Min Shen

Anti-obesity drugs, used to suppress appetite and reduce fat absorption, have been circulated and traded illegally worldwide. The traditional methods of liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography-mass spectrometry (GC-MS) for analyzing these drugs in in vivo samples require complex sample pretreatment and time-consuming procedures. To address this issue, a thermal-assisted paper spray mass spectrometry (PS-MS) method was developed in this study to analyze anti-obesity drugs in raw urine. By incorporating a heat source and optimizing the spray solvent and paper substrate, this technique demonstrates reduced matrix effect and higher sensitivity compared to traditional PS-MS methodology for direct analysis of anti-obesity drugs in urine samples. A temperature range of 100–200 °C can be set for screening anti-obesity drugs in urine samples, with the flexibility to adjust the temperature according to the specific drug being analyzed. The limits of detection (LODs) for these 15 anti-obesity drugs in urine ranged between 1 and 500 ng mL⁻¹. Furthermore, the thermal-assisted PS-MS method exhibited good linearities (R², 0.9903–0.9997) within the range from 10–100 to 1000 ng mL⁻¹ for the direct quantitation of anti-obesity drugs in urine samples with an internal standard. Therefore, the thermal-assisted PS-MS technique may provide a novel approach for the direct analysis of drugs in complex samples.

Introduction: Hair testing is well established for the assessment of past drug exposure; however, more research is needed to understand drug incorporation mechanisms and drug entry pathways into hair.Method: In this study, a micro-segmental LC–MS/MS method was used to analyze a 0.4 mm segment of hair after a single oral administration of zolpidem. Five single hairs were plucked at 1 day, 3 days, 7 days, and 28 days after administration from the vertex posterior of three subjects, and 5 single hairs were also plucked from the parietal, left temporal, and right temporal regions of the head at 28 days.Results and discussion: Proximal S1 (0–0.4 mm) in hair plucked at 1 day had the highest level of zolpidem at 1.5–2.4 pg/mm; much lower concentrations (< 1 pg/mm) were detected at proximal S2–S8 (0.4–3.2 mm). The drug concentration decreased gradually in S1 for 7 days after drug intake and disappeared by 28 days, suggesting that the drug from the bloodstream initially combined with the hair follicle and then gradually moved to the hair tip as the hair grew. The zolpidem concentration–hair segment profiles exhibited a large peak (root side) and a small peak (tip side) for the four sampling times in all three subjects, indicating that drug incorporation in the hair bulb occurred mainly from the blood but probably also entered the hair through sweat and sebum. Zolpidem was also detected in all hairs from the vertex posterior in all three subjects but was not detected in 1 hair from the parietal region and 2 hairs from the left temporal region. The consistency in drug detection, drug concentration level, and peak position was better in hair from the vertex posterior than from the other three regions, indicating that the vertex posterior is a suitable sampling region for estimating drug intake.

Purpose An analytical method using liquid chromatography with tandem mass spectrometry (LC–MS/MS) was established and validated for screening 425 drugs and poisons in dried blood spots (DBSs). Methods Blood (20 μL) was spotted on Whatman FTA™ classic card to prepare DBS sample, then extracted with 150 μL methanol and analyzed by LC–MS/MS using a multiple reaction monitoring method. Results The limit of detection of the compounds were 0.1–10 ng/mL. The values for recovery and matrix effect were 40.3–114.9% and 40.2–118.4%, respectively. This method was successfully applied to DBS samples from 105 humans suspected of drug poisoning, which was stored for 3–5 years at room temperature. Thirty-three kinds of drugs, including benzodiazepines, antipsychotics, antidepressants, antipyretic analgesics, non-steroidal anti-inflammatory drugs, antibiotics, antiepileptic drugs, new psychoactive drugs were confirmed in 102 cases, while no compound was detected in the other 3 cases. Estazolam, a benzodiazepine widely used in clinical practice as a sedative, hypnotic, and anti-anxiety drug, was the most frequently detected substance, occurring in 34.2% of the cases. Conclusions Most drugs in DBS could still be detected after storage for 3–5 years, but ambroxol, zopiclone, carbofuran, chlorpyrifos, and valproic acid were not detectable after 3–5 years of storage at room temperature. The components measured in DBS were consistent with those measured in whole blood at the collection time, thereby confirming that DBS samples have the advantage of stable storage at room temperature.

