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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., (© 2024 John Wiley & Sons Ltd.)

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., (© 2023 John Wiley & Sons Ltd.)

Driving under the influence (DUI) of alcohol is a road safety problem. Driving license regranting is based on the evaluation of medicolegal and toxicological variables that may include serum carbohydrate-deficient transferrin (CDT) and hair ethyl glucuronide (hEtG). The aim of the study was to compare the diagnostic performance of CDT and hEtG in a population of DUI offenders. Other factors potentially associated with heavy alcohol use were explored. The population included DUI offenders examined during the period of January 1, 2019, through June 30, 2022. Sociodemographic, medicolegal, and toxicological variables were collected. CDT in serum and EtG in head hair were determined in all subjects. Excessive alcohol intake (hEtG ≥30 pg/mg) was considered cause for unfitness to drive. Cohen's kappa coefficient was calculated. Descriptive analyses were performed using chi-square and Mann-Whitney tests. Variables significantly different between the groups were included in a multivariate binary logistic regression model. The sample encompassed 838 subjects (case group: 179, comparison group: 689). CDT exhibited poor agreement (κ = 0.053) with hEtG as the reference test. Lower education, age at DUI, heavy smoking, and GGT levels associated with heavy alcohol consumption differentiated the two groups. For DUI offenders, the use of CDT to assess heavy alcohol consumption is limited, possibly due to the time-window assessed, the time required for normalization, and the different amount of ethanol needed to reach higher CDT levels, in comparison to hEtG; thus, hEtG assessment is strongly recommended for this population. Heavy smoking, GGT, education, and age could be related to heavy alcohol consumption and higher risk of DUI., (© 2022 John Wiley & Sons Ltd.)

Letrozole is an aromatase inhibitor, used to treat postmenopausal women with hormone receptor-positive or unknown advanced breast cancer. It is prohibited in sport because it is used together with androgen anabolizing steroids to avoid their adverse effects. In the case of an adverse analytical finding, it may be important to distinguish between repetitive use due to voluntary administration and occasional use, possibly due to involuntary intake. With the objective to identify the dose capable of producing a positive hair test, and to apply these results to the scenarios of inadvertent letrozole ingestion by an athlete, this study investigates the urinary excretion and incorporation into hair of single doses of letrozole. Seven subjects were recruited for an excretion study of letrozole and its metabolite bis(4-cyanophenyl) methanol (M1) in urine, after the consumption of 0.62 mg, 1.25 mg, and 2.5 mg of letrozole, and to investigate the incorporation in hair after ingestion of 0.62 mg and 2.5 mg of letrozole. Urine and hair samples were also obtained from two women in chronic therapy. Urinary concentrations of letrozole and its metabolite M1 were lower in subjects administered once with 0.62 mg, 1.25 mg, or 2.5 mg letrozole than in women in regular therapy with 2.5 mg/day. In hair collected after a single dosage, concentrations of 16-60 pg/mg were detected while in women in chronic therapy concentrations were higher than 160 pg/mg all along the hair shaft. Hair analysis turned to be a promising possibility for the discrimination of letrozole repetitive use vs occasional/inadvertent administration., (© 2018 John Wiley & Sons, Ltd.)

