Evaluation of a rapid oral fluid point-of-care test for MDMA.

Cozart Bioscience Limited has developed novel lateral flow technology that allows the detection of drugs of abuse in biological fluids and suspect powders. This paper describes the application of this technology for the detection of 3,4-methylenedioxymethamphetamine (MDMA) in oral fluid. Samples (N = 370) were obtained from the analytical laboratory at Cozart Bioscience Limited following their routine analysis for drugs of abuse. Oral fluid samples were screened for the presence of MDMA and methamphetamine using the Cozart RapiScan System (CRS) and then confirmed for the presence of amphetamines (amphetamine, methamphetamine, MDA, MDMA, MDEA, and MBDB) by gas chromatography-mass spectrometry (GC-MS). In addition to the detection of MDMA and methamphetamine, the CRS cross-reacts with high levels of amphetamine to give a positive result. One hundred and twenty-one samples screened positive using the CRS. Six of these samples were confirmed negative for MDMA and methamphetamine, but contained very high levels of amphetamine. Employing a screening cutoff of 45 ng/mL for the CRS and a confirmation cutoff of 30 ng/mL for GC-MS, the sensitivity, specificity, and accuracy were 96.6, 96.8, and 96.8%, respectively. When applying the Substance Abuse and Mental Health Services Administration recommended confirmation cutoff for amphetamines of 50 ng/mL, the sensitivity, specificity, and accuracy increased marginally to 98.3, 96.9, and 97.3%, respectively.

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.

Evaluation of the Cozart RapiScan drug test system for opiates and cocaine in oral fluid.

The purpose of this study was to evaluate the efficiency of the Cozart RapiScan (CRS) drug test system for detecting opiates and cocaine in oral fluid. Oral fluid samples were collected using the Cozart RapiScan collection system from 358 donors who were receiving treatment for their addiction and were monitored for drug misuse. A further 103 oral fluid samples were collected from volunteer donors who were not drug users. The samples were analyzed in the laboratory using the two-panel Cozart RapiScan cartridge for opiates and cocaine and confirmed using gas chromatography-mass spectrometry (GC-MS). The samples were stored frozen at -20 degrees C until analysis by GC-MS. The overall accuracy of the CRS for both opiates and cocaine was 100%. Samples spiked at 50% above and below the cut-off consistently gave negative and positive results respectively. A total of 88 samples were positive for various opiates and 111 samples were positive for cocaine and/or its metabolites. The CRS for opiates and cocaine in oral fluid, using a cut-off of 30 ng/mL morphine or benzoylecgonine equivalents in neat oral fluid, had overall efficiencies of 98% and 99%, respectively, versus GC-MS. 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 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%.

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.

Performance characteristics of the Cozart RapiScan Oral Fluid Drug Testing System for opiates in comparison to ELISA and GC/MS following controlled codeine administration.

Oral fluid is an interesting alternative matrix for drug testing in many environments, including law enforcement, workplace drug testing, and drug treatment facilities. Performance characteristics of the FDA-cleared, qualitative, Cozart RapiScan Opiate Oral Fluid Drug Testing System (Opiate Cozart RapiScan System or Opiate CRS) were compared to the semi-quantitative Cozart Microplate EIA Opiate Oral Fluid Kit (Opiate ELISA) and to gas chromatography/mass spectrometry (GC/MS). The following oral fluid opiate cutoffs were evaluated: the GC/MS limit of quantification (LOQ) of 2.5 mg/l; 15 microg/l currently used for oral fluid testing in the United Kingdom (UK); 30 microg/l (Opiate CRS cutoff); and 40 microg/l, the proposed Substance Abuse and Mental Health Services Administration (SAMHSA) cutoff. Subjects provided informed consent to participate in this IRB-approved research and resided on the closed research ward throughout the study. Three oral codeine doses of 60 mg/70 kg were administered over a 7-day period. After a 3-week break, subjects received three doses of 120 mg/70 kg within 7 days. Oral fluid specimens (N = 1273) were analyzed for codeine (COD), norcodeine (NCOD), morphine (MOR) and normorphine (NMOR) by GC/MS with an LOQ of 2.5 microg/l for all analytes. MOR and NMOR were not detected in any sample; 26.5% of the specimens were positive for COD and 13.7% for NCOD. Opiate CRS uses a preset, qualitative cutoff of 10 microg/l; this is equivalent to 30 microg/l in undiluted oral fluid as the oral fluid collection process involves a 1:3 dilution with buffer. Sensitivity, specificity, and efficiency of Opiate CRS compared to Opiate ELISA were 98.6, 98.1, and 98.2% at a 30 microg/l cutoff and 99.0, 96.2, and 96.6% at a 40 microg/l cutoff. Compared to the much lower GC/MS LOQ of 2.5 microg/l, sensitivity, specificity and efficiency were 66.8, 99.3 and 90.7%. Increasing the GC/MS cutoff to the current UK level yielded performance characteristics of 81.5% (sensitivity), 99.3% (specificity), and 95.4% (efficiency). Using a GC/MS cutoff identical to the preset Opiate CRS cutoff yielded sensitivity, specificity, and efficiency of 88.5, 99.2, and 97.5%, respectively. At the proposed SAMSHA confirmation cutoff of 40 microg/l, sensitivity increased with little change in specificity and efficiency (91.3% sensitivity, 98.9% specificity, and 97.5% efficiency). Oral fluid is a suitable matrix for detecting drugs of abuse. Opiate CRS, with a 30 microg/l cutoff, is sufficiently sensitive, specific and efficient for oral fluid opiate analysis, performing similarly to Opiate ELISA at the same cutoff, and having performance characteristics >91% when compared to GC/MS at the proposed SAMHSA cutoff.

