Determination of Traditional and Designer Benzodiazepines in Urine through LC-MS/MS.

BACKGROUND: The detection of new designer benzodiazepines in biological fluids and tissues, together with the traditional ones, could represent an important analytical update for laboratories performing clinical and forensic toxicological analysis. OBJECTIVE: A liquid chromatography tandem mass spectrometry method (LC-MS/MS) has been developed, fully validated, and applied to a cohort of real urine samples collected from patients under withdrawal treatment and from intoxication cases. METHODS: 100 µL urines were added to a buffer solution containing deuterated internal standards; the samples were then extracted through a liquid/liquid procedure, dried under a nitrogen stream, and reconstituted in mobile phase. The chromatographic separation was performed in reverse phase through a C18 column with gradient elution. Mass spectrometry operated in positive polarization and multiple reaction monitoring mode. RESULTS: 25 molecules were optimized for instrumental analysis: 9 designer benzodiazepines and 16 traditional compounds (parent drugs and main metabolites). Sensitivity, specificity, linearity, accuracy, imprecision, recovery, matrix effects, and carry-over have been evaluated for all molecules. Only cinazepam did not satisfy all acceptance criteria for validation. 10 among the 50 analyzed samples tested positive for at least one of the monitored molecules. In particular, two different samples collected from the same case provided positive results for flubromazepam, a designer benzodiazepine. CONCLUSION: The method was proven to be useful in detecting not only traditional benzodiazepines but also new designer ones. The identification of a New Psychoactive Substance in real samples confirmed that analytical procedures should be updated to include as many substances as possible.

Ethyl glucuronide in hair: A 5-year retrospective cohort study in subjects sanctioned for driving under the influence of alcohol and psychoactive substances.

The evaluation of drinking behaviors can help in limiting high-risk situation, such as driving under the influence (DUI). We investigate ethyl glucuronide in hair (hEtG) levels to evaluate alcohol consumption behavior in subjects followed up after having been charged for DUI of psychoactive substances and/or alcohol. We performed a retrospective observational cohort study on 4328 subjects over 18 years old who underwent hEtG analysis in the period 2015-2019 in the Italian Province of Pavia. hEtG level was used as a proxy for the alcohol consumption behavior. Effects of age, sex, and district on alcohol drinking behavior were investigated with an ordinal logit model. A state sequence analysis was used to study people's alcohol consumption behavior over time. hEtG was found ≥7.0 pg/mg in 22.2% of the drivers (of which 7% has an hEtG ≥30.0 pg/mg). Among positive cases, a prevalence of males (96.3%) aged 35-44 (32.6%), coming from main city and hinterland (38.2%), was observed. The propensity to drink was higher for males (odds ratio [OR] ≈ 2.28, p < 0.001) and for subject coming from the district devoted to the cultivation of vineyards. Young age classes have a reduced drinking risk if compared to the drivers over 55 years old (p < 0.001). A general decreasing trend over time in hEtG values was observed. Being male, age ≥ 55 years, and coming from rural areas are potential risk factors related to alcohol drinking habits among drivers. Ethyl glucuronide in hair test in the driving license reissuing protocol contributed to decrease alcohol misuse behaviors.

New Synthetic Cathinones and Phenylethylamine Derivatives Analysis in Hair: A Review.

The analysis of psychoactive substances in hair is of great importance for both clinical and forensic toxicologists since it allows one to evaluate past and continuative exposure to xenobiotics. In particular, a new challenge is represented by new psychoactive substances: Among this new class of drugs of abuse, synthetic cathinone and phenethylamine derivatives are often detected in biological samples. Hence, there is a growing need to develop new analytical procedures or improve old ones in order to conduct evaluations of these emerging substances. This study is a systematic review of all the instrumental and experimental data available in the literature. A total of 32 articles were included in the review. Acidic solvents proved to be the most reliable solutions for extraction. Gas chromatography and liquid chromatography coupled to tandem mass spectrometric and high-resolution mass spectrometric systems represent the majority of the involved instrumental techniques. Sensitivity must be maintained at the pg/mg level to detect any occurrences up to occasional consumption. In total, 23 out of 32 articles reported real positive samples. The most frequently detected substance in hair was mephedrone, followed by butylone, methylone, MDPV, and α-pyrrolidinophenone-type substances.

