Validation of the Cozart Amphetamine Microplate EIA for the analysis of amphetamines in oral fluid.

The purpose of this study was to determine the performance characteristics of the Cozart Amphetamine Microplate EIA for detecting amphetamine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan Collection System from 135 volunteer donors from drug treatment clinics. A further 35 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the Cozart Amphetamine Microplate EIA and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart Amphetamine Microplate EIA for amphetamine in oral fluid over forty assays was 2.74-7.1% CV (within assay) and 3.4-7.0% CV (within day). A total of 78 samples were positive for various amphetamines and related designer drugs. The Cozart Amphetamine Microplate EIA, using a cutoff of 45 ng/ml amphetamine equivalents in neat oral fluid, had a sensitivity of 91.7+/-3.3% and a specificity of 95.9+/-1.9% versus GC-MS using a cutoff of 30 ng/ml. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Validation of the Cozart microplate EIA for analysis of opiates in oral fluid.

The purpose of this study was to determine the performance characteristics of the Cozart microplate EIA for detecting opiates in oral fluid from patients in a drug misuse treatment program. Oral fluid samples were collected using the Cozart RapiScan Collection System from 216 donors who were receiving treatment for their addiction and were monitored for drug abuse. A further 40 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory by using the Cozart microplate EIA for opiates and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen until analysis by GC-MS. The intra-assay precision for the Cozart microplate oral fluid EIA for opiates over 40 assays was 0.43% to 9.13% CV (within assay) and 2.9% to 9.1% CV (within day). A total of 109 samples were positive for various opiates. The Cozart microplate EIA for opiates in oral fluid, using a cut-off of 30 ng/ml morphine equivalents in neat oral fluid, had a sensitivity of 99.1 +/- 2.1% and a specificity of 94.4 +/- 2.2% versus GC-MS. A series of potential adulterants of oral fluid were evaluated and shown not to alter the outcome of the test result.

Comparison of GC-MS and EIA results for the analysis of methadone in oral fluid.

The purpose of these studies was to evaluate the performance characteristics of the Cozart Microplate Enzyme Immunoassay (EIA) for the determination of methadone in oral fluid from patients in a drug misuse treatment program. Oral fluid specimens were collected using the Cozart RapiScan Collection system from 198 donors who were receiving treatment for their addiction and were monitored for drug misuse. Oral fluid specimens were also collected from forty volunteer donors who were not drug users. The specimens were analyzed in the laboratory by EIA and then analysed for methadone and its main metabolite EDDP by gas chromatography-mass spectrometry (GC-MS). A total of 103 samples were confirmed positive for methadone. The Cozart Microplate EIA for d-Methadone in oral fluid using a cutoff of 30 ng/mL in diluted oral fluid had a sensitivity of 91.3% +/- 2.8% and a specificity of 100% +/- 1.0% vs. GC-MS.

Comparison of Cozart microplate ELISA and GC-MS detection of methadone and metabolites in human hair.

The purpose of this study was to determine the performance characteristics of the Cozart Methadone Microplate ELISA assay for the detection of methadone and methadone metabolites in hair specimens. One hundred and ten hair specimens were collected from volunteers (n=46) with a history of drug use and from drug-related deaths (n=64). The hair samples (approximately 20 mg) were extracted by sonication in methanol followed by overnight extraction in methanol at 60 degrees C. The methanol extract was evaporated to dryness, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture [methadone-d9 and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)-d3] and 0.1M HCI were added to approximately 20 mg of specimen or spiked blank hair and sonicated for 1 h. The pH was adjusted to neutral, and methadone and its primary metabolite, EDDP, were analyzed by GC-MS following solid-phase extraction using Bond Elute Certify columns and pH 7.4 phosphate buffer (0.1M). Forty hair specimens were confirmed positive for methadone by GC-MS. Concentrations ranged from 0.10 to 8.3 ng/mg for methadone and 0.1 to 1.2 ng/mg for EDDP. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results with a cutoff of 0.1 ng/mg for both methadone and EDDP as the reference method. The optimum cutoff for the Cozart Methadone Microplate ELISA was determined to be between 200 and 300 pg methadone equivalents/mg hair using a 20 mg hair sample. The Cozart Methadone Microplate EIA for methadone and metabolites in hair using a cut-off of 200 pg/mg hair with a 20 mg hair sample had a sensitivity of 95 +/- 2% and a specificity of 100 +/- 3.5% (vs GC-MS) and an accuracy of 98.2 +/- 1.3%.

