Gas chromatography-mass spectrometry confirmation of Cozart RapiScan saliva methadone and opiates tests.

The object of this study was to determine the sensitivity and specificity of the Cozart RapiScan onsite saliva test for methadone and opiates versus laboratory-based enzyme immunoassay (EIA) and gas chromatography-mass spectrometry (GC-MS) confirmation. Fifty saliva specimens were obtained from 28 volunteers among persons entering a substance abuse clinic. Specimens were tested onsite using the Cozart RapiScan Saliva test and Cozart RapiScan Reader. Specimens were retested by Cozart Microplate EIA assays on receipt at the laboratory and then frozen for later confirmation by GC-MS. For GC-MS, deuterated internal standards were added to specimen aliquots which were extracted using solid-phase columns at pH 6 and eluted with dichloromethane/isopropanol/ammonia (80:19:2). The dry residues were derivatized with PFOH and PFPA and dried, and the reconstituted extract was injected and quantitated by GC-MS. The Cozart RapiScan Methadone Saliva Assay was found to have a sensitivity and specificity of 100% +/- 12% versus GC-MS (2-ng/mL cutoff) and a sensitivity of 100% +/- 11% and a specificity of 95% +/- 2.4% versus the Microplate EIA for methadone (30-ng/mL cutoff). The Cozart RapiScan Saliva Opiate test had a sensitivity of 100% +/- 12% and a specificity of 92% +/- 3.2% versus GC-MS (2-ng/mL cutoff) and a sensitivity of 96% +/- 2.2% and specificity of 95% +/- 2.4% versus the Microplate EIA for opiates (30-ng/mL cutoff).

Blind trials of an onsite saliva drug test for marijuana and opiates.

The objective of these clinical trials was to calculate the performance, limit of detection, specificity and sensitivity of a novel, semi-quantitative immunoassay for drugs of abuse in saliva and to determine operator bias when measured blind by four different operators. The test is based on lateral flow gold particle technology coupled with digital photography to provide a semi-quantitative end point. The performance of the test was compared with that of enzyme immunoassays and GC/MS methods. Volunteers consumed marijuana or codeine and their saliva was collected 0.25 to 24 h later with the Cozart RapiScan collection device. The sensitivity and specificity of the opiate test were both 100%+/-10.4% for codeine for 9 h after dosing. The cutoff of the marijuana test at 10 ng/mL THCA was too high to detect marijuana use for more than a few hours after smoking. There was no operator bias because the results were presented in written form either as "positive" or "negative" for each of the five drug classes on the screen of the hand-held reader.

Hair analysis by immunological methods from the beginning to 2000.

Immunoassays for hair testing must satisfy three requirements: (1) They must have cross-reactivity with parent drug and lipophilic metabolites actually found in hair (2) they must not experience interference from the dissolved hair matrix and (3) they must be titered for cutoffs appropriate to the drug concentrations found in hair. Because the analytes found in hair after drug use are generally the parent drug or its lipophilic metabolites, immunoassays developed and intended for urine testing are not suitable for hair. Immunoassays whose antibodies are bound to a solid support, such as coated-tube radioimmunoassay or coated-plate ELISA tests, experience less matrix interference than those which use other means of separation of bound and free fractions. Homogenous assays are not suitable for hair testing because the hair matrix frequently interferes in the detection of the signal. Historically radioimmunoassays for drugs of abuse were first used for detecting drugs in hair. Currently ELISAs and coated-plate 96 well microplate EIAs are employed for screening hair digests or extracts for drugs. The optimum cutoffs for immunoassays for drugs in hair should be chosen based on the analyte concentration which produces the fewest false positive or false negative results when applied to tests of hair from known users and non-users of drugs. A hair immunoassay test at these cutoffs should have a sensitivity and specificity of better than 90%. The predictive value of the test will depend on the prevalence of drug use in the tested population. Cutoffs or decision thresholds for immunoassays used for screening for drugs should not be at the limit of detection of the assay because that produces a very large incidence of false positives. Because immunoassays are ligand-binding assays, they have a short range of linearity with low precision at both ends of the range. In the future, immunoassays will continue to be used for screening hair and other matrices for drugs of abuse because they provide rapid, inexpensive automated procedures for separating negative specimens from those which are suspected of containing drugs. For forensic purposes, all positive results must be confirmed by an independent analysis using a procedure based on a different property of the analyte. An immunoassay test should not be confirmed by a second immunoassay test but by a chromatographic test performed on a different dissolved or extracted aliquot of the original specimen.

