An LC-MS/MS method for the determination of drugs of abuse included THC-COOH, EtG, and NPS, using a single hair extraction sample.

Hair analysis plays an important role in the determination of drugs of abuse in both forensic and clinical toxicology investigations. The analysis of different substances often requires the use of different sample preparation methods, thereby increasing the amount of hair sample and time required. In the present study, a fast method involving a combination of a single 25 mg hair extraction procedure and four liquid chromatography-tandem mass spectrometry methods using the same chromatographic phases and column was developed and validated. The target was the identification and quantification of various commonly abused drugs and their metabolites, including amphetamines, cocaine, opioids, cannabinoids, THC-COOH and EtG, and more than 140 new psychoactive substances, including synthetic cannabinoids, phenethylamines, synthetic opioids, methylphenidate, cathinone, piperidine, and tryptamines.

Comparison of two methods for the extraction of ethylglucuronide from hair.

The aim was the comparison between the Society of Hair Testing (SoHT) consensus for the use of alcohol markers which powdering hair for the extraction of ethylglucuronide (EtG) in water and extraction using the patented M3 Reagent Test kit on cut hair. Hair samples were cut into small segments and washed twice with methanol and diethyl ether. The SoHT-Consensus entails the extraction of pulverised hair in water. This is obtained by incubation of 25 mg of hair at room temperature overnight and 2 h sonication, even if the overnight incubation is not mandatory. The M3 method entails incubation of 25 mg of cut hair with the M3-Reagent at 100°C for 60 min. After centrifugation, the supernatant is injected into a liquid chromatography-tandem mass spectrometry (LC-MS/MS). Samples (191) were collected in the APSS laboratory in Trento, Italy, between 2021 and 2022. The limit of quantification (LOQ) was set at 5 pg/mg for the pulverised and M3-Reagent methods. Assays showed good linearity above the range of LOQ-300 pg/mg. Precision (within 20%) values were also obtained using both methods. In the Passing-Bablock linear regression, the final regression curve between M3 (y) and the pulverising method (x) showed good agreement; the Bland-Altman analysis did not show any significant bias between the two methods. The M3-Reagent method, due to cut hair use, is easy to perform, saves time and allows for a smaller sample quantity loss with use of nondisposable grinding jars for the ball mill to obtain the extraction of EtG.

Post-mortem investigation into a death involving doping agents: The case of a body builder.

INTRODUCTION: A young male was found dead on the bed of a hotel room. He was expected to take part in a bodybuilding competition the day after. During the site inspection, drugs of different types were found. The next day, an autopsy was performed. The evidence of cardiomegaly with organ congestion involving lung, liver, kidneys, adrenal glands, spleen and brain was confirmed by both the autoptic and the histopathological exam. However, the cause of death needed to be investigated. METHODS: A thorough toxicological investigation was undertaken by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-high resolution mass spectrometry (LC-HRMS) and liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) on samples of urine, blood and hair. RESULTS AND DISCUSSION: Clenbuterol, a long-acting selective beta2 agonist, was found in both blood (1 ng/ml) and urine (1 ng/ml), and evidence of its use was provided by the analysis of the 3-cm hair (25 pg/mg). The main metabolite of drostanolone (2 alpha-methyl-androsterone), an anabolic steroid, was found in the urine (202 ng/ml), where an increased ratio of testosterone/epitestosterone (T/E = 11) emerged. Due to the results of the hair analysis, a long-term use of various anabolic steroids was supposed. The integrated analysis of the results and the absence of other possible causes (such as trauma or cardiac conduction anomalies) led to the identification of the abuse of doping substances as the underlying cause of death. CONCLUSION: Hair analysis has proven to be crucial in identifying drug misuse and the contributing cause of death.

Does Eumelanin Oxidation Play a Role on the Photostability of Ethyl Glucuronide in Hair Exposed to Simulated Solar Radiation?