Analysis of Cannabinoids and Metabolites in Dried Urine Spots (DUS).

Dried urine spots (DUS) represent a potential alternative sample storage for forensic toxicological analysis. The aim of the current study was to develop and validate a liquid chromatographic tandem mass spectrometric procedure for the detection and quantitative determination of cannabinoids and metabolites in DUS. A two-step extraction was performed on DUS and urine samples. An LC-MS/MS system was operated in multiple reaction monitoring and positive polarization mode. The method was checked for sensitivity, specificity, linearity, accuracy, precision, recovery, matrix effects and carryover. The method was applied to 70 urine samples collected from healthy volunteers and drug addicts undergoing withdrawal treatment. The method was successfully developed for DUS. LODs lower than 2.0 ng/mL were obtained for all the monitored substances. All the validation parameters fulfilled the acceptance criteria either for DUS or urine. Among the real samples, 45 cases provided positive results for at least one compound. A good quali-quantitative agreement was obtained between DUS and urine. A good stability of THC, THCCOOH and THCCOOH-gluc was observed after a 24 h storage, in contrast to previously published results. DUS seems to provide a good alternative storage condition for urine that should be checked for the presence of cannabinoids and metabolites.

A case report on fatal intoxication by tapentadol: Study of distribution and metabolism.

We report a case in which a tapentadol acute intoxication was suspected as the cause of death of a 39-year-old man: approximately two days after death, cardiac and femoral blood, as well as urine, bile, gastric content and chest hair, were collected during the autopsy. Tapentadol was detected before and after hydrolysis in femoral (530 ng/mL unconjugated and 1570 ng/mL conjugated) and cardiac (680 ng/mL unconjugated and 3440 ng/mL conjugated) blood, and additionally in bile (3200 ng/mL), urine (9300 ng/mL), chest hair (2850 pg/mg) and gastric content. LC-QTOF screening analysis confirmed the presence of five different tapentadol metabolites (tapentadol-O-glucuronide, tapentadol-O-sulfate, N-desmethyltapentadol, N-desmethyltapentadol-glucuronide and N-desmethyltapentadol-O-sulfate), in urine, bile, cardiac and femoral blood. Positivity of body hairs allowed us to conclude that the man had used tapentadol in the last weeks/months. Autopsy and toxicological results (also positive for clotiapine, diazepam and chlordesmethyldiazepam) suggested that tapentadol could have caused, even at low concentrations, a severe respiratory depression, which contributed to the death of the subject. This is one of the few cases in literature where tapentadol was detected in blood, together with its metabolites, and the only one in which the parent drug was identified in hairs.

Analysis of 14 endocannabinoids and endocannabinoid congeners in human plasma using column switching high-performance atmospheric pressure chemical ionization liquid chromatography-mass spectrometry.

The endocannabinoid system (ECS) is a complex cell-signaling system. To address the growing need of analytics capturing endocannabinoid levels to investigate the ECS, we developed and validated an assay for the quantitative analysis of 14 endocannabinoids and congeners. A simple extraction using protein precipitation with acetonitrile followed by online-trapping high-performance liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) was used to monitor the levels of 14 endocannabinoids in plasma. The assay was validated and intra-run and inter-run accuracies and imprecisions as well as matrix effects, recoveries, and sample stabilities were determined. As a proof of concept, a subset of study samples after naturalistic administration of Cannabis flower and concentrate was analyzed. With the exception of N-oleoyl dopamine and oleamide, all endocannabinoids fulfilled the predefined acceptance criteria. Reproducible recoveries and no significant matrix effects were observed. Sample stability was an issue. Analysis of the proof-of-concept study samples revealed a significantly (p = 0.006) higher concentration of docosatetraenoyl ethanolamide in concentrate users (300 ± 13 pg/mL) compared to flower users (252 ± 11 pg/mL). A robust, sensitive high-throughput assay for the quantitation of 14 endocannabinoids and congeners was successfully validated. Our study showed that it is mandatory to (A) appropriately stabilize samples and (B) separate and separately quantify 1-AG and 2-AG; otherwise, study results are unreliable. The analysis of study samples from Cannabis flower users versus Cannabis concentrate users revealed higher levels of docosatetraenoyl ethanolamide and anandamide (n.s.) in high THC concentrate users in accordance with the existing literature, supporting the validity of the assay measurements. Graphical abstract.