The mechanism of estazolam incorporation into hair was investigated by studying the time course of estazolam along single-strand hair after two oral administration of estazolam at 28 days interval. Estazolam in single hair segments 0.4 mm in length was verified and quantified by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). The distributions of estazolam within a strand of hair (collected at 12 h, 28 days, and 56 days post-administration) were visualized by micro-segmental analysis. The highest estazolam concentration (1.5–9.9 pg/mm) was detected in the hair bulb region (S1), and it then decreased through the hair shaft to the distal end, with a small fluctuation (0.3–3 pg/mm) near the junction of the hair roots and shafts (S4–S7) 12 h after drug intake. These findings suggested that the incorporation of estazolam occurred in two regions, mainly in the hair bulb and to a lesser extent in the upper dermis zone. Models using internal temporal markers (TIMs) and temporal intervals (TIs) were constructed to estimate the day of estazolam ingestion. The estimation accuracy was within an average error of 1.7 mm and 3.0 mm between the calculated and actual positions, based on the TIMs and TIs 56 days after estazolam intake. These findings can help in further elucidation of the drug incorporation mechanism, which is crucial for interpreting hair analysis results used to reveal individual drug-use history.

The emergence of novel drugs and the continuous expansion of the scope of the types of drugs under control have greatly increased requests for screening of a range of drugs in hair. Here, a multi-analyte method for the detection and quantification of 88 psychotropic drugs in the hair of addicts in drug abstinence was developed and fully validated using liquid chromatography–tandem mass spectrometry (LC–MS-MS). Hair samples (25 mg) were washed, cut into pieces, cryogenically ground and extracted in methanol. The extracted analytes were separated on an Allure PFP Propyl column (100 × 2.1 mm, 5 mm inside diameter, Restek, USA) and analyzed by LC–MS-MS in multiple reaction monitoring modes. The limits of detection and the limits of quantification ranged from 0.1 to 20 pg/mg and 0.2 to 50 pg/mg, respectively. The intra- and inter-assay precisions (relative standard deviation (RSD)) of all analyses ranged from 0.9% to 14.9% and 1.9% to 15.9%, respectively. Accuracy values were 100 ± 20%. The extraction recovery of quality control samples ranged from 50.9% to 99.6% for all analytes. The matrix effects for all analytes ranged from 46.8% to 99.7%. The method was successfully used to analyze 1,865 hair samples from addicts in drug rehabilitation at their own communities. Among the samples, 129 cases were positive; the majority of positive cases were from males (78.29%), 92.25% of whom were >35 years old. Traditional drugs, like methamphetamine and opioids, accounted for most positive cases, and 27 of the abstinence cases with a use history of methamphetamine were still positive. In addition to abused drugs, like methamphetamine, morphine and cocaine, the sedative-hypnotic and psychotherapeutic drugs, including clonazepam, alprazolam, estazolam, zolpidem and quetiapine, were detected in 26% of the hair samples, suggesting that these addicts may have insomnia and mental problems such as depression and psychosis, probably due to the long-term effects of drugs and withdrawal reactions. Three synthetic cannabinoids were also detected in four (2.7%) cases. A total of 37 cases were positive for methadone, tramadol and dextromethorphan, reflecting a new trend of alternative drug use when traditional drugs were not easy to obtain during the coronavirus disease 2019 outbreak.

Hair analysis has been mainly used to document drug use history in abusers, drug-facilitated crime cases, doping control analysis and postmortem toxicology in the fields of forensic toxicology, clinical toxicology, and doping control. Hair analysis has also gained more attention in the last 30 years in China. Relevant technology has been promoted as more research has appeared concerning hair analysis, and consensus has been sought among forensic toxicologists regarding aspects such as hair decontamination treatment, detection of abused substances in hair, segmental hair analysis and interpretation of analytical results. However, there are still some limitations in the estimation of drug intake time and frequency by segmental hair analysis due to the different growth cycles evident within a bundle of hairs, the drug incorporation mechanism and sampling errors. Microsampling and imaging mass spectrometry (IMS) technology based on a single hair may be a good choice to estimate drug intake time more accurately. Analysis of hair root samples may also be used to document acute poisoning in postmortem toxicology, and the analysis of the hair shaft can document long-term use of drugs depending on the length of the hair being evaluated.