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Abuse of new psychoactive substances (NPS) has become a health and social issue of global concern. p-Methoxyamphetamine (PMA)/p-methoxymethamphetamine (PMMA) with fluoro- or chloro-derivatives of amphetamine and methamphetamine were among the most common drugs found in specimens from fatal cases in Taiwan during the January 2011 to December 2018 period. A liquid–liquid extraction sample preparation protocol with highly sensitive ultra-high performance liquid chromatography–tandem mass spectrometry approach was developed for the simultaneous analysis of seven phenethylamine-type drugs—PMA, PMMA, p-methoxyethylamphetamine, 4-fluoroamphetamine (4-FA), 4-fluoromethamphetamine (4-FMA), 4-chloroamphetamine (4-CA) and 4-chloromethamphetamine (4-CMA)—in postmortem blood and urine specimens. Separation by liquid chromatography was performed by Agilent Zorbax SB-Aq column. Tandem mass spectrometry was operated in Agilent Jet Stream Technology electrospray ionization in positive-ion multiple reaction monitoring mode. An analytical methodology was evaluated using drug-free blood and urine after fortification with 100–2,000 ng/mL of the seven target analytes. Average extraction recoveries were >80%; slightly higher ion suppression was observed for PMA and 4-CA; intra-/inter-day precision (% coefficient of variation) and accuracy were in the ranges of 0.52–12.3% and 85–110%, respectively. Limit of detection and lower limit of quantitation for these seven analytes were both in the 0.5–5 ng/mL range. Interference and carryover were not significant. This relatively simple methodology was found effective and reliable for routine identification and quantitation of these seven analytes in postmortem and antemortem blood and urine specimens received in 2018. Analytical data obtained from these actual cases indicated the following: (i) compared to findings reported during the 2007–2011 period, the use of substituted phenethylamine-type drugs decreased in 2018; (ii) ketamine and 7-aminonimetazepam (the main metabolite of nimetazepam) were the most common co-ingested substances in specimens containing PMA/PMMA, 4-FA/4-FMA, or 4-CA/4-CMA; and (iii) in drug fatalities, the concentration of PMA was significantly higher than the concentration of PMMA in both urine and blood, while the reverse was true in urine specimens from antemortem cases.

Heroin, methamphetamine and ketamine have been the most commonly abused drugs in Taiwan. The presence of these drugs and their metabolites in postmortem specimens has been routinely monitored in our laboratory mostly by gas chromatographic-mass spectrometric methods. This study aimed to evaluate a more effective approach to simultaneously quantify these analytes (i.e., amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine (MDMA), morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, ketamine and norketamine) in postmortem urine and blood specimens by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Samples (1 mL) were extracted via solid-phase extraction, evaporated and reconstituted in the mobile phase for injection into the LC-MS-MS system. Respective deuterated analogs of these analytes were used as internal standards. Chromatographic separation was achieved by an Agilent Zorbax SB-Aq analytical column at 50°C. Mass spectrometric analysis was performed by electrospray ionization in positive-ion dynamic multiple reaction monitoring mode with optimized collision energy for respective precursor ion selected for each analyte, and the monitoring of two transition ions. Performance characteristics were assessed using drug-free samples that were fortified with 50-1,000 ng/mL of the 10 analytes. Analytical parameters evaluated and resulting data are as follows: (i) average extraction recoveries (n= 3) were better than 80%, except for MDMA (71%) and morphine (74%); (ii) inter-day and intra-day precision ranges (%CV) were 1.59-8.80% and 0.57-3.89%, respectively; (iii) calibration linearity (r2), detection limit and quantitation limit for all analytes were >0.999, 1 and 5 ng/mL, respectively; (iv) matrix effects (ion suppression) were observed for three analytes, but were satisfactorily compensated for by the deuterated internal standards adopted in the analytical protocol. This method was successfully applied to the analysis of specimens collected from unknown death cases from various district prosecutors' offices in Taiwan, and was also found helpful to understanding whether the detected opiates were derived from heroin or legal morphine/codeine-containing medications.