A comparison between two different dried blood substrates in determination of psychoactive substances in postmortem samples.

Purpose: Whatman™ 903 cards represent a valid type of support for collection, storage, and analysis of dried blood spots (DBS). Whatman™ FTA (Flinders Technology Associates) are a type of cards soaked in chemicals that cause denaturation of proteins, while preserving DNA and ensuring the safe handling of DBS; to date, these cards are still rarely employed in forensic toxicology. The aim of this study was to analyze several psychoactive substances on not-dried blood on the two different cards and to compare the qualitative and quantitative results. Methods: Twenty cardiac postmortem blood samples were collected and deposed on Whatman™ 903 and Whatman™ FTA cards. Spots and not-dried blood were analyzed following our validated and previously published liquid chromatography-mass spectrometry methods. Results: We were able to identify: eight drugs of abuse and their metabolites (15 cases), five benzodiazepines and their metabolites (3 cases), six antidepressants (6 cases) and two antipsychotics (3 cases). We observed a perfect qualitative correspondence and a general good quantitative correlation between data obtained from not-dried blood and the two different DBS cards, except for alprazolam, diazepam, desmethyldiazepam, fluoxetine and sertraline, that showed a lower concentration on FTA. Additional experiments suggest that the chemicals, adsorbed on FTA, are not the cause of the loss of signal observed for the substances previously mentioned and that methanol should be preferred as extraction solvent. Conclusions: This study proved that FTA cards are a good and a hazard-free alternative sample storage method for analysis of several psychoactive substances in postmortem blood. Supplementary Information: The online version contains supplementary material available at 10.1007/s11419-020-00567-2.

Comparison of Two Immunoassay Screening Methods and a LC-MS/MS in Detecting Traditional and Designer Benzodiazepines in Urine.

Sensitive and specific immunoassay screening methods for the detection of benzodiazepines in urine represent an important prerequisite for routine analysis in clinical and forensic toxicology. Moreover, emerging designer benzodiazepines force labs to keep their methodologies updated, in order to evaluate the reliability of the immunochemical techniques. This study aimed at evaluating the sensitivity and specificity of two different immunoassay methods for the detection of benzodiazepines in urine, through a comparison with the results obtained by a newly developed liquid chromatographic tandem mass spectrometric (LC-MS/MS) procedure. A cohort of authentic urine samples (N = 501) were processed, before and after a hydrolysis procedure, through two immunoassays and an LC-MS/MS method. The LC-MS/MS target procedure was optimized for monitoring 25 different molecules, among traditional and designer benzodiazepines, including some metabolites. At least one of the monitored substances was detected in 100 out of the 501 samples. A good specificity was observed for the two immunoassays (>0.99), independently of the cut-offs and the sample hydrolysis. The new kit demonstrated a fairly higher sensitivity, always higher than 0.90; in particular, a high cross-reactivity of the new immunoassay was observed for samples that tested positive for lorazepam and 7-aminoclonazepam. The two immunoassays appeared adequate to monitor not only traditional benzodiazepines but also new designer ones.

Distribution of Fluvoxamine and Identification of the Main Metabolite in a Fatal Intoxication.

