presumptive positive specimens (9.3 %) were LC-HRMS positive, with 72 positive for 1-(3-chlorophenyl)piperazine (mCPP), a designer piperazine and antidepressant trazodone metabolite. Of 206 presumptive negative specimens, one confirmed positive for mCPP (3.3 µg/L) and one for BZP (3.6 µg/L). BAT specificity (21.1 to 91.4 %) and efficiency (27.0 to 91.6 %) increased, and sensitivity slightly decreased (97.5 to 93.8 %) with optimized cutoffs of 25 µg/L PNPI, 42 µg/L PNPI, and 100 µg/L BZP. A high-throughput screening method is needed to identify piperazine NPS. We evaluated performance of the Randox BAT immunoassay to identify urinary piperazines and documented improved performance when antibody cutoffs were raised. In addition, in randomized workplace urine specimens, all but two positive specimens contained mCPP and/or trazodone, most likely from legitimate medical prescriptions. Graphical Abstract Biochip array technology (BAT) immunoassay for designer piperazines detection in urine. In chemiluminescent immunoassay, the labeled-drug (antigen) competes with the drug in the urine. In the absence of drug, the labeled-drug binds to the antibody releasing an enzyme (horseradish peroxidase) to react with the substrate and producing chemiluminescence. The higher the drug concentration in urine, the weaker the chemiluminescent signal is produced. All presumptive positive specimens and randomly selected presumptive negative specimens were analyzed and confirmed by a liquid chromatography high-resolution mass spectrometry with limit of quantification of 2.5 or 5 µg/L.

Stabilization of urinary THC solutions with a simple non-ionic surfactant.

To stabilize urinary solutions against adsorptive loss of metabolites of Delta9-tetrahydrocannabinol (THC), a non-ionic surfactant, Tergitol, was investigated to reduce the need for special handling and storage of such solutions. Addition of surfactant up to 20 times the critical micelle concentration (CMC) did not adversely affect the analytical process. Yet, at only two times CMC, surfactant was found to mitigate adsorptive loss of THC analytes under a variety of storage and handling conditions including exposure to glass and plastic surfaces, after storage in a refrigerator or freezer, and at reduced pH, where adsorptive losses were expected to be significant. On average, micellar solubilization of analyte increased the assayed concentration by 10% with a range of 3 to 20%, depending on condition, relative to solutions without surfactant. Solutions with surfactant did not fail (i.e., deviate in concentration by +/-20%) over a 49-week period, whereas those without surfactant failed by 21 weeks. These results indicate that addition of small amounts of non-ionic surfactant to solutions of urinary THC metabolites is a simple method to improve both the accuracy and precision of analyte concentrations, as determined by gas chromatography-mass spectrometry, in such solutions by mitigating adsorptive losses during storage and handling events.

Complete automation of solid-phase extraction with subsequent liquid chromatography-tandem mass spectrometry for the quantification of benzoylecgonine, m-hydroxybenzoylecgonine, p-hydroxybenzoylecgonine, and norbenzoylecgonine in urine--application to a high-throughput urine analysis laboratory.

A fully automated system utilizing a liquid handler and an online solid-phase extraction (SPE) device coupled with liquid chromatography-tandem mass spectrometry (LC-MS-MS) was designed to process, detect, and quantify benzoylecgonine (BZE), meta-hydroxybenzoylecgonine (m-OH BZE), para-hydroxybenzoylecgonine (p-OH BZE), and norbenzoylecgonine (nor-BZE) metabolites in human urine. The method was linear for BZE, m-OH BZE, and p-OH BZE from 1.2 to 10,000 ng/mL with limits of detection (LOD) and quantification (LOQ) of 1.2 ng/mL. Nor-BZE was linear from 5 to 10,000 ng/mL with an LOD and LOQ of 1.2 and 5 ng/mL, respectively. The intrarun precision measured as the coefficient of variation of 10 replicates of a 100 ng/mL control was less than 2.6%, and the interrun precision for 5 replicates of the same control across 8 batches was less than 4.8% for all analytes. No assay interference was noted from controls containing cocaine, cocaethylene, and ecgonine methyl ester. Excellent data concordance (R2 > 0.994) was found for direct comparison of the automated SPE-LC-MS-MS procedure and an existing gas chromatography-MS procedure using 94 human urine samples previously determined to be positive for BZE. The automated specimen handling and SPE procedure, when compared to the traditional extraction schema, eliminates the human factors of specimen handling, processing, extraction, and derivatization, thereby reducing labor costs and rework resulting from batch handling issues, and may reduce the number of fume hoods required in the laboratory.

