Journal of Analytical Toxicology

Bogusz, Maciej J.; Maier, Rolf-Dieter; Krüger, Klaus-Dieter; Kohls, Ulrike

doi: 10.1093/jat/22.7.549pmid: 9847004

A method for determining opiate agonists (morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-monoacetylmorphine, codeine, codeine-6-glucuronide, dihydrocodeine, dihydromorphine, buprenorphine, methadone, tramadol, and ibogaine), cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester) and lysergic acid diethylamide in serum, blood, urine and other biological matrices is presented. Aliquots (0.5–1.5 mL) of biological fluids were spiked with appropriate deuterated internal standards and extracted using a common solid-phase extraction method (C18 cartridges). The extracts were subjected to liquid chromatographic-atmospheric-pressure chemical-ionization mass spectrometric examination using selected ion monitoring procedures. These procedures were developed after analysis of full-scan mass spectra of examined compounds. The extraction method appeared very universal; the recoveries were high for almost all drugs and the extracts were very clean. The procedure was applied for routine forensic casework.

Divanon, F.; Debruyne, D.; Moulin, M.; Leroyer, R.

doi: 10.1093/jat/22.7.559pmid: 9847005

A total of 588 blood specimens collected in an emergency unit were screened for benzodiazepines (BZDs) using enzyme-multiplied immunoassay and gas chromatography. Two-hundred eighty-five samples were positive for BZDs, and 303 samples that were negative by EMIT® included 20 samples with BZDs detectable by gas-liquid chromatography. A total of 15 BZDs were identified, and the most frequently occurring were nordiazepam, bromazepam, diazepam, and alprazolam. Individual BZDs were found in 74% of cases, but some samples contained two, three, or even four BZDs. There is a risk of missing intoxication by BZDs with low therapeutic range and/or low cross-reactivity (alprazolam, bromazepam, flunitrazepam). There is a risk of misinterpreting a positive result for some BZDs with high therapeutic range and/or high cross-reactivity (nordiazepam), which may reflect a pharmacologically ineffective concentration. A semiquantitative analysis is inappropriate even when the identity of BZD is known. Immunoassays are the only methods presently available for use in emergencies, but physicians must be clearly informed of their limitations and interpret results with caution.

Ishida, Takashi; Yano, Masashi; Toki, Satoshi

doi: 10.1093/jat/22.7.567pmid: 9847006

Codeinone-glutathione adduct (CO-GSH) in the bile of guinea pigs given a subcutaneous injection of codeine was isolated and identified. Synthesized authentic CO-GSH was characterized by the mass and nuclear magnetic resonance spectra and used as the standard sample. The metabolite was isolated by preparative high-performance liquid chromatography on a C18 column. The fractions containing the conjugated metabolite were purified using Sep-Pak C18 cartridges. For further purification of the metabolite CO-GSH, a Radial Pak CN column was used. Structure assignment of the metabolite was then performed by fast-atom-bombardment mass spectrometry and 500 MHz Fourier-transform-NMR spectrometric analysis and identified as S-[4,5-epoxy-3-methoxy-17-methyl-6-oxomorphinan-(8S)-yl] glutathione.

Spiehler, Vina R.; Collison, Ines B.; Sedgwick, Paul R.; Perez, Susan L.; Le, Sam D.; Farnin, Dawn Ann

doi: 10.1093/jat/22.7.573pmid: 9847007

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.

Preston, Kenzie L.; Goldberger, Bruce A.; Cone, Edward J.