The action of solar radiation on the concentration of the ethanol metabolite ethyl glucuronide (EtG) in 40 hair samples of nonabstinent subjects was investigated. Hair samples of different colors were analyzed before and after irradiation with artificial sunlight under a light dose corresponding to 3 months of sun exposure. After irradiation, an increase of EtG concentration was detected in 55% of the samples ranging from 5% to 141%. In 16 cases, a concentration reduction ranging from -2% to -74% was observed. The measure of the level of pyrrole-2,3,5-tricarboxylic acid (PTCA), a marker of eumelanin oxidation, demonstrated the largest increase in oxidation in light brown hair where the greatest degradation of EtG was observed after irradiation. However, the rise of PTCA in all hair tested was accompanied by increase in EtG concentration in 8/10 samples and by decrease in 2/10, suggesting no correlation between the two markers. To verify if hair structure was modified by light, scanning electron microscopy (SEM) analysis was performed on irradiated hair of different colors and compared with the dark samples. SEM revealed modification of hair structure in all samples showing partial shaft exfoliation and reduction of hair thickness under the treatment with solar radiation.

A Study on Photostability of Ethyl Glucuronide in Hair Irradiated under Artificial Sunlight.

Drugs incorporated into hair are exposed to the environment, and cosmetic and chemical treatments, with possible decreases in their content. Knowledge concerning the effect of sunlight on drug content in hair can be helpful to forensic toxicologists, in particular, when investigating drug concentrations above or below pre-determined cut-offs. Twenty-eight authentic positive hair samples were selected which had previously tested positive for ethyl glucuronide (EtG). Washed hair were divided into two identical tufts, with the former exposed at 13,219 J/cm2 (300-800 nm spectrum of irradiance) for 48 h in a solar simulator, and the latter kept in the dark. Hair samples were extracted and analyzed by ultra performance liquid chromatography-tandem mass spectrometry. The percentage of photodegradation was calculated for EtG. In parallel, photodegradation processes of standard molecule dissolved in aqueous and organic solutions were studied. In 28 hair samples, positive for the targeted analyte, exposure to artificial sunlight induced an appreciable increase in EtG concentrations. The concentration range in the non-irradiated hair samples was 6.0-772.0 pg/mg, and 64.3% of samples exhibited an increase in post-irradiation samples, ranging from 7% to 255%. In seven cases, a decrease was observed ranging from -5.0% to -36.0%. Thus, either a decrease or an increase of EtG may be observed post-irradiation, depending on hair color and/or hair thickness. Because the denaturation status of hair fibers and the thickness of hair before irradiation could play a role, a scanning electron microscope study should be envisaged.

Non-fatal Overdose with U-47700: Identification in Biological Matrices.

BACKGROUND/OBJECTIVE: We report on a case of severe intoxication after insufflation of U-47700, a synthetic opioid that acts as a selective agonist of the µ-opioid receptor, and is several times more potent than morphine. A man in his 30s was found irresponsive in his apartment and was brought to the emergency department of a local hospital. A comatose state and severe respiratory depression were present. Hetero anamnesis revealed that the patient could have taken the substance named "U-47700", bought on the Internet. After supportive care, the patient fully recovered. METHOD: Urine, blood and a white powder found at his home were collected during his hospital stay and sent for testing using liquid chromatography-high resolution mass spectrometry (LC-HRMS) on an Orbitrap instrument. Later, his pubic hair was also collected. A standard comprehensive toxicology screening was performed. RESULTS: U-47700 was identified in all biological samples and in the seized white powder. Using liquid chromatography-high resolution mass spectrometry (LC-HRMS) the presence of U-47700 and its phase I and phase II metabolites in blood, urine and pubic hair was confirmed. U-47700 was determined at 94 ng/mL and 5.2 ng/mL in blood at the admission and the day after, respectively, and 3.02 ng/mg in pubic hair, together with its metabolites. No other opioid nor designer drug could be detected in blood and urine, while in pubic hair Cocaine, Benzoylecgonine, Norcocaine, Mephedrone, Ketamine, Norketamine, 3,4-Methylenedioxymethamphetamine, Tetrahydrocannabinol and Cannabinol were also detected. CONCLUSION: The toxicological findings confirmed the use of U-47700 in the intoxicated patient and also revealed a history of a poly-drug use. The use of LC-HRMS allowed the easy identification of the NPS and its metabolites in fluids and hair.