Fluvoxamine is a selective serotonin reuptake inhibitor, with a half-life of about 30 hours, that is commonly prescribed in the treatment of depression and obsessive and compulsive disorders. Though its more favorable adverse effect profile in comparison to tricyclic antidepressants, overdosages could lead to severe central nervous system depression. We hereby report the case of a 48-year-old woman with psychiatric disorders, who died in the Protected Community where she lived. An autopsy, during which multiorgan congestion and aspiration of gastric content were found, was performed 9 days after the death. Femoral and cardiac blood, urine and bile were collected for toxicological analysis. GC-MS, LC-MS-MS and LC-HRMS screenings were performed on blood samples. The analysis allowed to identify the following drugs: fluvoxamine, clotiapine, 7-aminoclonazepam, propranolol, gabapentin and haloperidol. Quantification of the detected drugs in blood was performed by means of a validated LC-MS-MS analytical procedure, and the following results were achieved: fluvoxamine (2.20 mg/L), gabapentin (41.00 mg/L), 7-aminoclonazepam (0.24 mg/L), clotiapine (0.07 mg/L), haloperidol (<0.01 mg/L) and propranolol (0.24 mg/L). Fluvoxamine concentration in blood exceeded ~10 times the upper limit of therapeutic blood levels (0.23 mg/L). Contributory causes of death, such as due to multiple drug use, however, cannot be excluded. The distribution of fluvoxamine in all biological fluids was evaluated and a postmortem redistribution effect was observed (C/P blood ratio: 1.86). Fluvoxamine acid metabolite was identified in urine, bile and in cardiac blood, through an LC-QTOF analytical procedure.

Fatal poisoning of four workers in a farm: Distribution of hydrogen sulfide and thiosulfate in 10 different biological matrices.

We evaluate the distribution of sulfide and thiosulfate (TS) in biological samples of four dairy farmers died inside a pit connected to a manure lagoon. Autopsies were performed 4 days later. Toxicological analyses of sulfide and TS were made using an extractive alkylation technique combined with gas chromatography/mass spectrometry (GC/MS). Autopsies revealed: multiorgan congestion; pulmonary edema; manure inside distal airways of three of the four victims. Sulfide concentrations were cardiac blood: 0.5-3.0 µg/mL, femoral blood: 0.5-1.2 µg/mL, bile: <0.1-2.2 µg/mL; liver 2.8-8.3 µg/g, lung: 5.0-9.4 µg/g, brain: 2.7-13.9 µg/g, spleen: 3.3-6.3 µg/g, fat: <0.1-1.5 µg/g, muscle: 2.6-3.5 µg/g. TS concentrations were cardiac blood: 2.1-4.9 µg/mL, femoral blood: 2.1-2.3 µg/mL, bile: 2.5-4.4 µg/mL, urine: <0.5-1.8 µg/mL; liver <0.5-2.6, lung: 2.8-5.4 µg/g, brain: <0.5-1.9 µg/g, spleen: 1.2-2.9 µg/g, muscle: <0.5-5.6 µg/g. The cause of death was assessed to be acute poisoning by hydrogen sulfide (H2S) for all the victims. Manure inhalation contributed to the death of three subjects. The measurement of sulfide and TS concentrations in biological samples contributed to better understand the sequence of the events. Subjects 3 provided the highest concentration of sulfide in brain, thus, supporting the hypothesis of a rapid loss of consciousness and respiratory depression. One by one, the other farmers entered the pit in attempts to rescue the coworkers but collapsed. Despite the rapid death, subject 3 was the only one with TS detectable in urine. This could be due to differences in metabolism of H2S.

Importance of segmental hair analysis in a suspected case of attempted homicide by flocoumafen and difenacoum.

The 4-hydroxycoumarin derivatives are the most used rodenticides and act as classical anticoagulants, interfering with the production of clotting factors in liver by antagonizing the action of vitamin K reductase, thereby inhibiting recycling of vitamin K1, involved in activation of blood clotting factors, resulting in massive bleeding. In this paper, we present the case of a 72-year old man providing abnormal coagulation parameters (PT-INR between 16.1 and 19.1) after hospitalization. Blood samples tested positive for flocoumafen and difenacoum, two superwarfarin rodenticides. Patient's hair specimens, sampled 19 days after his hospitalization, showed that traces of both difenacoum and flocoumafen were detected in the first 1 cm; in the intermediate segments (1-2 and 2-3 cm), both difenacoum and flocoumafen were absent, while in the distal segment (3-4.5 cm), only difenacoum was found, but in significant amounts (140 pg/mg). Exposure to difenacoum in the past months, at least 4-5 before hospitalization, was confirmed by the presence of the rodenticide in the distal segment. Moreover, among the seized material, two specimens resulted compatible with the two rodenticides.

Determination of fentanyl and 19 derivatives in hair: Application to an Italian population.