Rapid quantification of urinary oxycodone and oxymorphone using fast gas chromatography-mass spectrometry.

Human urine specimens that were determined to be presumptively positive for oxycodone and its metabolite, oxymorphone, by immunoassay screening were assayed using fast gas chromatography-mass spectrometry to positively identify and quantify the oxycodone and oxymorphone present. Urine specimens were first spiked with deuterated internal standards, oxycodone-d(3) and oxymorphone-d(3), subjected to acid hydrolysis, and then extracted using a positive-pressure manifold and mixed-bed solid-phase cartridge extraction methodology. Extracts were derivatized using methoxylamine and acetic anhydride. The acetylated-oxime derivatives of oxycodone and oxymorphone were identified and quantified using a selective ion monitoring (SIM). The method was found to be linear for both analytes to 1600 ng/mL, and limits of detection for oxycodone and oxymorphone were found to be 40 ng/mL and 20 ng/mL, respectively. Interlaboratory data comparisons (n = 40) showed correlation coefficients of 0.9999 and 0.9997 for oxycodone and oxymorphone, respectively. Twelve semisynthetic, structurally similar compounds at concentrations of 5000 ng/mL were assayed in the presence of oxycodone and oxymorphone and found not to interfere with identification and quantitation by this method. Finally, exact mass and tandem mass spectrometry techniques were employed to elucidate the structures of the SIM ions.

Prevalence of use study for amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-methamphetamine (MDMA), and 3,4-methylenedioxy-ethylamphetamine (MDEA) in military entrance processing stations (MEPS) specimens.

The Roche Abuscreen Onlinetrade mark Amphetamine immunoassay (IA), modified to include sodium periodate, and the Microgenics DRI Ecstasy IA were used to determine the prevalence of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) in urine specimens from applicants seeking to join the United States Armed Forces. Over a 4-month period, a total of 85,658 specimens were IA screened using the Department of Defense 500 ng/mL administrative cutoff level for AMP and MDMA. All presumptively positive specimens were confirmed using a solid-phase extraction procedure coupled with simultaneous analysis of AMP, MAMP, MDA, MDMA, and MDEA by fast gas chromatography-mass spectrometry using the same cutoff levels as the IA. The Roche Online Amphetamine IA identified 216 specimens as presumptively positive; of these, 70 specimens confirmed positive for AMP and 87 specimens confirmed positive for AMP and/or MAMP, resulting in a confirmation rate of 73%. The Microgenics DRI Ecstasy IA identified eight specimens as presumptively positive; of these, five specimens confirmed positive for MDMA and/or MDA, resulting in a confirmation rate of 63%. The total use prevalence for AMP, MAMP, MDA, MDMA, and/or MDEA in military entrance processing stations specimens over the testing period was determined to be 0.19%.

Rapid analysis of benzoylecgonine in urine by fast gas chromatography-mass spectrometry.

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.

Urine pH, container composition, and exposure time influence adsorptive loss of 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid.

11-nor-delta9-Tetrahydrocannabinol-9-carboxylic acid (11-nor-delta9-THC-COOH) is the primary cannabinoid present in the urine of individuals who have used marijuana and is the target analyte identified at forensic urinalysis drug testing laboratories. The preparation, storage, transport, and processing of control materials for gas chromatography-mass spectrometric (GC-MS) analysis of human urine specimens is critical to accurate compound identification and quantification. Previous studies have suggested that adsorptive loss of 11-nor-delta9-THC-COOH is influenced by container composition and storage temperature. In this study, urine solutions of 11-nor-delta9-THC-COOH (7.5, 15, 60, and 500 ng/mL) at three physiologically-relevant pHs (4.6, 6.5, and 8.4) were prepared and subjected to storage and processing in containers of different compositions (polypropylene and borosilicate glass). Analyte identification and quantification were achieved using tetramethylammonium hydroxide/iodomethane-based derivatization followed by GC separation and electron-impact MS. These analyses demonstrate that adsorptive loss of 11-nor-delta9-THC-COOH is a phenomenon found in acidic urine solutions and is relatively absent in urine solutions that are near-neutral or basic. Furthermore, the data indicate that the adsorptive loss of 11-nor-delta9-THC-COOH is dependent on solution-container exposure time and is similar between containers of two distinct compositions. These results suggest that for optimal analytical control performance, solution pH and control processing times are critical elements.

