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.

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.

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.

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.

Method validation of the biochip array technology for synthetic cannabinoids detection in urine.

BACKGROUND: Synthetic cannabinoids (SC) are widely-abused cannabimimetic drugs that do not screen positive in traditional cannabinoids immunoassays, making detection difficult. METHODS AND RESULTS: The first commercially-available immunoassay for urinary SC was validated. Limits of detection (5-20 µg/L), imprecision (<13.1% intra-, <37.7% inter-assay), and cross-reactivity profiles of 22 SC and 37 metabolites were obtained. A large negative bias (-80.8 to -28.0%) was observed. Sensitivity (98.3%), specificity (48.1%) and efficiency (53.9%) were determined from screening 20,017 urine specimens and confirming 1432 presumptive positive and 1069 selected negative specimens by LC-MS/MS. Cutoff optimization improved performance to 87.6% sensitivity, 85.2% specificity, and 85.4% efficiency. CONCLUSION: This high-throughput urine SC assay has good sensitivity and improved specificity and efficiency at modified cutoff concentrations.

Use and abuse of prescribed opioids, central nervous system depressants, and stimulants among U.S. active duty military personnel in FY 2010.

OBJECTIVES: This study establishes rates of use/abuse of Schedule II-IV prescription medications in U.S. active duty military personnel, and characterizes correlates of such use/abuse. METHODS: All active duty personnel serving for 12 months during fiscal year 2010 were included. Data were obtained from medical and pharmacy claims and drug screening results. Logistic regression models were used to examine predictors of drug use, along with bivariate analyses to compare abuse of prescribed and illegal drugs. RESULTS: Nearly one-third of active duty service members received at least one prescription for opioids, central nervous system depressants, or stimulants, with 26.4% having received at least one prescription for opioids. About 0.7%, 1.4%, and 0.6% of the total force received >90-day prescriptions for opioids, central nervous system depressants, or stimulants, respectively. Battlefield injury, receipt of psychotropic medications, and substance abuse adverse events were predictive of >90-day supply of opioids. About 0.7% of the total force had documented known drug abuse for prescribed drugs compared to 0.4% for illegal drug abuse. CONCLUSIONS: We recommend systematic monitoring of prescriptions for controlled substances which may carry serious consequences, evaluation of the impact of controlled substances on military readiness, and examination of the rationale for prescribing controlled drugs.

Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine.

INTRODUCTION: The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. METHODS: 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semi-quantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 µg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. RESULTS: Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 µg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1-10 µg/L. Performance at 5 µg/L was 92.2%, 98.1% and 97.4%, and for the 20 µg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 µg/L, and documented acceptable linearity from 5-25 µg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 µg/L); 4 showed moderate cross-reactivity (10-50% at 10 µg/L), 30 low cross-reactivity (<10% at 500 µg/L), and 27 <1% cross-reactivity at 500 µg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 µg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 µg/L) but below the manufacturer's recommended cutoff concentration (10 µg/L). CONCLUSION: The Immunalysis HEIA K2 Spice kit required no sample preparation, had a high-throughput, and acceptable sensitivity, specificity and efficiency, offering a viable method for screening synthetic cannabinoids in urine that cross-react with JWH-018 N-pentanoic acid antibodies.

Validation of the only commercially available immunoassay for synthetic cathinones in urine: Randox Drugs of Abuse V Biochip Array Technology.

Deterrence of synthetic cathinone abuse is hampered by the lack of a high-throughput immunoassay screen. The Randox Drugs of Abuse V (DOA-V) Biochip Array Technology contains two synthetic cathinone antibodies: Bath Salt I (BSI) targets mephedrone/methcathinone and Bath Salt II (BSII) targets 3',4'-methylenedioxypyrovalerone (MDPV)/3',4'-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP). We evaluated DOA-V synthetic cathinones performance and conducted a full validation on the original assay with calibrators reconstituted in water, and the new assay with calibrators prepared in lyophilized urine; both utilized the same antibodies and were run on the fully automated Evidence® Analyzer. We screened 20 017 authentic military urine specimens and confirmed positives by liquid chromatography-tandem mass spectrometry (LC-MS/MS) for 28 synthetic cathinones. Limits of detection (LOD) for the original and new assays were 0.35 and 0.18 (BSI), and 8.5 and 9.2 µg/L (BSII), respectively. Linearity was acceptable (R(2) >0.98); however, a large negative bias was observed with in-house prepared calibrators. Intra-assay imprecision was <20% BSI-II, while inter-assay imprecision was 18-42% BSI and <22% BSII. Precision was acceptable for Randox controls. Cross-reactivities of many additional synthetic cathinones were determined. Authentic drug-free negative urine pH <4 produced false positive results for BSI (6.3 µg/L) and BSII (473 µg/L). Oxidizing agents reduced BSI and increased BSII results. Sensitivity, specificity, and efficiency of 100%, 52.1%, and 53.0% were obtained at manufacturer's proposed cut-offs (BSI 5 µg/L, BSII 30 µg/L). Performance improved if cut-off concentrations increased (BSI 7.5 µg/L, BSII 40 µg/L); however, there were limited confirmed positive specimens. Currently, this is the first and only fully validated immunoassay for preliminary detection of synthetic cathinones in urine. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Prescription drug misuse among U.S. active duty military personnel: a secondary analysis of the 2008 DoD survey of health related behaviors.