Nowadays fentanyl and its analogs represent the most numerous group among synthetic opioid and, due to their higher potency in comparison to traditionl opioids, the main cause of the critical increase of fatal intoxications opioids-intake related in the USA. We developed an LC-MS/MS method for the detection and quantification of fentanyl and its analogs in hair, then applied to 117 real samples, 97 collected from drugs users and 20 from postmortem cases of drugs addicts. The ionization and MRM parameters have been optimized for 27 molecules: 20 reached the acceptance criteria for identification and quantification. LODs and LOQs of 0.2 and 0.5 pg/mg, respectively, were reached for most of the substances, except for five compounds for which were set at 0.5 and 1.0 pg/mg. 2 out of the 97 samples collected from drug users tested positive; one for carfentanil, butyryl fentanyl, THFF and ocfentanil; the other one for 3-methyl norfentanyl. 2 out of the 20 postmortem samples show positive results: one only for fentanyl, the other for furanyl fentanyl, acetyl fentanyl, methoxyacetyl fentanyl, methoxyacetyl norfentanyl, ocfentanil and 4-ANPP. Despite the relatively small number of samples, the results suggest that the method should be included in routine hair analyses for monitoring the new synthetic opioids potential intake by drug users.

Distribution of quetiapine and metabolites in biological fluids and tissues.

Quetiapine is an atypical antipsychotic drug, frequently found in post-mortem samples. The quantitative determination of active metabolites may help in the interpretation of the potential toxic effects of the parent drug and its role in death. A fully validated LC-MS/MS method was developed for the identification and quantification of quetiapine and two main metabolites (N-desalkylquetiapine and 7-hydroxyquetiapine) in blood, biological fluids and tissues. Then, the distribution of analytes in different matrices was evaluated. LODs of 0.9, 0.3 and 0.3ng/mL were calculated for quetiapine, N-desalkylquetiapine and 7-hydroxyquetiapine respectively; while a LOQ at the concentration of 10.0ng/mL was defined for the three analytes. 13 post-mortem positive real cases have been included in the experiment. The results revealed that quetiapine and N-desalkylquetiapine might undergo a significant post-mortem redistribution, while 7-hydroxyquetiapine is less affected by this factor. N-desalkylquetiapine could be found in blood in relatively high concentrations in comparison to those of quetiapine; therefore, it should be always advisable to measure both the analytes. The analysis of tissues could provide additional data on potential intoxication with quetiapine.

Determination of Antidepressants and Antipsychotics in Dried Blood Spots (DBSs) Collected from Post-Mortem Samples and Evaluation of the Stability over a Three-Month Period.

An LC-MS/MS method for the identification and quantification of antidepressants and antipsychotics was developed on dried blood spots (DBSs). Moreover, analyte stability on DBSs within a 3-month period was monitored. Aliquots of 85 µL of blood from autopsy cases were pipetted onto DBS cards, which were dried and stored at room temperature. DBSs were analyzed in triplicate immediately, within the following 3 weeks, and after 3 months. For each analysis, a whole blood stain was extracted in phosphate buffer and purified using Solid Phase Extraction (SPE) cartridges in order to avoid matrix effects and injected in the LC-MS/MS system. Thirty-nine molecules were screened. Limits of detection (LODs) ranged between 0.1 and 3.2 ng/mL (g) and 0.1 and 5.2 ng/mL (g) for antidepressants and antipsychotics, respectively. Limits of quantification (LOQs) varied from 5 to 10.0 ng/mL for both. Sixteen cases among the 60 analyzed resulted positive for 17 different analytes; for 14 of these the method was fully validated. A general good agreement between the concentrations on DBSs and those measured in conventional blood samples (collected concurrently and stored at -20 °C) was observed. The degradation/enhancement percentage for most of the substances was lower than 20% within the 3-month period. Our results, obtained from real post-mortem cases, suggest that DBSs can be used for routine sample storage.

A case report on potential postmortem redistribution of furanyl fentanyl and 4-ANPP.