Rapid quantification of urinary 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid using fast gas chromatography-mass spectrometry.

Human urine specimens that were determined to be presumptively positive for metabolites of delta9-tetrahydrocannabinol by immunoassay screening were assayed using a novel fast gas chromatography-mass spectrometry (FGC-MS) analytical method to determine whether this method would improve the efficiency of specimen processing without diminishing the reliability of metabolite identification and quantification. Urine specimens were spiked with deuterated internal standard, subjected to solid-phase extraction, and derivatized using tetramethylammonium hydroxide and iodomethane. The methyl ester/methyl ether derivatives were identified and quantified using both a traditional GC-MS method and the newly developed FGC-MS method. The FGC-MS method was demonstrated to be linear between 3.8 and 1500 ng/mL 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid (11-nor-delta-THC-COOH). The intrarun precision of 15 replicates of a 15 ng/mL control and the interrun precision of 161 sets of 7, 15, and 60 ng/mL controls were acceptable (coefficients of variation < 5.5%). The FGC-MS method was demonstrated to be specific for identifying 11-nor-delta9-THC-COOH and none of 43 tested substances interfered with identification and quantification of 11-nor-delta9-THC-COOH. Excellent data concordance (R2 > 0.993) was found for two specimen sets assayed using both methods. The FGC-MS method, when compared with a traditional GC-MS method, reduces total assay time by approximately 40% with no decrease in data quality.

Rapid simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine in urine by fast gas chromatography-mass spectrometry.

The use of fast gas chromatography-mass spectrometry (FGC-MS) was investigated to improve the efficiency of analysis of urine specimens that previously screened presumptively positive for amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), and/or 3,4 methylenedioxyethylamphetamine (MDEA) by immunoassay testing. Specimens were pretreated with basic sodium periodate, extracted using a positive-pressure manifold/cation-exchange solid-phase cartridge methodology, and derivatized using 4-carbethoxyhexafluorobutyryl chloride (4-CB). The analytical method was compared to traditional GC-MS analysis and evaluated with respect to assay chromatography, linearity, sensitivity, precision, accuracy, and reproducibility. The limits of detection were 62.5 ng/mL for MDA and 31.25 ng/mL for AMP, MAMP, MDMA, and MDEA. All of the target analytes were linear to 12,000 ng/mL with the exception of MAMP which was linear to 10,000 ng/mL. The intra-assay precision of a 500 ng/mL multiconstituent control (n=15) ranged from 522.6 to 575.9 ng/mL with a coefficient of variation of less than 3.8%. Authentic human urine specimens (n=187) previously determined to contain the target analytes were re-extracted and analyzed by both FGC-MS and the currently utilized GC-MS method. No significant differences in specimen concentration were observed between these analytical methods. No interferences were seen when the performance of the FGC-MS method was challenged with ephedrine, pseudoephedrine, phenylpropanolamine, and phentermine. When compared to traditional GC-MS analysis, FGC-MS analysis provided a dramatic reduction in retention time for amphetamine (1.8 min vs. 4.12 min). For example, the FGC-MS method reduced overall run time for a batch of 56 specimens from 12.0 h to 7.25 h. This reduction in analysis time makes FGC-MS an attractive alternative to traditional GC-MS by allowing a laboratory greater flexibility in the purchase and use of capital equipment and in the assignment of laboratory personnel, all resulting in greater overall efficiency by decreasing reporting times for AMP, MAMP, and designer amphetamine positive specimens.

Urine benzodiazepine screening using Roche Online KIMS immunoassay with beta-glucuronidase hydrolysis and confirmation by gas chromatography- mass spectrometry.