Diuretic agents are prohibited in sports in- and out-of-competition according to the regulations of the World Anti-Doping Agency (WADA) because of their possible masking effects on other doping agents in urine samples, and their ability to produce fast acute weight losses. Despite previous studies reported adverse analytical findings (AAFs) resulting from contaminations at ppm level (μg/g) of medicinal products, and recommended to introduce reporting limits for diuretics in doping controls, these are not adopted in analyses performed by WADA-accredited laboratories. We report the case of an athlete with two AAFs for hydrochlorothiazide (HCTZ) at low urinary concentrations (<10 ng/mL), who declared the use of nutritional supplements prepared in a compounding pharmacy. His nutritional supplements were analyzed revealing HCTZ presence in different concentrations, at the ppm level (μg/g and ng/mL). With the aim of testing the plausibility of the observed urinary HCTZ concentrations with the nutritional supplement ingestion, a urinary excretion study with three healthy volunteers was performed. HCTZ-contaminated powder (6.4 μg/g of HCTZ) was administered to each subject in different dosages, reproducing the possible ingestion pattern occurred. Urine specimens were collected before and after ingestion of the powder, up to 24 hours, and underwent liquid-liquid extraction and liquid chromatography-tandem mass spectrometry determination. Post-administration specimens were found to contain HCTZ at concentrations of 5-230 ng/mL, which supported the accidental inadvertent intake of the prohibited substance by the athlete. This study makes the argument that the introduction of reporting limits for diuretics are warranted in doping control samples, in order to protect against inadvertent AAFs due to contaminated products., (© 2018 John Wiley & Sons, Ltd.)

Fatty acid ethyl esters (FAEEs) are minor ethanol metabolites that can accumulate in hair. The performance of hair FAEEs as a biomarker that can discriminate null or moderate drinking from risky, excessive drinking was verified by evaluating the relationship between self-reported daily alcohol intake and FAEE concentration in hair. The study subjects were 160 healthy volunteers (52% female) that included teetotallers, moderate/social drinkers (< 60 g of ethanol per day), and heavy drinkers (≥ 60 g/day).The estimated daily alcohol intake (EDAI) was assessed by a specific written questionnaire aimed at estimating the measure and the frequency of alcohol drinking and at excluding confounding factors. FAEEs (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate) were extracted from the hair matrix by overnight incubation in n-hexane/dimethylsulphoxide, purified by solid-phase extraction (SPE) and analyzed by gas chromatography-mass spectrometry (GC-MS) in selected ion monitoring and Electron ionization (EI) mode, using pentadeuterated internal standards. Hair samples exhibited FAEE concentrations (expressed as the sum of the four esters, CFAEE ) ranging from 0.01 to 10.78 ng/mg (average 1.16 and median 0.60 ng/mg). The EDAI was from 0 to 246 g of ethanol per day, average 28 g/day and median 15 g/day. A cut-off of 0.5 ng/mg in 3 cm of a proximal hair segment was adopted to discriminate social drinking from excessive ethanol consumption. False positive samples were identified in subjects using ethanol-containing hair lotions and women on estroprogestin therapy. Specificity of 87% was reached when the identified false positives were excluded from data elaboration. CFAEE in hair at a predetermined cut-off can be used to discriminate between moderate and excessive drinking only when confounding factors are meticulously removed., (Copyright © 2014 John Wiley & Sons, Ltd.)

The drug content of hair may be affected by washing, chemical or thermal treatments, the use of cosmetics, or exposure to the environment. Knowledge concerning the effect of natural or artificial light on drug content in hair can be helpful to the forensic toxicologist, in particular when investigating drug concentrations above or below pre-determined cut-offs. The photodegradation of methadone and its metabolite, 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was studied in authentic positive hair samples by comparing drug concentrations determined by liquid chromatrography-high resolution mass spectrometry before and after exposure to UVB light (in vivo study). The same approach was applied in order to investigate the light sensitivity of opiates (6-monoacetylmorphine and morphine) and cocainics (cocaine and benzoylecgonine) in true positive hair. The yields of photodegradation were calculated for each drug class in eight different positive hair samples irradiated by UVB at 300 J/cm(2) obtaining averages, ranges and standard deviations. In parallel, the photostability of all the compounds as 10(-5) -10(-4)  M standard solutions in methanol were examined by means of UVB light irradiation in the range 0-100 J/cm(2) followed by UV/Vis spectroscopic analysis and direct infusion electrospray ionization-high resolution mass spectrometry (in vitro study). In hair, methadone was shown to be significantly affected by light (photodegradation of 55% on average), while its metabolite EDDP proved to be more photostable (17%). 6-monoacetylmorphine, morphine, benzoylecgonine, and cocaine were more photostable than methadone in vivo (on average, 21%, 17%, 20%, and 11% of degradation, respectively). When irradiated in standard solutions, the target molecules exhibited a larger photodegradation than in vivo with the exception of cocaine (photodegradation for methadone up to 70%, 6-monoacetylmorphine and morphine up to 90%, benzoylecgonine up to 67%, cocaine up to 15%). Some factors possibly affecting the yields of photodegradation in hair and partially explaining the differences observed between the in vivo and the in vitro studies were also investigated, such as the colour of hair (the role of melanin) and the integrity of the keratin matrix., (Copyright © 2014 John Wiley & Sons, Ltd.)

