Pitfalls of toxicological investigations in hair, bones, and nails in extensively decomposed bodies: illustration with two cases.

It is difficult to carry out toxicological investigations in biological samples collected from extensively decomposed bodies and to interpret obtained results as several pitfalls should be considered: redistribution phenomena, degradation of xenobiotics during the postmortem period, contamination by putrefaction fluids, and external contamination. This work aims to present two cases in order to illustrate and discuss these difficulties in this tricky situation. Case#1: the body of a 30-year-old woman was found in a wooded area (1 month after she has been reported missing by her family): hair and a femur section were sampled. Case#2: the decomposed corpse of a 52-year-old man was found in a ditch: hair and nails were sampled. After decontamination steps, toxicological investigations were performed using liquid chromatography with high-resolution mass spectrometry and tandem mass spectrometry detection methods. In case#1, the same drugs or metabolites (benzodiazepines, propranolol, tramadol, acetaminophen, paroxetine, and oxetorone) were detected in hair and in bone specimens. This result combination strongly suggests intakes close to the time of death for three of them (oxazepam, lormetazepam, and propranolol). In case#2, results of toxicological investigations in hair and nails [(hair/nail concentration in ng/mg) nordiazepam (1.12/1.06), oxazepam (0.113/0.042), zolpidem (0.211/< 0.01), hydroxyzine (0.362/< 0.01), and cetirizine (0.872/1.110)] were both consistent with several drug intakes but were not contributory to cause of death determination. In case of positive toxicological results in biological samples collected from extensively decomposed bodies (such as hair, bones, or nails), it is challenging to determine the time, and even more, the level of the dose of exposure(s).

Hair Analysis: Contamination versus Incorporation from the Circulatory System-Investigations on Single Hair Samples Using Time-of-Flight Secondary Ion Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

Contamination is a highly controversial issue in hair analysis. Therefore, hair testing protocols typically include wash steps to remove contamination. However, recent studies claim that washing could also lead to permanent incorporation of contaminants into hair, thus questioning the validity of hair testing at all. In the present study, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) with longitudinal sectioning of single hairs and different decontamination protocols was used to reveal differences between the incorporation of a substance into hair from external sources and an incorporation via bloodstream. Single hairs were longitudinally sectioned using a custom-made sample holder. Data were acquired with MALDI-MS by rastering each hair individually. Single hair samples from drug users, blank hairs, and zolpidem- and zolpidem-D6-soaked hairs were investigated. Different published washing protocols were tested, and an in-house washing protocol was developed. For images with higher spatial resolution, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used. Longitudinal sectioning of hairs dramatically increased sensitivity; even single-dose administrations of zolpidem in single hairs could thus be detected using MALDI-MS. Zolpidem from external sources could be detected in large quantities in superficial hair structures. Zolpidem from consumer hairs, proposed to be strongly bound to inner hair structures, could not be completely removed even by the strongest tested decontamination protocol, whereas zolpidem-soaked hairs could be cleared almost completely with the developed in-house wash protocol. The applied methods allowed a first insight into the connection of decontamination protocols and wash-in phenomena in hair analysis. Further studies with other drugs are necessary to assess the general validity of these findings.

Simultaneous determination of 75 abuse drugs including amphetamines, benzodiazepines, cocaine, opioids, piperazines, zolpidem and metabolites in human hair samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 75 abuse drugs and metabolites, including 19 benzodiazepines, 19 amphetamines, two opiates, eight opioids, cocaine, lysergic acid diethylamide, zolpidem, three piperazines and 21 metabolites in human hair samples, was developed and validated. Ten-milligram hair samples were decontaminated, pulverized using a ball mill, extracted with 1 mL of methanol spiked with 28 deuterated internal standards in an ultrasonic bath for 60 min at 50°C, and purified with Q-sep dispersive solid-phase extraction tubes. The purified extracts were evaporated to dryness and the residue was dissolved in 0.1 mL of 10% methanol. The 75 analytes were analyzed on an Acquity HSS T3 column using gradient elution of methanol and 0.1% formic acid and quantified in multiple reaction monitoring mode with positive electrospray ionization. Calibration curves were linear (r ≥ 0.9951) from the lower limit of quantitation (2-200 pg/mg depending on the drug) to 2000 pg/mg. The coefficients of variation and accuracy for intra- and inter-assay analysis at three QC levels were 4.3-12.9% and 89.2-109.1%, respectively. The overall mean recovery ranged from 87.1 to 105.3%. This method was successfully applied to the analysis of 11 forensic hair samples obtained from drug abusers.