Performance of the Roche Online KIMS (kinetic interaction of microparticles in solution) benzodiazepine (BZD) immunoassay (IA) with and without beta-glucuronidase treatment was evaluated on a Hitachi Modular automated IA analyzer calibrated using nordiazepam at 100 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences were evaluated. Precision of the assay (percent coefficient of variation (%CV)) with and without addition of the enzyme was less than 6% and 9%, respectively, with linearity (r(2) value of 0.9578 and 0.9746), respectively. Between-run precision of a 125 ng/mL nordiazepam control (n = 287) over 67 days, produced a %CV of 13.6% for the hydrolytic assay. Modification of the BZD assay to include automated hydrolysis of urinary BZD glucuronide conjugates was evaluated using three glucuronidated BZD standards prepared at concentrations ranging from 250 to 10,000 ng/mL. With hydrolysis, temazepam, oxazepam, and lorazepam glucuronides, produced cross-reactivities of 25%, 15%, and 20%, respectively. Without hydrolysis, the glucuronidated BZD standards produced less than 1% cross-reactivity in the assay. The ability of the assay to differentiate between positive and negative samples was evaluated by assaying 20 negative urine samples and serial dilutions of certified drug-free urine fortified with 28 different BZDs. All of the negative and positive urine samples produced the appropriate screening result. Cross-reactivities of 27 different BZDs, calculated as the normalized IA response divided by the BZD concentration that produced a response approximately equivalent to the response of a 100 ng/mL nordiazepam standard and multiplied by 100, ranged from 15% to 149%. Human urine samples (n = 28) that were previously found to contain BZDs by gas chromatography-mass spectrometry (GC-MS) also produced a positive BZD IA result. The IA was challenged with 78 potentially interfering compounds, and none produced a positive BZD response. As a part of the validation, a large number of human urine samples (29,500) were assayed using the modified Online BZD IA method to evaluate the performance of the method in production. Of the 29,500 samples tested, 80 produced a positive IA result. Analysis by GC-MS confirmed the presence of at least 1 BZD compound in 61 of the samples corresponding to a confirmation rate of 76%. The Online BZD IA modified by the automatic addition of beta-glucuronidase appears well adapted for the rapid detection of BZDs and their metabolites in human urine.

Quantitative urine confirmatory testing for synthetic cannabinoids in randomly collected urine specimens.

Synthetic cannabinoid intake is an ongoing health issue worldwide, with new compounds continually emerging, making drug testing complex. Parent synthetic cannabinoids are rarely detected in urine, the most common matrix employed in workplace drug testing. Optimal identification of synthetic cannabinoid markers in authentic urine specimens and correlation of metabolite concentrations and toxicities would improve synthetic cannabinoid result interpretation. We screened 20 017 randomly collected US military urine specimens between July 2011 and June 2012 with a synthetic cannabinoid immunoassay yielding 1432 presumptive positive specimens. We analyzed all presumptive positive and 1069 negative specimens with our qualitative synthetic cannabinoid liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which confirmed 290 positive specimens. All 290 positive and 487 randomly selected negative specimens were quantified with the most comprehensive urine quantitative LC-MS/MS method published to date; 290 specimens confirmed positive for 22 metabolites from 11 parent synthetic cannabinoids. The five most predominant metabolites were JWH-018 pentanoic acid (93%), JWH-N-hydroxypentyl (84%), AM2201 N-hydroxypentyl (69%), JWH-073 butanoic acid (69%), and JWH-122 N-hydroxypentyl (45%) with 11.1 (0.1-2,434), 5.1 (0.1-1,239), 2.0 (0.1-321), 1.1 (0.1-48.6), and 1.1 (0.1-250) µg/L median (range) concentrations, respectively. Alkyl hydroxy and carboxy metabolites provided suitable biomarkers for 11 parent synthetic cannabinoids; although hydroxyindoles were also observed. This is by far the largest data set of synthetic cannabinoid metabolites urine concentrations from randomly collected workplace drug testing specimens rather than acute intoxications or driving under the influence of drugs. These data improve the interpretation of synthetic cannabinoid urine test results and suggest suitable urine markers of synthetic cannabinoid intake.

Performance characteristics of an ELISA screening assay for urinary synthetic cannabinoids.