doi: 10.1093/jat/22.7.580pmid: 9847008

As part of ongoing research efforts to improve methods of monitoring drug use in treatment patients, the presence of cocaine in urine specimens was evaluated as a possible marker for recent illicit cocaine use. A total of 2327 urine specimens collected during a 17-week clinical trial of a cocaine-abuse treatment study were tested. Cocaine was measured by gas chromatography-mass spectrometry, and benzoylecgonine (BZE) equivalents were determined by fluorescence polarization immunoassay (FPIA). More than one-third of the specimens were positive (> 25 ng/mL) for cocaine (36.8%), and nearly two-thirds were positive (> 300 ng/mL) for cocaine metabolite by FPIA (62.7%). Median concentrations of cocaine and BZE equivalents were 235 and 14,900 ng/mL, respectively, and maximum concentrations were 112,025 and 1,101,190 ng/mL in cocaine- and BZE-positive specimens, respectively. There were 52 specimens that contained cocaine in equal or higher concentrations than BZE equivalents. No significant differences in cocaine or BZE concentrations between Caucasian and African-American or between male and female patients were found. Cocaine was present less frequently and at lower concentrations than BZE but more frequently than expected based on an average half-life of approximately 1 h, which suggests that cocaine may exhibit a longer terminal half-life and/or that accumulation of cocaine can occur in chronic, heavy users.

Bailey, David N.

doi: 10.1093/jat/22.7.587pmid: 9847009

The relative binding of acetaminophen, lidocaine, phenobarbital, procainamide, quinidine, and theophylline to sera of seven mammalian species was studied. Pooled commercial sera from cow, goat, horse, human, pig, rabbit, and sheep were supplemented with 5 and 10mM concentrations of each drug. For each serum, each drug, and each drug concentration, equilibrium dialysis was performed in duplicate against phosphate buffer (pH 7.4, 0.1M, 4°C). Percent drug bound to serum was calculated. Phenobarbital demonstrated more than 20% binding to goat, horse, human, and sheep serum at both 5 and 10mM concentrations; more than 20% binding to bovine serum at a concentration of 10mM; and more than 20% binding to pig and rabbit serum at 5mM. Quinidine (studied only at 5mM concentration) bound more than 20% to cow, goat, horse, human, pig, and rabbit serum. In contrast, procainamide at both the 5 and 10mM concentrations showed no binding to cow, horse, pig, rabbit, or sheep serum. Acetaminophen (studied only at 5mM concentration), lidocaine, and theophylline demonstrated less than 20% binding to each serum. Acetaminophen at 5mM did not bind to human serum, and lidocaine at 10mM did not bind to horse or pig serum. Although some interspecies variation in drug binding to the seven sera was noted, the overall magnitude of binding of each drug to each serum was, for the most part, similar. Phenobarbital and quinidine showed stronger (> 20%) binding; procainamide showed negligible binding; and acetaminophen, lidocaine, and theophylline demonstrated intermediate (< 20%) binding.

Tsuchihashi, Hitoshi; Nishikawa, Mayumi; Igarashi, Kazuo; Tatsuno, Michiaki; Katagi, Munehiro; Kasuya, Fumiyo; Fukui, Miyoshi

doi: 10.1093/jat/22.7.591pmid: 9847010

A simple, rapid, and sensitive method which allows us to simultaneously determine bromvalerylurea (BVU) and its three metabolites (3-methylbutyrylurea [MVU], α-(cystein-S-yl)isovalerylurea [CVU], and α-(N-acetylcystein-S-yl)isovalerylurea [AcCVU]) was investigated by frit-fast atom bombardment liquid chromatography-mass spectrometry (frit-FAB LC-MS). The LC-MS analysis was performed after the solid-phase extraction from tissue and urine samples with a Sep-Pak C18 cartridge. Tissue homogenates and urine were adjusted to pH 4.0 and applied to the cartridges. The retained BVU and its metabolites were eluted from the cartridge with 2 mL of acetonitrile/10mM ammonium acetate buffer (pH 3.5, 50:50, v/v). The eluate was analyzed by LC-MS, which employs a semimicro type L-column ODS column. The proposed conditions are as follows: mobile phase A, 0.4% glycerol in acetonitrile/10mM ammonium acetate buffer (pH 3.5) (5:95, v/v); mobile phase B, 0.4% glycerol in acetonitrile; elution mode, linear gradient, 100% A (5 min) to 100% B in 15 min; flow rate, 0.2 mL/min; split ratio, 1:40. Extraction recoveries of BVU and its metabolites were 91.90–97.79% from the spiked liver homogenate and 89.68–96.13% from the spiked urine. The detection limits ranged from 10 to 25 ng/g in selected ion monitoring mode.