Fatal intoxications due to accidental or voluntary intake of synthetic opioids represent an actual emerging issue. We report a case where we have analyzed furanyl fentanyl and its metabolite 4-anilino-N-phenetyl-piperidine (4-ANPP) in blood, urine, gastric content, bile and cerebrospinal fluid. In this case, a 53-year-old man was found dead at home with a needle still inserted in a vein; a plastic bag containing a white powder (later identified as a furanyl fentanyl-based product) was discovered in the room. Biological samples were collected during autopsy and extracted/purified onto a SPE cartridge before instrumental analysis. Qualitative and quantitative analyses were performed by LC-MS/MS on peripheral and cardiac blood, urine, cerebrospinal fluid (CSF), bile and gastric content. Furanyl fentanyl was identified and quantified in all the biological fluids collected. Interestingly, gastric content revealed an unexpected high amount of furanyl fentanyl; yet, cardiac blood and femoral blood provided significantly different concentrations (11.8 and 2.7 ng/g respectively). The concentration of furanyl fentanyl in CSF was similar to that measured in femoral blood (2.6 ng/mL), thus confirming that CSF could be a good alternative biological fluid whenever a postmortem redistribution is suspected. Concentrations of 93.5, 50.4, 171.7, 41.9, 10.2 ng/mL(g) were measured for 4-ANPP in cardiac blood, femoral blood, urine, bile and cerebrospinal fluid, respectively. The outcomes from the presented case report suggest that the two substances have been not only injected intravenously, but probably also ingested by the man. Fentanyl derivative and its precursor seemed to undergo an extensive postmortem redistribution.

Determination of benzodiazepines in blood and in dried blood spots collected from post-mortem samples and evaluation of the stability over a three-month period.

We successfully developed and validated an LC-MS/MS method for the identification of 27 and quantification of 9 benzodiazepines and metabolites in whole blood and DBSs. The results provided a good qualitative and quantitative correlation between DBSs stored at room temperature and whole blood stored at -20°C. A good stability for a three-month period was observed for most of the compounds detected in real post-mortem samples.

Ethyl glucuronide hair testing: A review.

Ethyl glucuronide (EtG) is a minor, non-oxidative ethanol metabolite that can be detected in several matrices (e.g. blood, urine, hair, meconium) for variable periods of time. Quantification of EtG in hair (hEtG) has established itself, over recent years, as one of the most reliable biomarkers of long-term alcohol consumption habits, with the Society of Hair Testing (SoHT) offering cut-off values for assessment of both abstinence and heavy drinking (>60 g/day). Despite its high diagnostic performance, however, issues concerning inter- and intra-laboratory variability as well as data interpretation are still being investigated and represent the ultimate barrier to widespread acceptance of hEtG in the forensic context. The aim of this review is to summarize currently available analytical methods of hEtG testing, provide a framework to understand current hEtG cut-offs and their possible upcoming changes (in particular, a lower abstinence cut-off has been proposed for the 2019 revision of the SoHT consensus), and offer a schematic but exhaustive overview of the pitfalls in result reproducibility and interpretation that may limit applications of hEtG testing in the forensic context. Ultimately, the purpose of the authors is not to undermine the reliability of hEtG as an alcohol use marker, but rather to enhance it by promoting familiarization with all aspects related to it, from ethanol pharmacokinetics and EtG incorporation into hair, to sample preparation and analytical methods, to specific cases warranting close attention and additional tests for correct interpretation of hEtG results.

A multi-analyte LC-MS/MS method for screening and quantification of 16 synthetic cathinones in hair: Application to postmortem cases.

