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.

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.

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.

Brain concentrations of cocaine and benzoylecgonine in fatal cases.

Since cocaine in blood rapidly hydrolyzes to benzoylecgonine, cocaine concentrations determined in postmortem blood may not reflect the presence or concentration of cocaine in the body at the time of death. The interpretative value of the determination of cocaine and benzoylecgonine in brain tissue was investigated. Cocaine and benzoylecgonine were quantitated by coextraction and formation of the propyl derivative of benzoylecgonine followed by selected ion monitoring gas chromatography/mass spectrometry (GC/MS) using electron ion impact ionization. Cocaine and benzoylecgonine were found to be evenly distributed throughout the brain. Cocaine and benzoylecgonine concentrations were stable in frozen brain tissue (-4 degrees C) on reanalysis after 1 to 3 months of storage, and in refrigerated tissue (10 degrees C) after 30 days of storage. Blood, brain, and liver concentrations of cocaine and benzoylecgonine in 37 cocaine overdose cases and 46 cases in which cocaine was incidental to the cause of death were reviewed. The ratios of cocaine/benzoylecgonine in the toxic cases (brain mean 14.7 and blood mean 0.64) were clearly different from those found in the incidental cases (brain mean 0.87 and blood mean 0.27). The brain/blood ratios of cocaine and benzoylecgonine concentrations generally were characteristic of the time elapsed since cocaine dosing. In cocaine overdose cases, the mean ratio was 9.6 for cocaine and 0.36 for benzoylecgonine. These are within the range found in animal studies for brain/blood ratios of cocaine and benzoylecgonine 0.5 to 2 h after cocaine administration. In incidental cases, the brain/blood ratios were mean 2.5 for cocaine and 1.4 for benzoylecgonine.(ABSTRACT TRUNCATED AT 250 WORDS)

Radioimmunoassay screening and GC/MS confirmation of whole blood samples for drugs of abuse.

From 1981 to 1984, an average of 300 radioimmunoassay screens on whole blood were performed each week in the authors' laboratory. Most samples were screened for opiates, phencyclidine and its analogs, barbiturates, and cocaine or its metabolite benzoylecgonine. A commercially available radioimmunoassay was used with modifications to facilitate screening of whole blood. Increasing sample size increased the sensitivity of the assay. Changing reagent concentration (1:1 dilution), incubation time, sample matrix (water, urine, or blood), or fraction counted (precipitate or supernatant) did not affect the utility of the standard curve or the sensitivity of the assay. All positive results for phencyclidine, opiates, cocaine, and related compounds were confirmed by GC/MS. Barbiturate positives were confirmed by UV spectrophotometry. The rate of confirmation in postmortem bloods from coroner's cases for 1981-84 was: cocaine/benzoylecgonine, 57%; opiates, 79%; phencyclidine, 49%; and barbiturates, 58%.

Digoxin-like immunoreactive substance in postmortem blood of infants and children.

A digoxin-like immunoreactive substance (DLIS) has been reported in the serum of infants not receiving digoxin. This study was undertaken to determine if DLIS is present in the postmortem blood and tissues of infants or children and whether the endogenous substance could interfere with forensic toxicological analysis in suspected overdose. Ninety blood specimens taken from the heart at autopsy of children or infants were screened for DLIS using commercial radioimmunoassay kits. The average age at death in these cases was 8.6 months, the median age was 2 months. DLIS equivalent to 0.25 to 2.0 ng/mL digoxin was found in one third of the cases. The incidence of positive findings was 5/6 stillborns, 10/45 Sudden Infant Death Syndrome (SIDS), 10/15 deaths as a result of infection, 4/7 homicides, 1/8 deaths caused by congenital defects, and 0/9 accidental deaths. The body distribution of DLIS was investigated and highest levels were found in the liver. Findings of DLIS in blood were correlated with renal failure, (elevated vitreous urea nitrogen), electrolyte imbalance, and liver trauma. Apparent concentrations were in the equivalent therapeutic range of digoxin and would not be confused with accidental or intentional overdose with digoxin.

Another fatal case involving hydroxyzine.

Blood and tissue concentrations of hydroxyzine, an antihistamine with sedative properties, were determined in a fatal case of accidental hydroxyzine ingestion. Hydroxyzine was extracted at alkaline pH and analyzed by gas chromatography.

Toxicological findings in amoxapine overdose.

Tissue concentrations of amoxapine, a new antidepressant with antipsychotic properties, were determined in two cases in which amoxapine was taken in overdose. Two extractions and GLC procedures, UV spectra, and TLC properties of amoxapine are described. The concentrations of amoxapine are described. The concentrations of amoxapine in brain tissue (52 micrograms/g and 53.2 micrograms/g) were about equal to concentrations found in liver (77 micrograms/g and 50 micrograms/g) and higher than blood concentrations (6 micrograms/mL and 1.5 micrograms/mL). The two metabolites of amoxapine, 8-hydroxyamoxapine and 7-hydroxyamoxapine, were not detected by TLC or GLC.

The use of brain digoxin concentrations to confirm blood digoxin concentrations.

Recent research suggests that the cardiotoxic as well as the neurotoxic effects of digitalis may be mediated by the central nervous system. Therefore brain regions implicated in the genesis of cardiac rhythm disorders were assayed for digoxin. An 125I-labeled radioimmunoassay was used to determine blood and tissue digoxin concentrations. Digoxin was found in the optic tract and optic chiasm in each of four persons who had been taking digoxin regularly. Digoxin is apparently concentrated from blood by the choroid plexus of the fourth ventricle but not by the choroid plexus of the lateral ventricle. However, digoxin was present in the area postrema and nucleus of the vagus only in the two digoxin overdose cases. Digoxin was not detected in any of the other brain regions analyzed. The presence of digoxin in the area postrema (the chemoreceptor trigger zone) and the nucleus of the vagus in the toxic but not in the therapeutic cases suggests a mechanism for the emesis and cardiac arrest brought about by digoxin toxicity in humans. The digoxin content of the medulla, especially the surface of the medulla under the obex, may be useful in confirmation of elevated blood digoxin concentrations.

Comparison of results for quantitative determination of morphine by radioimmunoassay, enzyme immunoassay, and spectrofluorometry.

The quantitative results (accuracy and precision) for determination of opiates by radioimmunoassay (RIA), enzyme immunoassay (EMIT), and spectrofluorometry on split samples are compared. A variety of physiological samples were studied, including random urine from a methadone maintenance clinic and postmortem urine, blood, bile, brain, and lung tissue from heroin-induced or heroin-related deaths. The opiate concentrations detected by the two immunoassay methods were in good agreement with each other in the absence of interfering substances which are believed to react with the antimorphine antibodies. The immunoassay results were in agreement within the relative standard deviation with the fluorometry results in 55% of the urine samples and 80% of the blood samples. The immunological methods are superior to fluorometry for quantitation of morphine in urine samples due to quenching interferences in fluorometry from urine. They were comparable to fluorometry for quantitation of morphine in blood samples.