A Study on Photostability of Amphetamines and Ketamine in Hair Irradiated under Artificial Sunlight.

Drugs incorporated into hair are exposed to the environment, and cosmetic and chemical treatments, with possible decreases in their content. Knowledge concerning the effect of sunlight on drug content in hair can be helpful to forensic toxicologists, in particular, when investigating drug concentrations above or below pre-determined cut-offs. Twenty authentic positive hair samples were selected which had previously tested positive for amphetamines and/or ketamine. Washed hair were divided into two identical strands, with the former exposed at 765 W/m² (300⁻800 nm spectrum of irradiance) for 48 h in a solar simulator, and the latter kept in the dark. Hair samples were extracted and analyzed by liquid chromatography high-resolution mass spectrometry detection. The percentage of photodegradation was calculated for each analyte (i.e., amphetamine, methamphetamine, methylendioxyamphetamine, ketamine, and norketamine). In parallel, photodegradation processes of standard molecules dissolved in aqueous and organic solutions were studied. In 20 hair samples positive for the targeted analytes, exposure to artificial sunlight induced an appreciable decrease in drug concentrations. The concentration ranges in the non-irradiated hair samples were 0.01⁻24 ng/mg, and 65% of samples exhibited a decrease in post-irradiation samples, with reduction from 3% to 100%. When more drugs were present in the same hair sample (i.e., MDMA and ketamine) the degradation yields were compound dependent. A degradation product induced by irradiation of ketamine in aqueous and methanol solutions was identified; it was also found to be present in a true positive hair sample after irradiation. Ketamine, amphetamines, and their metabolites incorporated in the hair of drug users undergo degradation when irradiated by artificial sunlight. Only for ketamine was a photoproduct identified in irradiated standard solutions and in true positive irradiated hair. When decisional cut-offs are applied to hair analysis, photodegradation must be taken into account since sunlight may produce false negative results. Moreover, new markers could be investigated as evidence of illicit drug use.

Sample preparation methods for determination of drugs of abuse in hair samples: A review.

Hair analysis has assumed increasing importance in the determination of substances of abuse, both in clinical and forensic toxicology investigations. Hair analysis offers particular advantages over other biological matrices (blood and urine), including a larger window of detection, ease of collection and sample stability. In the present work, an overview of sample preparation techniques for the determination of substances of abuse in hair is provided, specifically regarding the principal steps in hair sample treatment-decontamination, extraction and purification. For this purpose, a survey of publications found in the MEDLINE database from 2000 to date was conducted. The most widely consumed substances of abuse and psychotropic drugs were considered. Trends in simplification of hair sample preparation, washing procedures and cleanup methods are discussed. Alternative sample extraction techniques, such as head-space solid phase microextraction (HS-SPDE), supercritical fluid extraction (SFE) and molecularly imprinted polymers (MIP) are also reported.

Monitoring haloperidol exposure in body fluids and hair of children by liquid chromatography-high-resolution mass spectrometry.