Synthetic cannabinoids are marketed as legal alternatives to cannabis, as routine urine cannabinoid immunoassays do not detect synthetic cannabinoids. Laboratories are challenged to identify these new designer drugs that are widely available and represent a major public health and safety problem. Immunoassay testing offers rapid separation of presumptive positive and negative specimens, prior to more costly and time-consuming chromatographic confirmation. The Neogen SPICE ELISA kit targets JWH-018 N-pentanoic acid as a marker for urinary synthetic cannabinoids. Assay performance was evaluated by analyzing 2469 authentic urine samples with the Neogen immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two immunoassay cut-off concentrations, 5 and 10 µg/L, classified samples as presumptive positive or negative, followed by qualitative LC-MS/MS confirmation for 29 synthetic cannabinoids markers with limits of detection of 0.5-10 µg/L to determine the assay's sensitivity, specificity and efficacy. Challenges at ±25% of each cut-off also were investigated to determine performance around the cut-off and intra- and inter-plate imprecision. The immunoassay was linear from 1 to 250 µg/L (r(2) = 0.992) with intra- and inter-plate imprecision of ≤5.3% and <9%, respectively. Sensitivity, specificity, and efficiency results with the 5 µg/L cut-off were 79.9%, 99.7%, and 97.4% and with the 10 µg/L cut-off 69.3%, 99.8%, and 96.3%, respectively. Cross-reactivity was shown for 18 of 73 synthetic cannabinoids markers evaluated. Good sensitivity, specificity, and efficiency, lack of sample preparation requirements, and rapid semi-automation documented that the Neogen SPICE ELISA kit is a viable method for screening synthetic cannabinoids in urine targeting JWH-018 N-pentanoic acid.

Comparison and evaluation of DRI methamphetamine, DRI ecstasy, Abuscreen ONLINE amphetamine, and a modified Abuscreen ONLINE amphetamine screening immunoassays for the detection of amphetamine (AMP), methamphetamine (MTH), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA) in human urine.

The performances of four immunoassays (DRI amphetamines, DRI ecstasy, Abuscreen ONLINE amphetamines, and a modified Abuscreen ONLINE amphetamines) were evaluated for control failure rates, sensitivity, and specificity for amphetamine (AMP), methamphetamine (MTH), 3,4-methylenedioxyamphetamine (MDA), and 3,4-methylenedioxymethamphetamine (MDMA). The two DRI reagents and the ONLINE reagents were run according to manufacturer specifications using a Roche Hitachi Modular DDP system. The modified ONLINE reagent was calibrated with MDMA and had 16mM sodium periodate added to the R2 reagent. These assays were run on approximately 27,500 human urine samples and 7000 control urine samples prepared at 350 and 674 ng/mL over the course of 8 days. All assays were calibrated using a single point, qualitative cutoff standard with the manufacturer-recommended compound at the Department of Defense cutoff (500 ng/mL). Gas chromatography-mass spectrometry (GC-MS) confirmation was conducted on screened-positive samples. Control performance for the manufacturer recommended assays was excellent, with a maximum qualitative control failure rate of 2.03%. The modified ONLINE reagent demonstrated poor control performance with a maximum failure rate of 38.3% and showed no improved MDMA sensitivity when compared with the ONLINE reagent; the confirmation rate (20%) was improved when compared with the production ONLINE reagent (8%). The DRI ecstasy reagent provided improved sensitivity for MDMA as compared with the ONLINE reagent, with approximately 23% more samples screening and confirming positive for MDMA and a confirmation rate of approximately 90%. The DRI methamphetamine reagent had a low confirmation rate (6% or less) and produced numerous positives for samples with only ephedrine or pseudoephedrine present.

Rapid simultaneous determination of amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and 3,4-methylenedioxyethylamphetamine in urine by solid-phase extraction and GC-MS: a method optimized for high-volume laboratories.

To facilitate analysis of high sample volumes, an extraction, derivatization and gas chromatographic-mass spectrometric analysis method was developed to simultaneously determine amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA) 3,4-methylenedioxymethamphetamine (MDMA), and 3,4-methylenedioxyethylamphetamine (MDEA) in urine. This method utilized a positive-pressure manifold cation-exchange polymer-based solid-phase extraction followed by elution directly into automated liquid sampler (ALS) vials. Rapid derivatization was accomplished using heptafluorobutyric anhydride (HFBA). Recoveries averaged 90% or greater for each of the compounds. Limits of detection were 62.5 ng/mL (AMP and MDEA), 15.6 ng/mL (MAMP), and 31.3 ng/mL (MDA and MDMA) using a 2-mL sample volume. The method was linear to 5000 ng/mL for all compounds using MDMA-d5 and MAMP-d14 as internal standards. Over 200 human urine samples previously determined to contain the target analytes were analyzed using the method. Excellent agreement was seen with previous quantitations. The method was challenged with 75 potentially interfering compounds and no interferences were seen. These interfering compounds included ephedrine, pseudoephedrine, phenylpropanolamine, and phenethylamine. The method resulted in dramatic reductions in processing time and waste production.