Screening postmortem whole blood for oxycodone by ELISA response ratios.

The objective of this study was to investigate the accuracy of screening postmortem whole blood for oxycodone using the ratio of the oxycodone immunoassay response to the response for the specimen obtained with a general opiate-class immunoassay. Fifty eight specimens which were negative for opiates and 158 postmortem whole blood specimens positive for opiates including 66 specimens known to contain oxycodone were assayed. Specimens were diluted 1:5 with assay buffer and analyzed by both the Neogen Oxymorphone/Oxycodone ELISA and the Neogen Opiate Group ELISA (Neogen Corporation, Lexington KY). The oxycodone equivalents in ng/mL from the Oxymorphone/Oxycodone ELISA were divided by the morphine equivalents in ng/mL from the Opiates ELISA to obtain an Oxycodone/Opiates Response Ratio. This ratio was compared with the GC/MS data for all specimens and for opiate positive specimens. Receiver Operating Characteristic (ROC) analysis suggested that optimum relative response ratio was 2.0. The sensitivity of the ELISA response ratio for the presence of oxycodone at a response ratio cutoff of 2.0 was 89.4% +/- 3.8% and the specificity was 88.1% +/- 3.2%. Specimens with a ratio of 2.0 or higher had a greater than 50% probability (positive predictive value) of containing oxycodone in a population with a greater than 15% prevalence of oxycodone.

The utility of drug testing in epidemiological research: results from a general population survey.

AIMS: To assess the utility of biological testing in a general population survey for estimating prevalence and evaluating self-report data quality. DESIGN: An audio computer-assisted interview was administered to subjects from June 2001 to January 2002. Immediately following the interview, subjects were requested to participate in hair, oral fluid and urine testing. SETTING: Subjects were from randomly selected households in the City of Chicago using multi-stage sampling methods. Interviews were conducted in subjects' homes. PARTICIPANTS: The data represent 627 randomly selected adult participants, ages 18-40 years. MEASUREMENTS: Prevalance, kappa, conditioned kappa, sensitivity, specificity, under-reporting, 'mixed model' and logistic regression. FINDINGS: Higher rates of marijuana use were generated from survey reports than from drug testing. Drug testing generated higher prevalence rates than survey reports for recent use of cocaine and heroin. Under-reporting of recent drug use was apparent for all three substances. Sensitivity was particularly low for cocaine and heroin. Race was related to under-reporting, with African Americans less likely to report marijuana use despite a positive test result. CONCLUSIONS: The utility of drug testing for surveys depends on the type of substance examined as well as on the type of test employed. Multiple tests have more utility than a single test. Drug testing is useful for identifying the levels and sources of under-reporting in a survey and provides a basis for adjusting prevalence estimates based on self-reports.

Performance of a microtiter plate ELISA for screening of postmortem blood for cocaine and metabolites.

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).

Validation of the Cozart microplate ELISA for detection of opiates in hair.

The purpose of this study was to determine the performance characteristics of the Cozart Opiates Microplate ELISA assay for the detection of opiates in hair specimens. One hundred and six hair specimens were collected from volunteers and from drug-related deaths. The hair samples were extracted by sonication followed by overnight extraction in methanol at 60 degrees C. The methanol extract was dried, reconstituted in ELISA negative calibrator, and then analyzed. For gas chromatography-mass spectrometry (GC-MS) analysis, deuterated internal standard mixture and 0.1M HCl were added to 20 mg of specimen or spiked blank hair and sonicated for 1 h. The opiates were extracted by solid-phase and derivatized with BSTFA + 1% TMS for GC-MS analysis. Fifty-one hair specimens were confirmed positive by GC-MS. The true positives, true negatives, false positives, and false negatives for different cutoffs with the ELISA were determined by comparison of the ELISA response (normalized to weight of hair extracted) to the GC-MS results as the reference method. The optimum cutoff for the Cozart Opiate Microplate ELISA was determined to be between 200 and 300 pg morphine equivalents/mg hair using a 20-mg hair sample. The Cozart Opiates Microplate EIA for opiates in hair using a cutoff of 200 pg/mg hair with a 20-mg hair sample had a sensitivity of 98% +/- 2% and a specificity of 92.7% +/- 3.5% versus GC-MS.