Validity of drug use reporting in a high-risk community sample: a comparison of cocaine and heroin survey reports with hair tests.

Hair specimens were collected from 322 subjects and analyzed as part of an experimental study administering household surveys during 1997 to a high-risk community sample of adults from Chicago, Illinois. Toxicologic results were compared with survey responses about recent and lifetime drug use. About 35% of the sample tested positive for cocaine, and 4% tested positive for heroin. Sample prevalence estimates of cocaine use based on toxicologic results were nearly five times the survey-based estimates of past month use and nearly four times the survey-based estimates of past year use. With the hair test results as the standard, cocaine and heroin use were considerably underreported in the survey. Underreporting was more of a problem for cocaine than for heroin. Among those who tested positive, survey disclosure of cocaine use was associated with higher levels of cocaine detected in hair. In general, when recent drug use was reported, it was usually detected in hair. When a drug was detected in hair, use was usually not reported in the survey. When heroin was detected in hair, cocaine was almost always detected as well.

Validation of an automated microplate enzyme immunoassay for screening of postmortem blood for drugs of abuse.

The objective of this study was to compare the sensitivity and specificity of an enzyme immunoassay employing antibodies bound to a microtiter plate (MPEIA) with those of two radioimmunoassays for screening postmortem blood from selected coroner's cases for drugs of abuse. The radioimmunoassays were a coated-tube radioimmunoassay (CTRIA) and a double antibody radioimmunoassay (DARIA). Specimens consisted of 260 postmortem blood specimens from coroner's cases. Immunoassay results (positive or negative) were compared with confirmed results on those cases by gas chromatography-mass spectrometry, alone or in combination with gas-liquid chromatography using either a nitrogen-phosphorus or flame-ionization detector. Sensitivity was calculated as the true-positive rate using chromatographic confirmation as the reference standard. Specificity was calculated as the true-negative rate. Sensitivity and specificity were calculated for 5-7 potential cutoff concentrations for the drug classes opiates, amphetamines, cocaine and metabolites, and barbiturates. For opiates, the sensitivity and specificity were 99% and 93%, respectively, for the MPEIA at a cutoff of 20-ng/mL morphine, compared with 94% and 96% for the CTRIA at a cutoff of 5-ng/mL morphine and > 99% and 96% for the DARIA at 20-ng/mL morphine. For cocaine and metabolites, the sensitivity and specificity were 96% and 93%, respectively, for the MPEIA at 50-ng/mL benzoylecgonine, compared with 93% and 96% for CTRIA at 50-ng/mL benzoylecgonine and 98% and 97% for the DARIA at 50-ng/mL benzoylecgonine. For amphetamines, the sensitivity and specificity were >99% and 91%, respectively, for the MPEIA at 25-ng/mL methamphetamine, compared with 93% and 86% for the CTRIA at 25-ng/mL methamphetamine and 83% and 89% for the DARIA at 50-ng/mL methamphetamine. For barbiturates, the sensitivity and specificity were > 99% and 92%, respectively, for the MPEIA at 50-ng/mL secobarbital, compared with 91% and 87% for the CTRIA at 500-ng/mL secobarbital and 79% and 95% for the DARIA at a cutoff of 1000-ng/mL phenobarbital.

Setting cutoff concentrations for immunoassay screening of postmortem blood.

The objective of this study was to establish the optimum immunoassay cutoff concentrations for screening postmortem blood from coroner's cases for drugs of abuse with a coated tube radioimmunoassay (RIA) to ensure that the results with the coated tube RIA would be equal to or better than those with the previously used double antibody RIA. Immunoassay results (positive or negative) blood were compared to confirmed results on those cases by GC/MS alone or in combination with GLC using either a NPD or FID detector. Four to seven potential cutoff concentrations were evaluated for the drug classes opiates, amphetamines, cocaine and metabolites, and barbiturates. Specimens were 350 postmortem blood specimens and liver homogenates. The cutoffs chosen for the coated tube RIA using this approach were 5 ng/mL morphine, 25 ng/mL methamphetamine, 500 ng/mL benzoylecgonine, and 500 ng/mL secobarbital. These cutoffs corresponded to a sensitivity and specificity of 94% and 96% for opiates, 93% and 86% for amphetamines, 91% and 96% for cocaine and metabolites and 91% and 87% for barbiturates. The double antibody RIAs were run on the same specimens with cutoffs of 20 ng/mL morphine, 50 ng/mL methamphetamine, 50 ng/mL benzoylecgonine and 1000 ng/mL phenobarbital. The sensitivity and specificity's for the double antibody immunoassay were: > 99% and 96% for opiates, 83% and 89% for amphetamines, 98% and 97% for cocaine, 79% and 95% for barbiturates.