BACKGROUND: Haloperidol, 4-[4-(4-chlorophenyl)-4-hydroxy-1-piperidinyl]-1-(4-fluorophenyl)-1-butanone (HP), one of the most widely used antipsychotics in the treatment of schizophrenia, mania, and other psychiatric disorders, is frequently encountered in cases of unintentional pediatric intoxication because the ingestion of a small amount can cause significant toxic effects in children. For monitoring HP in suspected ingestions, a liquid chromatography-high-resolution mass spectrometry method has been developed and validated in urine, blood, and hair samples. METHODS: The analyte was extracted from 1 mL blood or urine by liquid/liquid extraction and from 5 mg of hair by micropulverized extraction; gradient elution on an Atlantis T3 column was realized using HP-d4 as an internal standard. Positive ion electrospray ionization and high-resolution mass spectrometry determination were performed in an Orbitrap mass spectrometer. RESULTS: The method exhibited a r > 0.999 in the studied ranges (0.1-50 ng/mL in urine and blood and 0.1-50 ng/mg in hair) and a limit of quantification of 0.1 ng/mL for urine and blood and 0.1 ng/mg for hair; intra-assay and interassay relative SDs were always more than 18%. The method was applied to determine haloperidol in 3 children who were admitted to emergency departments. HP concentrations ranged from 2 to 21 ng/mL in urine, from not detected to 4.9 ng/mL in blood, and from 0.37 to 0.73 ng/mg in hair samples. CONCLUSIONS: The utilization of high-resolution/high-accuracy mass spectrometry in full scan mode allowed the identification of HP metabolites in urine and blood, thus unequivocally documenting the exposure to the drug. HP metabolites were structurally characterized by high-resolution multiple mass spectrometry. For the first time, a HP metabolite was detected in hair.

Determination of ketamine and norketamine in hair by micropulverized extraction and liquid chromatography-high resolution mass spectrometry.

Ketamine (KT), primarily used as a general anaesthetic agent in clinical practice, has become very popular in recent years as a recreational drug, due to its dissociative and hallucinogenic effects. A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method has been developed and validated for the quantification of KT and its main metabolite norketamine (NK) in 2.0mg of hair. Sample preparation consisted of a rapid, simultaneous pulverization and extraction step in acidic solution, followed by centrifugation and filtration. Gradient elution was performed by an Atlantis T3 analytical column, and deuterated KT was used as the internal standard. Positive ion electrospray ionization and HRMS determination in full-scan mode were achieved with an Orbitrap mass spectrometer. The method has a linear range of 0.05-50 ng/mg, a limit of quantisation of 0.05 ng/mg and a limit of detection of 0.02 ng/mg for both KT and NK. The validated method was applied for the determination of KT and NK in two authentic hair samples from subjects suspected of taking psychoactive substances. The detection of the metabolite at low concentration gave proof for systemic drug origin and an investigation into the possible presence of further metabolites was performed by means of retrospective screening.

High performance liquid chromatography-high resolution mass spectrometry and micropulverized extraction for the quantification of amphetamines, cocaine, opioids, benzodiazepines, antidepressants and hallucinogens in 2.5 mg hair samples.

A high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) method for simultaneous screening and quantification of 28 drugs was developed and validated for 2.5 mg hair samples. Target drugs and their metabolites included amphetamines, cocaine, opioids, benzodiazepines, antidepressants, and hallucinogens. After decontamination, hair samples were extracted with 200 µL of a mixture of water: acetonitrile:1 M trifluoroacetic acid (80:10:10, v/v) using a 5 min simultaneous pulverization/extraction step. The extracts were analysed by HPLC-HRMS in an Orbitrap at a nominal resolution of 60,000, with concomitant in source collisional experiments (in source CID). Gradient elution on an Atlantis T3 column resolved 28 target compounds and 5 internal standards. Total chromatographic run time was 26 min. Calibration was achieved by linear regression analysis utilizing six calibration points; R2 ranged from 0.9964 to 0.9999, the limits of quantification were 0.1 ng/mg for 8 compounds, 0.2 ng/mg for 16 compounds and 0.5 ng/mg for 4 compounds; mean relative errors from -21% to +23% were obtained; relative standard deviation, used to estimate repeatability and intermediate reproducibility at three concentrations, was always less than 20%. Process efficiency and recoveries for all analytes were better than 65 and 73%, respectively, at any concentration. The method was applied to hair samples from forensic investigations that contained a broad assortment of drugs of abuse and pharmaceuticals. The use of concomitant HRMS full scan and CID afforded the possibility of retrospective analysis for discovering untargeted drugs.

Simultaneous LC-HRMS determination of 28 benzodiazepines and metabolites in hair.

A liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the simultaneous identification and quantification of 28 benzodiazepines, including 6 metabolites, in 50 mg of hair has been validated. Positive ion electrospray ionization and HRMS determination in full-scan mode were realized on an Orbitrap mass spectrometer at a nominal resolving power of 60,000. In-source collisional experiments were conducted to obtain additional information for a more reliable identification of the investigated drugs. HRMS in full-scan mode allowed the exact determination of molecular masses of all analytes eluting in the HPLC run, so that both the immediate and retrospective screening of results for drugs and their metabolites were available. Sample preparation consisted of an overnight incubation in phosphate buffer pH 8.4 and a subsequent liquid/liquid extraction with methylene chloride/diethyl ether (90:10). Gradient elution was performed on a Luna C18 analytical column and four deuterated analogues were used as internal standards (IS). Validation was performed using both spiked hair samples and hair samples from subjects treated with benzodiazepines. Selectivity was evaluated by analysis of 20 certified blank hair samples. Extraction efficiency and matrix effects were evaluated by analysis of true positive samples. The lowest limits of quantification (LLOQs) ranged from 1 to 10 pg/mg. Linearity was investigated in the range from LLOQ to 1,000 pg/mg, for each compound (R(2) 0.998-0.999). Mean relative errors, calculated at three concentration levels, ranged from 1 to 20% (absolute value). Precision, at concentrations higher than the LLOQs, was always less than 15% expressed as percentage relative standard deviation. After validation, the procedure was applied to real samples collected for clinical and forensic toxicology purposes from subjects who were assumed to have taken benzodiazepines.

Neonatal hair analysis by liquid chromatography-high-resolution mass spectrometry to reveal gestational exposure to venlafaxine.

Venlafaxine (VEN) is a second generation antidepressant drug, belonging to the class of selective serotonine and norepinephrine reuptake inhibitors, widely used in the treatment of depression and anxiety disorders. Though its pharmacological profile is considered safe, treatment with VEN can cause several nervous, gastrointestinal, cardiovascular and genitourinary adverse effects. Therapeutic drug monitoring for VEN could be useful in specific situations, including exposure to the drug during pregnancy. A liquid chromatography-high-resolution mass spectrometry method was developed and validated for the assay of VEN in 2.5-mg hair samples from 2 newborn identical twin sisters. The analyte was extracted by a rapid, simultaneous pulverization and extraction step, allowing analysis when tiny amounts of hair are available, such as in the case of a newborn. Gradient elution on an Atlantis T3 column was performed using nordiazepam-d5 as an internal standard. Positive ion electrospray ionization and high-resolution full scan determination were performed in an Orbitrap mass spectrometer. The method was linear range in the range 0.2-25 ng/mg, and had a quantification limit of 0.2 ng/mg, a relative standard deviation in the range 0.7%-1.4% (intra-assay) and 2.9%-5.9% (interassay), and was accurate (as % relative error) in the range -9% to + 2%, using a hair sample size as low as 2.5 mg. The utilization of high-resolution/high accuracy mass spectrometry in full-scan mode allowed both the quantitative determination of VEN in the hair of the 2 newborns and the straightforward identification of 4 VEN metabolites, namely O-desmethylvenlafaxine, N-desmethylvenlafaxine, N,N-didesmethylvenlafaxine, and N,O-didesmethylvenlafaxine, by means of retrospective screening, thus unequivocally documenting in utero exposure.

Soil sampling strategies: evaluation of different approaches.

The National Environmental Protection Agency of Italy (APAT) performed a soil sampling intercomparison, inviting 14 regional agencies to test their own soil sampling strategies. The intercomparison was carried out at a reference site, previously characterised for metal mass fraction distribution. A wide range of sampling strategies, in terms of sampling patterns, type and number of samples collected, were used to assess the mean mass fraction values of some selected elements. The different strategies led in general to acceptable bias values (D) less than 2sigma, calculated according to ISO 13258. Sampling on arable land was relatively easy, with comparable results between different sampling strategies.