Evaluation of Dissolution Profile between Original and Generic Products of Zolpidem Tartrate by Microdialysis-HPLC.

The evaluation of the dissolution profile of hypnotic drugs is important to promote switching from original products to generic products by removing distrust in generic hypnotics. In this study, we investigated differences in the dissolution profiles between original and generic products (GE-D, GE-S, and GE-T) in commercially available zolpidem tartrate (ZOL) products using the HPLC method using a connected microdialysis probe (microdialysis-HPLC method). Although the degree of hardness and the disintegration time were not different among the original, GE-S, and GE-T, GE-D was 1.4 times harder than the other products. The disintegration time of GE-D was approximately twice as long as that of the original product. Generic products dissolved rapidly as compared with the original product, however, the dissolution rate in the ZOL powder (milled ZOL product) was not different between the original and generic products. Macrogol 6000 (polyethylene glycol (PEG)-6000) was used in the generic products, and this additive was the only PEG difference from the original product. We investigated whether the PEG in the product affected the solubility of ZOL and found that the addition of PEG-4000 or PEG-6000 significantly increased the dissolution rate. These results suggest that the solubility of ZOL may be increased by PEG when the product is disintegrated, resulting in the increased dissolution rate in the generic products. In conclusion, we found that the difference of PEG affected the dissolution profile in the disintegration process using the microdialysis-HPLC method. This finding can help ensure the safety of milled products and the selection of additives.

Influences of hair dyeing on the distribution shapes of zolpidem and methoxyphenamine in hair.

In order to investigate the influences of hair dyeing on the distribution shapes of drugs in hair, different hair dyeing processes ("semi-permanent coloring without bleaching" and "permanent coloring with bleaching") were performed in vitro on black hair specimens collected from two subjects (Asians) who took a single dose of zolpidem (ZP, 10 mg of ZP tartrate) or methoxyphenamine (MOP, 50 mg of MOP hydrochloride). Under the following three different dyeing conditions, (1) semi-permanent coloring, (2) permanent coloring (once), (3) permanent coloring (twice), drug distributions in single hair specimens were investigated using a 2-mm segmental analysis by liquid chromatography-tandem mass spectrometry. Distribution shapes of drugs changed significantly only under the permanent coloring (twice) condition, resulting in reduced peak concentration and extended distribution width. There was, however, no significant difference in the amounts of drugs in hair between non-treated and dyed specimens, suggesting the drugs hardly leaked out of hair or were only slightly degraded during dyeing. In addition, while assuming contact with aqueous environment such as daily hair washing after dyeing, dyed hair specimens were individually immersed in ultrapure water for 20 hours, then the outflow of drugs in ultrapure water as well as the distribution shapes of drugs remaining in hair were determined. The drug outflow after permanent coloring (once and twice) was significantly larger than those after semi-permanent coloring, and the outflow ratios, [outflow]/([outflow] + [amount remaining in hair]), ranged over 9.8-24% (n = 3) for ZP and 68-71% (n = 3) for MOP after permanent coloring (once), and 54-72% (n = 3) for ZP and 86-91% (n = 3) for MOP after permanent coloring (twice). The distribution shapes of drugs after 20 h of immersion tended to flatten as outflow ratios increased, resulting in no change in the shapes after semi-permanent coloring, and complete collapse of their shapes after permanent coloring (twice). Thus, the present results indicated that hair dyeing involving bleaching and subsequent contact with aqueous environment after dyeing could significantly influence distribution shapes of drugs in hair.

Estimation of the time of zolpidem intake and differentiation between consumption and external contamination using MALDI-MSI for investigations on single hair samples.