Validation of a microtiter plate ELISA for screening of postmortem blood for opiates and benzodiazepines.

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for opiates and benzodiazepines for screening of postmortem blood. Ninety postmortem whole blood specimens were obtained from drug-involved deaths which had been screened and confirmed positive for opiates and/or benzodiazepines. Forty negative specimens were obtained from non-opiate-involved deaths. Specimens were tested using the Neogen Opiates Group and Neogen Benzodiazepines Group microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of opiates and benzodiazepines encountered in medical examiner specimens. True positive, true negatives, false positives, and false negatives were determined and graphed for the ELISA results against gas chromatography-mass spectrometry (GC-MS), gas chromatography-nitrogen-phosphorus detection and case histories. From these graphs and the ROC curves, the optimal cut-off for the Neogen Opiates Group ELISA was found to be between 20 and 50 ng/mL morphine equivalents and the optimum cut-off for the Neogen Benzodiazepines Group ELISA was between 20 and 50 ng/mL temazepam equivalents. The Neogen Opiates Group ELISA had a sensitivity of 95.2% +/- 2.7% and a specificity of 92.2% +/- 3.4% versus GC-MS at a cut-off of 20-ng/mL cut-off and a sensitivity of 88.8% +/- 3.9% and specificity of 96.8% +/- 2.1% versus GC-MS at a 50-ng/mL morphine equivalents cut-off. The Neogen Benzodizepines Group ELISA had a sensitivity of 100% +/- 1.3% and a specificity of 94.6% +/- 2.9% versus GC-MS (20-ng/mL temazepam equivalents cut-off) and a sensitivity of 95.8% +/- 2.5% and specificity of 98.2% +/- 1.8% versus GC-MS at a 50-ng/mL cut-off.

Choice of an ELISA assay for screening postmortem blood for amphetamine and/or methamphetamine.

The object of this study was to evaluate the suitability of the Neogen Corp. microtiter plate enzyme-linked immunoassays (ELISA) for the screening of postmortem blood for amphetamine and methamphetamine and to choose the more appropriate assay for screening. Forty-seven postmortem whole blood specimens were obtained from drug-involved deaths, which had been screened and confirmed positive for methamphetamine and/or amphetamine. Eighty-five negative specimens were obtained from non-amphetamines-involved deaths, 17 of which involved decomposition. Specimens were tested using the Neogen Amphetamine Ultra and Neogen Methamphetamine/MDMA microtiter plate ELISA assays. No matrix effects were found for whole blood in these assays, and a dilution of 1:5 was chosen to facilitate pipetting and to bring the IC50 of the microtiter plate ELISA assay within the range of amphetamines concentrations encountered in medical examiner specimens. True positives, true negatives, false positives, and false negatives were determined relative to gas chromatography-mass spectrometry (GC-MS) and graphed for the ELISA. From these graphs and the receiver operating curves (ROC), the optimal cut-off for the Neogen Methamphetamine/MDMA ELISA was 50 ng/mL methamphetamine equivalents and the optimum cut-off for the Neogen Amphetamine Ultra ELISA was 100 ng/mL amphetamine equivalents. The Neogen Methamphetamine ELISA had a sensitivity of 93.6% +/- 3.5% and a specificity of 77.6% +/- 4.5% versus GC-MS at the cut-off of 50-ng/mL methamphetamine equivalents. The Neogen Amphetamine Ultra ELISA had a sensitivity of 95.7% +/- 3.0% and a specificity of 72.9% +/- 5.2% versus GC-MS at the 100-ng/mL amphetamine equivalents cut-off. The areas under the ROCs were equivalent for the two ELISA assays.