Screening and confirming the presence of drugs and toxic compounds in various matrices are important and challenging tasks routinely faced by forensic and clinical laboratories. Recent advances in the liquid chromatographic and mass spectrometric technologies have provided an opportunity for the development of more specific and effective approaches to achieve the "screening" and "confirmation" goals in a single analytical step. The objectives of this study are: (i) the establishment of an ultra-high performance liquid chromatographic, quadrupole time-of-flight mass spectrometric mass spectrometric and MS-MS spectral database, including 1,200 compounds of interest; and (ii) the development of an effective protocol, using this database and three searching algorithms, for general unknown screening of these compounds. The established database and protocol were evaluated through the analysis of 30 external proficiency test and 100 postmortem samples and found to be significantly more effective than the LC-IT-MS and GC-MS approaches previously established in our laboratory.

A simple method, incorporating protein-precipitation/organic backwashing and liquid chromatography-tandem mass spectrometry (LC-MS-MS), has been successfully developed for the simultaneous analysis of four highly water-soluble and less volatile herbicides (paraquat, diquat, glufosinate and glyphosate) in ante- and postmortem blood, urine and gastric content samples. Respective isotopically labeled analogs of these analytes were adopted as internal standards. Acetonitrile and dichloromethane were used for protein precipitation and organic solvent backwashing, respectively, followed by injecting the upper aqueous phase into the LC-MS-MS system. Chromatographic separation was achieved using an Agilent Zorbax SB-Aq analytical column, with gradient elution of 15 mM heptafluorobutyric acid and acetonitrile. Mass spectrometric analysis was performed under electrospray ionization in positive-ion multiple reaction monitoring mode. The precursor ions and the two transition ions (m/z) adopted for each of these four analytes were paraquat (185; 169 and 115), diquat (183; 157 and 78), glufosinate (182; 136 and 119) and glyphosate (170; 88 and 60), respectively. Analyte-free blood and urine samples, fortified with the analytes of interest, were used for method development/validation and yielded acceptable recoveries of the analytes; interday and intraday precision and accuracy data; calibration linearity and limits of detection and quantitation. This method was successfully incorporated into an overall analytical scheme, designed for the analysis of a broad range of compounds present in postmortem samples, helpful to medical examiners' efforts to determine victims' causes of death.

A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed, validated and applied to simultaneous analysis of oral fluid samples for the following 10 analytes: methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), buprenorphine, norbuprenorphine, morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, amphetamine, and methamphetamine. The oral fluid sample was briefly centrifuged and the supernatant was directly injected into the LC-MS-MS system operated under reverse-phase chromatography and electrospray ionization (ESI). Deuterated analogs of the analytes were adopted as the internal standards and found to be effective (except for buprenorphine) to compensate for potential matrix effects. Each analytical run took10 min. Linearity range (r(2)0.99) established for buprenorphine and the other nine analytes were 5-100 and 1-100 ng/mL. Intra- and interday precision (% CV) ranges for the 10 analytes were 0.87-12.2% and 1.27-12.8%, while the corresponding accuracy (%) ranges were 91.8-113% and 91.9-111%. Limits of detection and quantitation established for these 10 analytes were in the ranges of 0.1-1.0 and 0.25-1.0 ng/mL (5 ng/mL for buprenorphine). The method was successfully applied to the analysis of 62 oral fluid specimens collected from patients participating in methadone and buprenorphine substitution therapy programs. Analytical results of methadone and buprenorphine were compared with data derived from GC-MS analysis and found to be compatible. Overall, the direct injection LC-MS-MS method performed well, permitting rapid analysis of oral fluid samples for simultaneous quantification of methadone, buprenorphine, opiate and amphetamine drug categories without extensive sample preparation steps.