Estimation of drug ingestion time (event time) and distinguishing between drug ingestion and external contamination are important for interpreting hair analysis results in forensics practice. Here, we present a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) method for in situ analysis of intact hair. We applied a longitudinal cutting method for a single hair to analysis authentic hair samples from a victim of a drug-facilitated sexual assault (DFSA) case and zolpidem-soaked hair. MALDI-MSI showed that zolpidem-positive segments distributed at 4-6 mm or 6-8 mm from the root in three single hairs of a DFSA victim collected 25 days after the event, at concentrations ranging from 0.1 to 5.7 pg mm-1, in agreement with the results from segmental analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS). The estimation of drug intake time was about 20-30 days before sampling, which was consistent with the known time of drug intake. This MALDI-MS method allows imaging analysis of trace substances in a single hair and can realize the intuitive reflection of drug taking time. In addition, zolpidem applied by soaking was mainly distributed on both sides of the longitudinal hair shaft, whereas ingested zolpidem was found only in the middle of the hair shaft of the DFSA victim. The MALDI-MS images of unwashed and washed hair suggested that the amount of externally applied drug was decreased by washing, it was still present on surface layer (cuticle) sides although. Visualization using MALDI-MSI could therefore distinguish between drug ingestion and contamination by reflecting the distribution and deposition site of the drug in hair.

Target and suspect screening of (new) psychoactive substances in South Korean wastewater by LC-HRMS.

New psychoactive substances (NPS) are a type of abused drug designed to mimic the effects of the currently known illicit drugs, whose structures are constantly changing to escape surveillance. The quick identification of NPS use in the community therefore demands immediate action. This study aimed to develop a target and suspect screening method using LC-HRMS to identify NPS in wastewater samples. An in-house database of 95 traditional and NPS was built using the reference standards, and an analytical method was developed. Wastewater samples were collected from 29 wastewater treatment plants (WWTP) across South Korea, representing 50 % of the total population. The psychoactive substances in waste water samples were screened using in-house database and developed analytical methods. A total of 14 substances were detected in the target analysis, including three NPS (N-methyl-2-AI, 25E-NBOMe, and 25D-NBOMe) and 11 traditional psychoactive substances and their metabolites (zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, phendimetrazine, phentermine, methamphetamine, codeine, morphine, and ketamine). Out of these, N-methyl-2-AI, zolpidem phenyl-4-COOH, ephedrine, ritalinic acid, tramadol, phenmetrazine, and phendimetrazine were detected with a detection frequency of over 50 %. Primarily, N-methyl-2-Al was detected in all the wastewater samples. Additionally, four NPSs (amphetamine-N-propyl, benzydamine, isoethcathinone, methoxyphenamine) were tentatively identified at level 2b in a suspect screening analysis. This is the most comprehensive study to investigate NPS using target and suspect analysis methods at the national level. This study raises a need for continuous monitoring of NPS in South Korea.

Occurrence of Z-drugs, benzodiazepines, and ketamine in wastewater in the United States and Mexico during the Covid-19 pandemic.

Z-drugs, benzodiazepines and ketamine are classes of psychotropic drugs prescribed for treating anxiety, sleep disorders and depression with known side effects including an elevated risk of addiction and substance misuse. These drugs have a strong potential for misuse, which has escalated over the years and was hypothesized here to have been exacerbated during the COVID-19 pandemic. Wastewater-based epidemiology (WBE) constitutes a fast, easy, and relatively inexpensive approach to epidemiological surveys for understanding the incidence and frequency of uses of these drugs. In this study, we analyzed wastewater (n = 376) from 50 cities across the United States and Mexico from July to October 2020 to estimate drug use rates during a pandemic event. Both time and flow proportional composite and grab samples of untreated municipal wastewater were analyzed using solid-phase extraction followed by liquid chromatography-tandem mass spectrometry to determine loadings of alprazolam, clonazepam, diazepam, ketamine, lorazepam, nordiazepam, temazepam, zolpidem, and zaleplon in raw wastewater. Simultaneously, prescription data of the aforementioned drugs were extracted from the Medicaid database from 2019 to 2021. Results showed high detection frequencies of ketamine (90 %), lorazepam (87 %), clonazepam (76 %) and temazepam (73 %) across both Mexico and United States and comparatively lower detection frequencies for zaleplon (22 %), zolpidem (9 %), nordiazepam (<1 %), diazepam (<1 %), and alprazolam (<1 %) during the pandemic. Average mass consumption rates, estimated using WBE and reported in units of mg/day/1000 persons, ranged between 62 (temazepam) and 1100 (clonazepam) in the United States. Results obtained from the Medicaid database also showed a significant change (p < 0.05) in the prescription volume between the first quarter of 2019 (before the pandemic) and the first quarter of 2021 (pandemic event) for alprazolam, clonazepam and lorazepam. Study results include the first detections of zaleplon and zolpidem in wastewater from North America.