A multi-analyte method for detection and quantification of 16 synthetic cathinones (known also as "bath salts") in human hair has been developed and fully validated using liquid chromatography-tandem mass spectrometry system. About 20 mg of hair samples, previously washed and homogenized, were ultrasonicated with 1 mL HCl 0.1 M solution. Samples were then extracted using a solid phase extraction procedure (SPE), taken to dryness and reconstituted in 100 µL mobile phase. Finally, they were directly injected into a liquid chromatographic system, coupled with tandem mass spectrometer detector. The validation criteria parameters were satisfactory according with the international guidelines. A LOQ of 5 pg/mg was obtained for 4-fluoromethcathinone (4-FMC), buphedrone, ethcathinone, methcathinone, mephedrone and naphyrone, while the method proved to be more sensitive for 4-methylethcathinone (4-MEC), methedrone, alpha-pyrrolidinopentiophenone (α-PVP), alpha-pyrrolidinohexiophenone (α-PHP), methylenedioxypyrovalerone (MDPV), butylone, ethylone, 3,4-dimethylmethcathinone (3,4-DMMC), pentedrone and pentylone, reaching a LOQ of 1 pg/mg. Potential use of bath salts was investigated in postmortem cases of young subjects previously tested positive at least to one traditional drug of abuse. Two samples out of 17 cases analyzed provided positive results for synthetic cathinones. One sample has been divided in two segments of 2.5 cm length each. Both segments were positive for 8 different cathinone derivatives, namely: 3,4-DMMC, 4-FMC, 4-MEC, α-PHP, α-PVP, methcathinone, methedrone and pentedrone. The second case provided positive results for ethcathinone.

Variability on ethyl glucuronide concentrations in hair depending on sample pretreatment, using a new developed GC-MS/MS method.

Quantitative determination of ethyl glucuronide in keratin matrix, particularly in hair samples, provides a significant contribution to the evaluation of the extent of ethanol intake. The first-choice method to carry out this analysis is LC-MS/MS, but other techniques may be used. The aim of this work is: a) to develop and validate a GC-MS/MS method for ethyl glucuronide determination in hair; b) to compare GC-MS/MS and LC-MS/MS in analysis of real samples; c) to compare EtG concentration obtained after hair cutting and pulverization. About 30 mg hair samples were washed, pulverized and soaked in 1 ml deionized water. After incubation, the solution was purified through a SPE anion exchange cartridge; the eluate was dried under nitrogen stream, derivatized with PFPA and reconstituted in n-hexane. Then, the sample was injected in the GC-MS/MS system, operating in negative chemical ionization mode and in selected reaction monitoring. The two most intense transitions were used to monitor ethyl glucuronide and deuterated internal standard. All the validation parameters fulfilled the international acceptance criteria. LOD and LOQ were set at 2.0 and 3.0 pg/mg respectively. This method was applied to 194 hair samples collected from teetotallers and alcohol consumers and represents a suitable alternative to LC-MS/MS for the determination of EtG in hair samples, in particular when scarce quantity of hair is available. This study confirmed that pulverization of hair increases the concentration of EtG, but some variability of EtG levels remains probably due to the presence of non-homogeneous material even though pulverization.

A liquid chromatography-tandem mass spectrometry method for the determination of cocaine and metabolites in blood and in dried blood spots collected from postmortem samples and evaluation of the stability over a 3-month period.

The aims of this study were (1) to identify and quantify cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and cocaethylene (CE) in DBS; (2) to compare dried blood spots (DBSs) analytical results with the routine blood analyses; (3) to monitor analytes stability on DBS within a 3-month period. Eighty-five µL of blood from postmortem cases were put on a card for DBS analysis and kept in the dark, at room temperature. Samples were extracted through solid-phase extraction (SPE) cartridges and injected in the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The analytical procedure is simple, sensitive, and specific. Limits of detection (LODs) and quantification (LOQs) were calculated at 1.0 and 5.0 ng/mL(g) for COC and CE, and at 0.5 and 2 ng/mL for EME and BE, respectively. Validation parameters fulfilled all the acceptance criteria. Fifty-five postmortem cases were evaluated. Eighteen cases were positive for COC (44-2456 ng/mL) and BE (228-4700 ng/mL), 12 for EME (92-1500 ng/mL), and 11 cases for CE (11-273 ng/mL). Stability was evaluated on 8 cases collected in the period January 2017-January 2018. For each case, 5 DBSs were collected at T0. Four DBSs were analyzed within the 4 following weeks and 1 sample was analyzed after 3 months. The concentrations on DBSs, stored at room temperature, always matched the ones obtained on blood samples kept at -20°C (<20% variation, both at T0 and after 3 months). BE and COC concentrations remained stable after a 3-month storage, EME concentrations slightly increased after 3 weeks in the 2 analyzed samples, while CE provided a less homogeneous stability depending on the sample.