Occupational cocaine exposure of crime laboratory personnel preparing training aids for a military working dog program.

The potential for passive cocaine exposure was evaluated in crime laboratory employees preparing training aids for a military working dog program (MWD). The primary goal of the study was to elucidate the routes of exposure and implement procedural changes that would minimize this risk. Several work environments and laboratory procedures were examined by monitoring personal breathing zones (PBZ), ambient airborne cocaine levels in the laboratory spaces, and urinary levels of the primary cocaine metabolite, benzoylecgonine. The study was performed initially using current laboratory procedures to establish a baseline and to identify potential sources of exposure. A subsequent study was performed to determine the effectiveness of the follow-up procedure in reducing exposure. As a result of the changes, the 8-h time weighted averages (TWAs) were 40 to 80% lower in the follow-up study as compared to the baseline assessment. Dermal absorption and PBZ inhalation of cocaine during manufacture were likely the most significant source of cocaine exposure. Ambient airborne cocaine may have also contributed to the total exposure, but for most observations, the concentrations were significantly less than those determined from PBZ monitoring. The maximum ambient cocaine concentration was 0.0144 mg/m(3) compared to a maximum of 0.4004 mg/m(3) observed during PBZ monitoring. Occupational exposure decreased in the follow-up study because of the proper use of personal protective equipment and improvements in engineering controls.

LC-MS analysis of 2-oxo-3-hydroxy LSD from urine using a Speedisk positive-pressure processor with Cerex PolyChrom CLIN II columns.

A rapid and sensitive solid-phase extraction method for the lysergic acid diethylamide (LSD) metabolite 2-oxo-3-hydroxy-LSD (O-H-LSD) was developed for use in a forensic laboratory. The method uses a positive-pressure manifold anion-exchange polymer-based solid-phase extraction with subsequent analysis by liquid chromatography-mass spectrometry (LC-MS). The average extraction efficiency was 92%. The limits of detection (LOD) and quantitation (LOQ) were calculated at 250 pg/mL. The assay was linear, precise, and accurate from 250 to 30,000 pg/mL with an r(2) of 0.999. Samples including 93 compounds with properties similar to O-H-LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse were analyzed and produced no significant interference. This method has increased our efficiency in analyzing O-H-LSD by reducing the overall extraction and analysis time. The increase in extraction efficiency enabled decreased assay LOD and LOQ values while lowering the volume required for injection.

Evaluation of a solid-phase extraction method for benzoylecgonine urine analysis in a high-throughput forensic urine drug-testing laboratory.

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.

Comparison of EMIT II, CEDIA, and DPC RIA assays for the detection of lysergic acid diethylamide in forensic urine samples.

In an effort to determine a practical, efficient, and economical alternative for the use of a radioimmunoassay (RIA) for the detection of lysergic acid diethylamide (LSD) in human urine, the performance of two photometric immunoassays (Dade Behring EMIT II and Microgenics CEDIA) and the Diagnostics Products Corp. (DPC) RIA were compared. Precision, accuracy, and linearity of the 3 assays were determined by testing 60 replicates (10 for RIA) at 5 different concentrations below and above the 500-pg/mL LSD cut-off. The CEDIA and RIA exhibited better accuracy and precision than the EMIT II immunoassay. In contrast, the EMIT II and CEDIA demonstrated superior linearity r2 = 0.9809 and 0.9540, respectively, as compared with the RIA (r2 = 0.9062). The specificity of the three assays was assessed using compounds that have structural and chemical properties similar to LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the 144 compounds studied, the EMIT II cross-reacted with twice as many compounds as did the CEDIA and RIA. Specificity was also assessed in 221 forensic human urine specimens that previously screened positive for LSD by the EMIT II assay. Of these, only 11 tested positive by CEDIA, and 3 were positive by RIA. This indicated a comparable specificity performance between CEDIA and RIA. This also was consistent with a previously reported high false-positive rate of EMIT II (low specificity). Each of the immunoassays correctly identified LSD in 23 out of 24 human urine specimens that had previously been found to contain LSD by gas chromatography-mass spectrometry at a cut-off concentration of 200 pg/mL. The CEDIA exhibited superior precision, accuracy, and decreased cross-reactivity to compounds other than LSD as compared with the EMIT II assay and does not necessitate the handling of radioactive materials.