Qualitative detection of opiates in sweat by EIA and GC-MS.

Sweat was collected with the PharmChek sweat patch, and drugs were eluted from the collection pad of the patch. A solid-phase enzyme immunoassay (EIA) using microtiter plates was modified for the analysis of opiates in sweat. After opiate administration, sweat contains primarily parent opiate (heroin, codeine) and lipophilic metabolites (6-monoacetylmorphine [6-MAM]). The immunoassay was determined to have a cross-reactivity with codeine of 588%, with hydrocodone of 143%, with diacetylmorphine of 28%, and with 6-MAM of 30% relative to 100% for the morphine calibrators. The optimum cutoff concentration for this modified assay was determined by receiver operator characteristic analysis using 215 patches from 95 subjects to be 10 ng/mL morphine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 86.9% and a diagnostic specificity of 92.8% versus gas chromatography-mass spectrometry (GC-MS), which was the reference method. The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 7.8%, and the interassay coefficient of variation (CV) was 39%. Analysis of spiked patches around the cutoff gave a percent positive threshold of approximately 50% between 10 and 15 ng/mL and a 95% confidence level for a positive result by the EIA between 20 and 25 ng/mL. Eighteen possible adulterants that could be injected into or under the patch were studied. Two (tile cleaner and detergent) can cause false-positive responses in the immunoassay. Two adulterants reduced response to spiked drug (Visine eye drops and Ben Gay ointment), which could cause a false-negative response. All results were confirmed by GC-MS. The clinical sensitivity and specificity for detecting drug use by analyzing sweat collected from human subjects following known doses of codeine (0, 30, and 60 mg orally) or heroin (20 mg intravenously) were 76 and 100%, respectively.

Detection of methamphetamine in sweat by EIA and GC-MS.

Sweat was collected with the PharmChekTM sweat patch and drugs were eluted from the collection pad of the patch. A solid phase, enzyme immunoassay using microtiter plates was modified for analysis of methamphetamine in sweat. After methamphetamine administration, sweat contains primarily parent methamphetamine. The immunoassay was determined to have crossreactivity relative to 100% for the methamphetamine (MA) calibrators; to 144% for methylenedioxymethamphetamine (MDMA); to 30% for d-amphetamine; to 21% for methylenedioxyamphetamine (MDA); and to 8% for I-methamphetamine. The optimum cutoff concentration for this modified assay was determined by receiver operating characteristic analysis to be 10 ng/mL amphetamine equivalents. At this cutoff concentration the assay had a diagnostic sensitivity of 84.5% and a diagnostic specificity of 93.2% versus gas chromatography-mass spectrometry (GC-MS). The positive predictive value at a prevalence of 50% was 86%. The intra-assay precision at 10 ng/mL was 9.9% (coefficient of variation, CV) and the interassay CV was 13%. Analysis of spiked patches at plus or minus 25 and 50% around the cutoff gave a percent positive threshold of approximately 50% at a cutoff of 10 ng/mL and a 95% confidence level for a positive result by the EIA between 15 and 20 ng/mL. Of 18 potential adulterants that might be injected into or under the patch, two (tile cleaner and cough syrup) caused a false-positive response by immunoassay. All results were confirmed by GC-MS. The clinical sensitivity and specificity of the overall analysis system (sweat collection and analysis) were 85 and 100%, respectively, using known methamphetamine dosing of volunteers (10, 20, and 25 mg) as the reference standard.

Enzyme immunoassay validation for qualitative detection of cocaine in sweat.

A solid-phase enzyme immunoassay (EIA) involving microtiter plates was modified for analysis of cocaine in sweat. Sweat was collected with the PharmChek sweat patch and drugs were eluted from the collection pad of the patch. The sweat contained primarily parent cocaine. The assay was determined to have cross-reactivity for cocaine of 102% relative to 100% for the benzoylecgonine (BE) calibrators and for cocaethylene of 148%. The optimum cutoff concentration for this modified assay, determined by receiver-operating characteristic curve analysis, was 10 micrograms/L cocaine or BE equivalents. At this concentration the assay had 94.5% sensitivity and 99.1% specificity vs gas chromatography-mass spectrometry (GC-MS) as an acceptable indicator of the true clinical state. The positive predictive value at a prevalence of 50% was 99%. Threshold analysis for positives suggested that the 95% confidence interval for a positive result by the EIA was between 12.5 and 15 micrograms/L and that quality-control samples at 5 and 15 micrograms/L could be run with each batch to certify the precision around the cutoff. All positive samples must be confirmed by GC-MS. The sensitivity and specificity of the overall analysis system (immunoassay screen and GC-MS confirmation) was 86% and 97%, with known cocaine dosing of volunteers as the acceptable indicator of the true clinical state.