OBJECTIVES: This study identifies predictors of prescription drug misuse among U.S. active duty service members (ADSM). The 2008 Department of Defense Survey of Health-Related Behaviors (HRB) Among Active Duty Military Personnel indicated that ADSM misuse pain relievers, tranquilizers, sedatives, and stimulants at levels ranging from 2% to 17%. METHODS: Secondary, multivariate analyses of HRB survey data examined predictors of self-reported prescription drug misuse for 4 distinct drug categories. RESULTS: Receipt of a pain reliever prescription in the past month, year, or previous year were strong predictors (adjusted odds ratio above 2.0) of misuse for all drug categories; receipt of a prescription for anxiety or depression medication in the past year was the strongest predictor of sedative misuse (adjusted odds ratio = 4.46, 95% confidence intervals 3.18-6.24). Absence of a drug testing program was significantly related to the likelihood of drug misuse for all drug categories. CONCLUSIONS: ADSM with a history of treatment for pain and mood disorders, and who self-report headaches, sleep disorders, and fatigue are at higher risk for misusing prescription drugs, perhaps in an effort to self-manage symptoms. The results should be interpreted as a starting place for future exploration, not as the sole basis for policy or program development.

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.

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.

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%.

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.

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.

Comparison of the Microgenics CEDIA heroin metabolite (6-AM) and the Roche Abuscreen ONLINE opiate immunoassays for the detection of heroin use in forensic urine samples.

Current Department of Defense (DoD) and Department of Health and Human Services (HHS) procedures for the detection of heroin abuse by testing urine utilize an initial opiate (codeine/morphine) immunoassay (IA) screen followed by gas chromatography-mass spectrometry (GC-MS) confirmation of 6-acetylmorphine (6-AM), if the morphine concentration is above established cutoff. An alternative to the current opiates screen for heroin abuse is the direct IA for the metabolite of heroin, 6-acetylmorphine. In this regard, the performance of the Microgenics CEDIA heroin metabolite (6-AM) screening reagent was assessed. This evaluation was conducted on the P module of a Hitachi Modular automated IA analyzer calibrated using 6-AM at 10 ng/mL. Reproducibility, linearity, accuracy, sensitivity, and interferences associated with use of the 6-AM IA reagent were evaluated. The IA reagent precision (percent coefficient of variation (%CV)) around each of seven standards was less than 0.63%, with a linearity (r(2)) value of 0.9951. A total of 37,713 active duty service members' urine samples were analyzed simultaneously using the CEDIA heroin metabolite (6-AM) reagent and the Roche Abuscreen ONLINE opiate reagent to evaluate both the prevalence rate of 6-AM in the demographic group and the sensitivity and specificity of the reagents for the detection of heroin use. Of the 37,713 samples tested using the CEDIA heroin metabolite (6-AM) reagent, three samples screened positive at the DoD and HHS cutoff of 10 ng/mL. One of the three samples confirmed positive for 6-AM by GC-MS above the cutoff of 10 ng/mL, the two remaining samples confirmed negative for 6-AM at a GC-MS limit of detection (LOD) of 2.1 ng/mL. In contrast, the Roche Abuscreen ONLINE opiate IA produced 74 opiate-positive results for codeine/morphine, with 6 of the 74 specimens confirming positive for morphine above the DoD cutoff concentration of 4000 ng/mL (8% DoD morphine confirmation rate), only one of the 74 opiate-positive screen specimens confirmed positive for 6-AM above the 10 ng/mL GC-MS cutoff concentration. As a further check of the sensitivity and specificity of the Microgenics 6-AM IA reagent, human urine samples (n = 87) known to contain 6-AM by GC-MS, were re-analyzed using both IA reagents. All 87 of the samples screened positive using the CEDIA heroin metabolite (6-AM) assay. However, using the Roche ONLINE opiate reagent, 12 of the known 6-AM positives screened negative at the DoD and HHS screening cutoff of 2000 ng/mL (morphine). Of the remaining 75 samples that screened positive by the ONLINE opiate reagent, five of the samples did not contain morphine above the DoD GC-MS cutoff concentration of 4000 ng/mL and would not have required 6-AM analysis. However, under the HHS GC-MS morphine cutoff concentration of 2000 ng/mL all 75 samples would have required 6-AM analysis. Furthermore, using the current DoD opiate screen, 17 out of 87 samples known to contain 6-AM would have gone undetected (19.5% false-negative rate); additionally, even under the more stringent HHS opiate screening standards 12 out of the 87 samples known to contain 6-AM would also have gone undetected (13.8% false-negative rate). The Microgenics CEDIA heroin metabolite (6-AM) reagent assay appears well adapted for the rapid and specific detection of heroin abuse as an alternative for, or an adjunct test to, the current opiates (codeine/morphine) IA screening procedure.