Urinary excretion of alpha-hydroxytriazolam following a single dose of halcion.

As an approved medicinal product and reportedly an abused substance that have been associated with death and "considered to be a factor...of impaired driving, sexual assault, and other violent crimes", triazolam is controlled at the same level (Level III) as flunitrazepam in Taiwan. Alleged misuses of this substance have been associated with case specimens submitted to this laboratory. A sample preparation (with and without enzymatic hydrolysis) and gas chromatography-mass spectrometry protocols were evaluated and applied to the analysis of free and total alpha-hydroxytriazolam (the main metabolite of triazolam) in urine. Ions designated for TMS-derivatized alpha-hydroxytriazolam and alpha-hydroxytriazolam-d4 are m/z 415, 417, and 430 and 419, 421, and 434, respectively. The overall protocol achieved the following results when applied to the analysis of 2-mL drug-free urine specimens fortified with 10-200 ng/mL alpha-hydroxytriazolam: recovery, 95%; interday and intraday precision ranges, 1.50-3.52% and 0.93-4.71%, respectively; linearity, r2 > 0.99; and limits of detection and quantitation, 0.05 and 0.1 ng/mL, respectively. This protocol was applied to the analysis of case specimens and urine samples collected from two patients (A and B) taking one oral dose of Halcion (0.25 mg triazolam). Excretion profiles of free and total alpha-hydroxytriazolam show that free alpha-hydroxytriazolam is detectable, but at very low levels (< 5 ng/mL). Peak excretion of total alpha-hydroxytriazolam occurs at approximately 5-10 h following the drug intake. Total alpha-hydroxytriazolam is excreted at detectable levels approximately 2-35 h following an oral dose of 0.25 mg triazolam. Total free and conjugated alpha-hydroxytriazolam excreted by A and B are 0.61% and 31.6%; and 0.36% and 57.2% of the dose, respectively.

Performance characteristics of 7-aminoflunitrazepam specific enzyme-linked immunosorbent assays.

With 7-aminoflunitrazepam (7-amino-FM2)-specific ELISAs now readily available from several commercial sources (e.g., Cozart Bioscience, Immunalysis, this study was conducted to evaluate the performance characteristics of these products when applied to the two-step testing protocol as commonly practiced in today's workplace drug-testing programs. Cross-reacting characteristics of these two assays toward a list of 25 benzodiazepines were evaluated. These assays were then applied to the analysis of urine specimens collected from patients treated with flunitrazepam (FM2) and/or other benzodiazepines. Resulting data were evaluated against gas chromatography-mass spectrometry (GC-MS) test data to ascertain corresponding cutoffs suitable for the two-step immunoassay/GC-MS testing strategy. Both Cozart and Immunalysis ELISAs are highly specific to 7-amino-FM2, with the latter reagent generating slightly higher responses. Diazepam and FM2 (parent compound) are the only compounds with significant cross-reacting characteristics. With the ELISA reagents' optimal dynamic ranges set between 0 and 25 ng/mL, urine specimens should be diluted by a factor of 5 prior to ELISA testing. If 30 ng/mL 7-amino-FM2 is adapted as the GC-MS cutoff, the corresponding ELISA cutoffs range is approximately 100-200 (or 20-40 when diluted by a factor of 5) ng/mL. Reagent lot and specimen characteristics (with or without the presence of cross-reacting compounds) affect the correlation of data derived from ELISA and GC-MS tests.

Effectiveness of multiple internal standards: deuterated analogues of methylenedioxymethamphetamine, methylenedioxyamphetamine, methamphetamine, and amphetamine.

With the increasing abuse of methylenedioxymethamphetamine (MDMA) thereby requiring analysis, we have undertaken a systematic evaluation on parameters associated with the analysis of MDMA and related compounds, including methylenedioxyamphetamine (MDA), methamphetamine (MA), and amphetamine (AM). Parameters studied included three solid-phase adsorbents, five derivatization reagents, and four deuterated internal standards (IS). This report examines whether differences in quantitation data derived from the use of four ISs (one for each analyte) and two ISs (one for AM and MA, one for MDA and MDMA) are statistically significant. Two types of samples were included in this study. The first type (Type I) included four replicate sets of standard solutions prepared in urine matrix. All analytes (AM, MA, MDA, and MDMA) were included in all samples, and these analytes' concentrations in each set were at five levels (100, 250, 500, 1000, and 2000 ng/mL). Four deuterated analogues (MA-d(8), AM-d(8), MDMA-d(5), and MDA-d(5)) at 500 ng/mL were also included in all solutions. The second type of samples (Type II) included 25 case urine specimens. Most of these specimens contained MA/AM and/or MDMA/MDA. The specific objective of this study is to determine whether the 4-IS approach can indeed generate better quantitative data than a less-costly 2-IS. For Type I samples, where the true concentrations of the analytes are known, two-sample t-test is adapted to examine whether the two sets of prediction errors (i.e., known concentration minus calculated concentration) resulting from the 4-IS and the 2-IS approaches are statistically different. For Type II samples, where the analytes' true concentrations are unknown, one-sample t-test was adapted to determine whether the difference of the quantitation results derived from the 4-IS and the 2-IS approaches is statistically significant. Statistical analysis of quantitation data derived from Types I and II samples indicates that differences in MDA and MDMA concentrations resulting from the use of one (MDA-d(5) or MDMA-d(5)) or two (MDA-d(5) and MDMA-d(5)) are statistically nonsignificant. On the other hand, similar analysis on data derived from Type I samples indicate the use of the analytes' respective deuterated analogues as the ISs appear to generate better quantitative data for AM and MA.

Deposition characteristics of methamphetamine and amphetamine in fingernail clippings and hair sections.

Fingernail clippings collected from 97 consenting females, who admitted amphetamines and/or opiates use and are currently under treatment, were quantitatively analyzed for the presence of methamphetamine and amphetamine. Sixty-two subjects were found positive for methamphetamine/amphetamine. Paired nail-hair specimens were collected from 6 of these subjects for a 12-week period and analyzed to determine the duration of detectability and deposition characteristics of amphetamines in fingernails; whether data derived from the analysis of nail clippings and hair sections are reflective of drug use patterns; and whether there is a relationship between the analytical data derived from the paired nail-hair specimens. Typical sample pre-treatment procedures and GC-MS protocols were evaluated to establish the validity of various analytical parameters and to ensure that the resulting data can be properly interpreted. Major findings include 1. Methamphetamine was found in the nails of 62 subjects collected in Week 0. The distribution of methamphetamine concentrations (ng/mg) in these nail samples are range, 0.46-61.50; mean, 9.96; and standard deviation: 13.33. The corresponding data for amphetamine are < 0.20-5.42, 0.93, and 1.01, respectively. 2. Sectional analyses of hair samples collected from 6 subjects in Week 0 show methamphetamine concentrations peak at different distances from the root. 3. The concentrations of methamphetamine and amphetamine in nail clippings are generally lower than the first 1.5-cm section of hair samples collected at the same time from the same individual. 4. Amphetamine/ methamphetamine concentration ratios in nail clippings and hair samples are comparable. 5. Methamphetamine concentration in the nail clippings collected at Weeks 0, 4, 8, and 12 decreases in a pattern similar to that exhibited by the first 1.5-cm sections of the hair samples collected at the same time.