Methylenedioxymethamphetamine (MDMA) has been one of most popular drugs in the "club" scene in Taiwan. This epidemic was studied through the examination of toxicological data obtained from the 59 fatalities tested positive for MDMA during the period of January 2001 to December 2008. Ketamine was found in 28 of these cases, signifying the popularity of this drug in Taiwan. The annual number of deaths in each of the 8 years in this period was 4, 7, 9, 14, 8, 9, 2, and 6, respectively. Among these 59 deaths, 39 (66.1%) were men, and the mean, median, and range of ages were 24.6, 23, and 14-46, respectively. Causes of death ruled by the attending pathologists and the distributions for these fatalities were acute intoxication, 40 and mechanical injury, 19, including 3 hanging and 2 drowning. The manners of death were ruled as accidental, 44; homicidal, 6; suicidal, 7; and undetermined, 2. In this study, postmortem whole blood was analyzed by gas chromatography-mass spectrometry with a limit of quantitation at 0.05 microg/mL for both MDMA and MDA. The mean, median, and range of MDMA concentrations in the cases, where MDMA acute intoxication was ruled as the cause of death, were 4.75, 2.60, and 0.12-40.41 microg/mL. MDA was found in 30 of these 40 cases with the following mean, median, and range data: 0.19, 0.13, and 0.05-1.81 microg/mL. The corresponding data of MDMA and MDA in the remaining 19 MDMA-related deaths were significantly lower: 1.25, 0.97, 0.08-3.05 and 0.11, 0.09, 0.06-0.24 microg/mL, respectively.

Parallel to the "poppy-seed defense" strategy commonly reported in the United States, donors of urine samples tested positive for opiates in Taiwan often claimed the consumption of Brown Mixture (BM) as the source of the observed morphine and codeine. Because BM contains opium powder (10.0-10.5% morphine), opium tincture (0.9-1.1% morphine), or camphorated opium tincture (0.045-0.055% morphine) and is a popular remedy, and heroin use is considered a serious criminal act, the claim of BM use has to be adequately addressed. In this study, BM from seven different manufacturers (5 tablets and 2 solutions) and urine samples from alleged heroin users and volunteers with various ingestion patterns and were analyzed for their morphine and codeine contents. The analytical procedure included hydrolysis, trimethylsilylation, and gas chromatography-mass spectrometry analysis. The contents of morphine and codeine in the tablets were found to be very consistent, but with significant differences in the two BM solutions. Morphine concentrations found in urine specimens collected from volunteers ingesting BM tablets (or solutions) were always4000 ng/mL. The following morphine-to-codeine ([M]/[C]) ratios were observed for urine specimens with morphine concentrationor = 300 ng/mL: (A)3.0 for volunteers ingesting BM solution and (B)3.0 (mostly5.0) for volunteers ingesting BM tablets and alleged heroin users. It appeared that (A) BM ingestion (tablet or solution) was unlikely to result in a morphine concentration4000 ng/mL; and (B) [M]/[C] ratio might not be an effective parameter to differentiate heroin use from BM tablet ingestion.

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.

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.

With increasing requests for the analysis of various specimens related to fatal and non-fatal abuse of methylenedioxymethamphetamine (ecstasy, MDMA), the toxicology laboratory of the Institute of Forensic Medicine has established protocols for the analysis of MDMA and related compounds in hair, urine, and various postmortem specimens. Analytical protocols include extraction, derivatization, and gas chromatographic-mass spectrometric analysis adapting deuterated analogs of the analytes as internal standards. Data resulting from these analyses and hereby reported include postmortem distribution of MDMA and methylenedioxyamphetamine (MDA) in heart blood, gastric content, urine, and bile specimens from 20 fatal cases; other drugs found in the heart blood from these 20 cases; and the distribution of MDMA and MDA in 25 antemortem urine and 6 hair specimens. The MDA/MDMA concentration ratio observed in a limited number of hair specimens (n = 6) are consistent and appear to be higher than those found in other specimens. Compared to other commonly abused drugs (e.g., cocaine and heroin), the "drug/metabolite" concentration ratio (MDMA/MDA) in hair is not significantly different from the ratios derived from other specimens, such as urine and blood. This observation is consistent with the relative drug/metabolite incorporation rates reported for cocaine/benzoylecgonine, tetrahydrocannabinol/tetrahydrocannabinoic acid, and MDMA/MDA.

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, r20.99; 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.

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.