Incorporation of five common hypnotics into hair after a single dose and application to a forensic case of drug facilitated crimes.

In order to obtain fundamental information on the disposition of hypnotics into hair after a single oral dose the quantitative hair analysis of triazolam (TZ), etizolam (EZ), flunitrazepam (FNZ), nitrazepam (NZ) and zolpidem (ZP) have been performed using a validated LC-MS/MS procedure. Hair specimens (straight, black) were collected from three subjects about one month and three months after a single 0.25 mg dose of TZ, 1 mg of EZ, 2 mg of FNZ, 5 mg of NZ and 10 mg of ZP tartrate. The subjects ingested just one out of five different hypnotics on each day, each of five days in turn. All ingested hypnotics have been detected in hair from each subject both one month and three months after intake, and their concentrations were in the range of 0.023-0.043 pg/hair strand (0.077-0.36 pg/mg) for TZ, 0.11-0.63 pg/hair strand (0.44-5.2 pg/mg) for EZ, 0.14-2.6 pg/hair strand (0.56-22 pg/mg) for FNZ, 0.33-1.7 pg/hair strand (1.3-17 pg/mg) for NZ and 20-40 pg/hair strand (120-270 pg/mg) for ZP. For FNZ and NZ, not only the parent drugs but also their metabolites, 7-amino-FNZ and 7-amino-NZ, were detected in the range of 2.3-9.2 pg/hair strand (9.2-82 pg/mg) and 2.4-9.1 pg/hair strand (8.0-55 pg/mg), respectively. The calculated incorporation ratios into hair against the dose were found to exhibit similarity between the four benzodiazepines. This finding suggests the ability to apply these quantitative data to approximately estimating the amounts of other benzodiazepines, which have similar chemical structures, in hair although it should be noted that the amounts of drugs in hair varies considerably depending on the hair color. On the other hand, the incorporation ratio of ZP showed 15-29 times higher than that of TZ, indicating that lipophilic ZP was more likely to incorporate into hair than benzodiazepines. In addition, the application of the present data to a drug-facilitated sexual assault was shown.

Alprazolam and Zolpidem in Skeletal Tissue of Decomposed Body Confirms Exposure.

In several medico-legal cases, bone samples analysis may provide the only source of toxicological information. This case study reports the analysis of a human bone specimen, belonging to a 46-year-old man, found 3 months after his death due to cervical-thoracic injuries in a motorcycle accident. Bone specimen was the only available material for toxicological analysis, among few skull hair and rotten skin. Analysis was performed by a newly developed and validated ultra-high-pressure liquid chromatography-mass spectrometry/mass spectrometry (UHPLC-MS/MS) method, following simple and efficient sample pretreatment. The results were in accordance with the man's medical record: Alprazolam and zolpidem were found at 2.2 and 5.4 ng/g of bone, respectively. Both these drugs were prescribed to the deceased.

Incorporation of zolpidem and methoxyphenamine into white hair strands after single administrations: Influence of hair pigmentation on drug incorporation.

In order to investigate the influence of pigmentation on the incorporation of drugs into hair, time-course changes in drug distribution along non-pigmented (white) hairs as well as pigmented (black) hairs plucked from the same subject was observed following single administrations of two basic drugs with different properties, zolpidem and methoxyphenamine. These drugs in 1-mm sections of single hair specimens were each determined by a liquid chromatography-tandem mass spectrometric procedure. During the early stage (12-36 h) after intake, for black hairs, both drugs were detected over the entire area of hair root (4-5 mm in length), in which notable concentration of these drugs in the hair bulb (0-1-mm segment from the bottom of hair root, Region 1) and lower concentrations in the upper dermis zone (1-2-mm to 3-4-mm or to 4-5-mm segments, Region 2) were commonly observed. Meanwhile, for white hairs, high drug concentrations in Region 1 as detected in black hairs were not observed although only small amounts of these drugs were detected over Region 2. Subsequent time-course changes in the concentration of drugs in hair demonstrated that the drugs once incorporated into white hair via Region 2 decreased gradually over the period from 24 h to 35 days after intake, but those of black hairs remained almost unchanged. These findings revealed here suggest that hair pigments have two important roles in the distribution of drugs: (1) incorporation of drugs into hair via Region 1, and (2) retention of already incorporated drugs in the hair tissue. These findings would be useful for discussing individual drug-use history based on hair analysis in the forensic fields.