The relationship of drug concentrations between oral fluid and whole blood was evaluated by studying the linear correlation of concentrations and calculating the oral fluid to blood concentration ratios (OF/B) for different substances. Paired oral fluid and whole blood samples were collected from volunteers and persons suspected of drug use in four European countries. Oral fluid samples were collected with the Saliva∙Sampler™ device. All samples were analyzed for drugs of abuse and psychoactive medicines with validated gas and liquid chromatography-mass spectrometric methods. The median OF/B ratios were, for amphetamines 19-22, for opioids 1.8-11, for cocaine and metabolites 1.7-17, for tetrahydrocannabinol (THC) 14, for benzodiazepines 0.035-0.33, and for other psychoactive medicines 0.24-3.7. Most of the these results were close to theoretical values based on the physicochemical properties of the drugs and to values presented earlier, but there was a lot of inter-individual variation in the OF/B ratios. For all substances, except for lorazepam (R(2)  = 0.031) and THC (R(2)  = 0.030), a correlation between the oral fluid and whole blood concentrations was observed. Due to large variation seen here, drug findings in oral fluid should not be used to estimate the corresponding concentrations in whole blood (or vice versa). However, detection of drugs in oral fluid is a sign of recent drug use and oral fluid can be used for qualitative detection of several drugs, e.g. in epidemiological prevalence studies. By optimizing the sampling and the analytical cut-offs, the potential of oral fluid as a confirmation matrix could be enhanced., (Copyright © 2013 John Wiley & Sons, Ltd.)

The objective of this study was to determine the presence of alcohol and drugs in drivers severely injured in traffic crashes in six European countries. Data were collected from 2492 seriously injured drivers of cars and vans in Belgium, Denmark, Finland, Italy, Lithuania, and the Netherlands, between 2007 and 2010. Toxicological analysis was performed with chromatographic techniques on whole blood for 23 substances. The percentage of drivers positive for at least one psychoactive substance ranged between 28% (Lithuania) and 53% (Belgium). Alcohol (≥0.1 g/L) was the most common finding with the highest percentage in Belgium (42.5%). Among the alcohol-positive drivers, 90.5% had a blood alcohol count (BAC) ≥0.5 g/L and 65.7% had a BAC ≥1.3 g/L. Benzodiazepines (0.0-10.2%) and medicinal opioids (0.5-7.8%) were the most prevailing medicinal drugs, but half of the concentrations were lower than therapeutic. Cannabis (0.5-7.6%) was the most prevailing illicit drug. Alcohol was found in combination with drugs in 2.3-13.2% of the drivers. Drug combinations were found in 0.5-4.3% of the drivers. This study confirms the high prevalence of psychoactive substances in injured drivers, but we observed large differences between the participating countries. Alcohol was the most common finding, followed by cannabis and benzodiazepines. Notable are the many drivers having a BAC ≥ 1.3 g/L. The majority of the substances were found in combination with another psychoactive substance, mostly alcohol. The high prevalence of high BACs and combinations (compared to roadside surveys) suggest that those drivers are most at risk and that preventive actions should target them preferentially., (Copyright © 2012 John Wiley & Sons, Ltd.)