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 the Cozart microplate EIA for cocaine and metabolites in oral fluid.

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

Sensitivity, specificity, and efficiency in detecting opiates in oral fluid with the Cozart Opiate Microplate EIA and GC-MS following controlled codeine administration.

Oral fluid specimens (N = 1406) were collected from 19 subjects prior to and up to 72 h following controlled administration of oral codeine. Volunteers provided informed consent to participate in this National Institute on Drug Abuse Institutional Review Board-approved protocol. A modification of Cozart Microplate Opiate EIA Oral Fluid Kit (Opiate ELISA), employing codeine calibrators, was used for semiquantitative analysis of opiates, followed by gas chromatography-mass spectrometry (GC-MS) for the confirmation and quantitation of codeine, norcodeine, morphine, and normorphine in oral fluid. GC-MS limits of detection and quantitation were 2.5 microg/L for all analytes. The Substance Abuse and Mental Health Services Administration (SAMHSA) has proposed a 40-microg/L opiate screening and a 40-microg/L morphine or codeine confirmation cutoff for the detection of opiate use. Oral fluid opiate screening and confirmation cutoffs of 30 micro g/L are in use in the U.K. Utilizing 2.5-, 20-, 30-, and 40-microg/L GC-MS cutoffs, 26%, 20%, 19%, and 18% of the oral fluid specimens were positive for codeine or one of its metabolites. Six Opiate ELISA/confirmation cutoff criteria (2.5/2.5, 10/2.5, 20/20, 30/20, 30/30, and 40/40 microg/L) were evaluated. Calculations for Opiate ELISA sensitivity, specificity, and efficiency were determined from the number of true-positive, true-negative, false-positive, and false-negative results at each screening/confirmation cutoff. Sensitivity, specificity, and efficiency for the lowest cutoff were 91.5%, 88.6%, and 89.3%. Application of the cutoff currently used in the U.K. yielded sensitivity, specificity, and efficiency results of 79.7%, 99.0%, and 95.4% and similar results of 76.7%, 99.1%, and 95.1% when applying the SAMHSA criteria. These data indicate that the Opiate ELISA efficiently detects oral codeine use. In addition, the data, collected following controlled oral codeine administration, may aid in the interpretation of opiate oral fluid test results and in the selection of appropriate oral fluid screening and confirmation cutoffs.

Cozart RapiScan Oral Fluid Drug Testing System: an evaluation of sensitivity, specificity, and efficiency for cocaine detection compared with ELISA and GC-MS following controlled cocaine administration.

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

Sensitivity and specificity of the Cozart microplate EIA cocaine oral fluid at proposed screening and confirmation cutoffs.

BACKGROUND: Oral fluid is currently being evaluated as an alternative matrix for monitoring illicit drugs in federally mandated workplace drug testing, for addiction treatment programs, and for driving under the influence testing. The sensitivity, specificity, and efficiency of the Cozart Microplate EIA Cocaine Oral Fluid Kit (COC ELISA) were determined by comparison with gas chromatography-mass spectrometry (GC/MS) results at screening and confirmation cutoffs proposed in the US and UK. METHOD: Oral fluid was collected by expectoration after citric acid candy stimulation or with Salivette neutral cotton swabs or Salivette citric acid-treated cotton swabs before and after cocaine (COC) administration. Specimens (n = 1468) were analyzed with the COC ELISA for screening and with solid-phase extraction followed by GC/MS for confirmation. Three screening cutoffs (10, 20, and 30 microg/L) and four GC/MS cutoffs (2.5, 8, 10, and 15 microg/L COC, benzoylecgonine, and/or ecgonine methyl ester) were evaluated. GC/MS limit of quantification was 2.5 micro g/L for all analytes. RESULTS: COC ELISA interassay imprecision (CV; n = 19) was 16% at 16.7 microg/L and 12% at 81.8 microg/L. With the 2.5, 8, 10, and 15 microg/L GC/MS cutoffs, 59.0%, 54.7%, 52.7%, and 48.7% of the oral fluid specimens were positive, respectively. Sensitivity, specificity, and efficiency were 92.2%, 84.7%, and 88.8%, respectively, for the suggested Substance Abuse and Mental Health Services Administration (SAMHSA) cutoffs and 90.2%, 89.2%, and 89.7% for cutoffs currently used in the UK. CONCLUSIONS: COC ELISA had suitable sensitivity, specificity, and efficiency for identifying COC exposure at both the proposed SAMHSA and UK cutoffs. Sensitivity, specificity, and efficiency were >84% for both cutoffs, but 92 additional true-positive samples were identified with the SAMHSA cutoffs.