Analytical requirements, perspectives and limits of immunological methods for drugs in hair.

The analytical requirements for analysis of drugs in hair are sensitivity in the range of picograms per milligram of hair, specificity for lipophilic drugs and absence of matrix effects with hair digests. These requirements are met by immunoassays which are also inexpensive, rapid and easy to use. However, in applying immunoassays to hair testing, certain limitations of the assay and of interpretation of assay results should be kept in perspective. These limitations are illustrated in this review with examples of the analysis of opiates in hair from patients and opiate addicts. The first requirement for immunological analysis of hair digests is that the digest must not denature the antibody proteins of the immunoassay reagents. For this reason enzymatic digests are better for immunological assay than chemical digests. Strongly acidic or alkaline digests must be brought to a neutral pH before immunoassay. Immunoassays used for analysis of hair should be calibrated with spiked hair digest standards to correct for possible matrix effects. The second requirement is that the immunoassay have the sensitivity and specificity to detect the drug in hair. Drugs of abuse are found in hair in the range of 10 pg-10 ng/mg hair. Radioimmunoassays are capable of detection and quantitation in this concentration range. Although the mechanism of drug incorporation into hair is not known, it is now apparent that primarily the parent drug and lipophilic metabolites are found in hair. For example, the ratio of cocaine/benzoylecgonine averages 10 (range 2-50) in published reports of analysis of hair from cocaine users. Therefore, immunoassays which are highly sensitive for the parent drug are required and results of immunoassays should be expressed as equivalents. When spiking standards for calibration of hair digest immunoassays, parent drug known to be present in hair should be used, e.g. cocaine not benzoylecgonine. With immunoassays which are specific for the lipophilic metabolite found in hair such as 6-MAM, differential radioimmunoassay can be used to discriminate between medical and illicit sources for the opiate drugs found in hair. Because of the low concentrations of drugs encountered in hair, immunoassays for hair have been used at cutoff concentrations at their limits of detection. The limit of detection (LOD) has been determined by calculating the mean and standard deviation (S.D.) for the assay response for a number of negative hair samples. The cutoff was then set at a distance of 2, 3, or 5 S.D.s from the mean response.(ABSTRACT TRUNCATED AT 400 WORDS)

Whole blood deproteinization for drug screening using automatic pipettors.

Automating the initial screening of postmortem blood and other tissues for drugs of abuse requires a pipetting procedure that is compatible with immunoassay screening tests. The deproteinization procedure uses a zinc sulfate-5-sulfosalicylic acid reagent to precipitate blood proteins. This procedure for deproteinization of whole blood and tissue homogenates is compatible with coated tube radioimmunoassays (RIA) and can therefore be used in automating the initial screening step.

A comparison of three computer models for prediction of dose in acute amitriptyline overdose.

The pharmacokinetics of amitriptyline in overdose have been reported not to fit conventional compartmental models. In this study, the dose-concentration-time relationships of amitriptyline in overdose were modeled with discriminant analysis, with an evolutionary heuristic search program, and with a decision-tree model based on the entropy of uncertainty of classification. The computer models all used the same data from dogs administered treatment (80 mg/kg), toxic (250 mg/kg), or fatal (500 mg/kg) doses directly into the surgically isolated duodenum. All the models achieved a high degree of success (77 to 93%) in assigning records to the high-, low-, or middle-dose groups. Two of the models gave a probability of the assignment. Results of this analysis suggest that blood amitriptyline and nortriptyline concentrations are most useful in estimating dose in acute amitriptyline overdose.

Whole blood quality assurance control samples for forensic toxicology.