Delahunty, Tom; Schoendorfer, Don

doi: 10.1093/jat/22.7.596pmid: 9847011

Caffeine and two metabolites (paraxanthine and theobromine) were quantitated by high-performance liquid chromatography (HPLC) using extracts from transdermal sweat patches that continously collected and stored analytes lost through the skin. Following caffeine consumption, remarkably clean chromatograms were obtained with minimal sample preparation. Caffeine and paraxanthine accumulated in the patch at comparable rates, and theobromine accumulated more slowly. A major urinary metabolite, 1-methylxanthine, was notably absent in sweat-patch and plasma extracts, a finding which favors a renal source for this metabolite. A simple, noninvasive approximation of N-demethylation can be made by calculating the paraxanthine/caffeine and theobromine/caffeine ratios in the patch extract. These ratios were significantly reduced in high-dose (600 mg) versus low-dose (200 mg) subjects, possibly reflecting a decreased clearance of caffeine. Because the sweat patches can he worn for several days, the technique gives a multiday historical record which reflects the fluctuating systemic concentration of caffeine and its hepatic metabolites and thus might be useful to noninvasively monitor compliance by caffeine-restricted patients or to assess drug-metabolizing status.

Verstraete, Alain G.; Steyaert, Sophia

doi: 10.1093/jat/22.7.601pmid: 9847012

The precision and the diagnostic performance of the Boehringer Mannheim CEDIA DAU LSD assay was evaluated. The assay was performed in the semi-quantitative mode on a Hitachi 917 analyzer. Within-run coefficients of variation (CVs) of the semiquantitative values for 0.25 and 1.0 ng/mL were 11.2 and 6.2%, respectively. Day-to-day CVs for the same concentrations were 12.6 and 8.6%. We analyzed 318 urine samples by CEDIA, DPC® Coat-A-Count® RIA and Behring EMIT®II. Confirmation was performed by GC-MS, after extraction on Bond Elut Certify® columns. Two hundred sixty-three samples were negative by all methods. Twenty-five samples were positive by all immunoassays, 19 of which were confirmed by gas chromatography-mass spectrometry (GC-MS). One sample was falsely negative by CEDIA. Three samples were positive by EMIT and CEDIA, but negative by RIA and GC-MS. Twenty-six samples were positive by EMIT alone, but they were not confirmed by GC-MS. The LSD CEDIA assay seems to be less specific than DPC RIA but more specific than the EMIT LSD assay.

Jenkins, Amanda J.; Sarconi, Krista M.; Raaf, Heather N.

doi: 10.1093/jat/22.7.605pmid: 9847013

Olanzapine, a new antipsychotic agent, was approved by the U.S. Food and Drug Administration in 1996 for use in the treatment of schizophrenia. It is structurally similar to clozapine, has a low incidence of extrapyramidal effects, and is effective in treating both the positive and negative symptoms of schizophrenia. This paper describes the determination of olanzapine in biological specimens obtained from the autopsy of a 35-year-old white male found dead in bed at a psychiatric facility. In the months prior to his death, the deceased was prescribed multiple medications, including olanzapine. Olanzapine was identified qualitatively by full scan gas chromatography-mass spectrometry, with quantitative analysis performed by liquid-liquid extraction followed by dual-column gas chromatography. The following concentrations were determined in the specimens analyzed: heart blood, 550 ng/mL; bile, 6346 ng/mL; and gastric contents, 157 ng/mL. Vitreous humor, cerebrospinal fluid, and urine specimens were negative. Although steady-state plasma concentrations of 10–25 ng/mL olanzapine have been reported, effective levels are known to be highly variable and a plasma concentration of 300 ng/mL has been tolerated without adverse effects. Based upon the autopsy, toxicological findings, and case investigation, the cause of death was determined to be intramyocardial arteriosclerosis with severe stenosis of the nodal artery due to hypertensive cardiovascular disease, and the manner was natural.