Pilot studies were undertaken to examine the feasibility of preparing freeze-dried whole blood samples containing drugs of interest to be used as assay controls. The availability of such samples will enable analysts and laboratory managers to monitor analytical performance. A pilot batch of freeze-dried blood containing five compounds of forensic significance and a blank blood sample were prepared for reconstitution to 2.5-mL aliquots. Samples of each of these preparations were distributed to 55 laboratories in the USA and UK for analysis. A summary of the results is presented along with comments on the feasibility of producing freeze-dried whole blood samples for use in forensic toxicology.

Methamphetamine in antemortem blood and urine by radioimmunoassay and GC/MS.

Methamphetamine abuse is increasing and methamphetamine is second only to alcohol as a positive finding in cases submitted to the San Diego Sheriff's Crime Laboratory. In general, whole blood specimens are submitted more often than urine. A modified version of a commercially available radioimmunoassay, Coat-A-Count (CAC) Methamphetamine, was investigated as a screen for methamphetamine in whole blood and urine. The assay was modified by using 100 microL of sample, making up standards in whole beef blood, extending the incubation time to 2 h or overnight, and using a cutoff reference of 50 ng/mL methamphetamine. The detection limit for the CAC Methamphetamine kit was 20 ng/mL methamphetamine in whole blood. The CAC Methamphetamine results were compared to Abuscreen Amphetamine High Specificity results and to gas chromatography/mass spectrometry (GC/MS) quantitation of amphetamine and methamphetamine for 157 positive and 48 negative blood specimens. With the CAC Methamphetamine assay there were 2 false negatives detected, both less than the 50 ng/mL cutoff level. There were 12 (6%) false positives with the CAC Methamphetamine assay and 29 (14%) false positives with the Abuscreen Amphetamine assay. Of the positive samples, 95% contained only methamphetamine, with an average concentration of 308 ng/mL, range 25-2030 ng/mL.

Computer-assisted interpretation in forensic toxicology: morphine-involved deaths.

Case data from 200 morphine-involved deaths (Spiehler, V. and Brown, R., Journal of Forensic Sciences, Vol. 32, No. 4, July 1987, pp. 906-916) were analyzed for patterns and relationships using artificial intelligence (AI) computer software. Case parameters were blood unconjugated morphine, blood, brain, and liver total morphine, sex, age, frequency of use, time of death after injection, cause of death, and presence of other drugs. The programs used were Expert 4 (Biosoft-Cambridge), BEAGLE (Warm Boot, Ltd.), and KnowledgeMaker (Knowledge Garden Inc.). Interpretation was defined as estimating the dose, response, and time after drug dosing. The AI programs were used to advise on time and response outcomes for cases, to calculate the probability of the estimate being true, to develop rules for interpretation of morphine-involved cases, and to diagram a decision tree. On known cases the AI programs were successful 70 to 90% of the time in classifying the cases as to response and time. No data on dose were available in this database. The success rate in individual cases was proportional to the program-estimated probability. All three programs found the case parameters of most value in predicting response to be blood unconjugated morphine, blood total morphine, and liver total morphine. The case data most useful in estimating time of death since drug injection were blood unconjugated morphine, percent unconjugated morphine in blood, and brain total morphine. The rule induction programs found that morphine overdoses were characterized by blood unconjugated morphine greater than 0.24 micrograms/mL, liver morphine greater than 0.50 to 0.75 micrograms/g, brain morphine greater than 0.08 micrograms/g or greater than blood unconjugated morphine, and percent blood unconjugated morphine greater than 37%. Rapid deaths were characterized by percent unconjugated morphine greater than 44 to 50%; blood unconjugated morphine, as a function of other drugs present, greater than 0.09 to 0.21 micrograms/mL; and brain total morphine greater than 0.16 to 0.22 micrograms/g. This work demonstrates that inexpensive AI programs commercially available for personal computers can be useful in interpretation in forensic toxicology.

Confirmation and certainty in toxicology screening.

Confirmation of presumptive positive urine drug screens, necessary to minimize the reporting of false-positive results, can be costly and time-consuming. The predictive value model can be used to select the confirming tests and to calculate the confidence of the result. The predictive value of a test result is the probability, based on the sensitivity and specificity of the test, that the result is a true positive or a true negative. The predictive value model applied to toxicology screening tests for drugs of abuse showed that prevalence, in addition to sensitivity and specificity, was the factor controlling the confidence level of a result. For example, the predictive value of a positive result for a screening test that has a sensitivity of 99% and a specificity of 99%, applied to screening in a population with a prevalence of 1% is 0.50; for a prevalence of 10%, it is 0.92. Confirmation with a second, chemically independent, test of equal sensitivity and specificity increases the predictive value to 0.99.