Effective validation of chromatographic analytical methods: The illustrative case of androgenic steroids.

The increasing need to develop quantitative chromatographic methods with upgradable multi-targeted approach, allowing flexible and reliable application on large daily workload makes the implementation of an efficient strategy of method's validation and maintenance crucial for the quality assurance policy. The expounding case of a gas chromatographic-mass spectrometric method for the urinary endogenous steroid profiling is presented to illustrate a validation strategy that combines rigorous estimation of validation parameters with highly efficient use of the collected data. The analysis of blank urine samples fortified at six concentration levels with 18 targeted steroids was replicated nine times in three working sessions along twelve days. This dataset of 54 analysis formed the groundwork on which the statistical evaluation of several validation parameters was founded, including calibration, intra- and inter-day accuracy and precision, limit of detection (LOD), limit of quantification, ion abundance ratio repeatability, selectivity, specificity, and carry-over. The preliminary comparison of the response variances at different concentration levels provided the evaluation for heteroscedasticity. Then, the most appropriate calibration model was determined for each steroid, in terms of order (linear vs. quadratic) and weighting, allowing to complete their quantitation in each solution. Intra- and inter-day accuracy and precision were calculated therefrom. LOD values were computed with the Hubaux-Vos method from the weighted linear segment of the calibration curves. Only the assessment of recovery and ionization suppression/enhancement required the execution of further independent experiments. The case study demonstrated that the application of adequate statistical testing typically produced non-homogeneous models of calibration curves, mostly arising from heteroscedastic and quadratic distribution of datasets, unlike what is reported in overly simplified approaches. The misleading information obtained from the regression coefficient R2 to evaluate linearity was evidenced. The strong dependence of calculated LOD and accuracy from the selected calibration parameters was highlighted, making the implementation of an adequate calibration maintenance policy highly advisable.

Toward the Interpretation of Positive Testing for Fentanyl and Its Analogs in Real Hair Samples: Preliminary Considerations.

The detection of new psychoactive substances (NPS) in hair has become extensively researched in recent years. Although most NPS fall into the classes of synthetic cannabinoids and designer cathinones, novel synthetic opioids (NSO) have appeared with increasing frequency in the illicit drug supply. While the detection of NSO in hair is now well documented, interpretation of results presents several controversial issues, as is quite common in hair analysis. In this study, an ultra-high-performance liquid chromatography-tandem mass spectrometry method able to detect 13 synthetic opioids (including fentanyl analogs) and metabolites in hair was applied to 293 real samples. Samples were collected in the USA between November 2016 and August 2018 from subjects who had reported heroin use in the past year or had already tested positive to hair testing for common opiates. The range, mean and median concentrations were calculated for each analyte, in order to draw a preliminary direction for a possible cut-off to discriminate between exposure to either low or high quantities of the drug. Over two-thirds (68%) of samples tested positive for fentanyl at concentrations between LOQ and 8600 pg/mg. The mean value was 382 pg/mg and the median was 95 pg/mg. The metabolites norfentanyl and 4-ANPP were also quantified and were found between LOQ and 320 pg/mg and between LOQ and 1400 pg/mg, respectively. The concentration ratios norfentanyl/fentanyl, 4-ANPP/fentanyl and norfentanyl/4-ANPP were also tested as potential markers of active use and to discriminate the intake of fentanyl from other analogs. The common occurrence of samples positive for multiple drugs may suggest that use is equally prevalent among consumers, which is not the case, as correlations based on quantitative results demonstrated. We believe this set of experimental observations provides a useful starting point for a wide discussion aimed to better understand positive hair testing for fentanyl and its analogs in hair samples.

Analysis of Drugs of Abuse in Hair Samples by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS).

The determination at low concentrations of common psychotropic drugs is increasingly requested in hair samples for the retrospective investigation of habitual drug abuse and dependence as well as in other toxicological investigations. The dramatic improvements of the instrumentation based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) make the detection of tiny amounts of almost whatever drug is in hair possible, even after single-dose intake. Therefore, LC-MS/MS is gradually replacing gas chromatographic techniques in both screening and confirmation procedures, and is increasingly acknowledged as the technique of choice for hair analysis. We describe a simple procedure for the quantitative determination in hair samples of 15 common drugs of abuse, or metabolites, based on methanol extraction and direct analysis by ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS).

Development and validation of a Partial Least Squares-Discriminant Analysis (PLS-DA) model based on the determination of ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair for the diagnosis of chronic alcohol abuse.

The chronic intake of an excessive amount of alcohol is currently ascertained by determining the concentration of direct alcohol metabolites in the hair samples of the alleged abusers, including ethyl glucuronide (EtG) and, less frequently, fatty acid ethyl esters (FAEEs). Indirect blood biomarkers of alcohol abuse are still determined to support hair EtG results and diagnose a consequent liver impairment. In the present study, the supporting role of hair FAEEs is compared with indirect blood biomarkers with respect to the contexts in which hair EtG interpretation is uncertain. Receiver Operating Characteristics (ROC) curves and multivariate Principal Component Analysis (PCA) demonstrated much stronger correlation of EtG results with FAEEs than with any single indirect biomarker or their combinations. Partial Least Squares Discriminant Analysis (PLS-DA) models based on hair EtG and FAEEs were developed to maximize the biomarkers information content on a multivariate background. The final PLS-DA model yielded 100% correct classification on a training/evaluation dataset of 155 subjects, including both chronic alcohol abusers and social drinkers. Then, the PLS-DA model was validated on an external dataset of 81 individual providing optimal discrimination ability between chronic alcohol abusers and social drinkers, in terms of specificity and sensitivity. The PLS-DA scores obtained for each subject, with respect to the PLS-DA model threshold that separates the probabilistic distributions for the two classes, furnished a likelihood ratio value, which in turn conveys the strength of the experimental data support to the classification decision, within a Bayesian logic. Typical boundary real cases from daily work are discussed, too.

Occupational Exposure to Alcohol-Based Hand Sanitizers: The Diagnostic Role of Alcohol Biomarkers in Hair.

Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) in hair are effective direct biomarkers of ethanol ingestion, whose analytical determination can be used to discriminate between chronic and occasional ethanol intake. Ethanol is a compound widely used in some workplaces (e.g., clinics, hospitals) and is present in considerable amounts in mouthwash for oral cleaning, medications, cosmetic products, hydro-alcoholic disinfectants and antiseptics for hands. This study examined the ethyl alcohol exposure derived from hand disinfectants (in gel form) by simulating the typical occupational situation of medical-health workers (healthcare workers, nurses, surgeons, etc.) who frequently wash their hands with antiseptic sanitizer. Two types of hand disinfectants with 62% w/w of ethanol content were daily applied to the hands of a teetotaler for 20 times a day, for 4 consecutive weeks, thus simulating a typical workplace situation and a cumulative dermal exposure to ethanol of ~1,100 g. Different matrices (head, chest and beard hair, urine) were regularly sampled and analyzed using a ultra high-performance liquid chromatography tandem massspectrometry validated method for EtG and a (HS)SPME-GC-MS validated technique for FAEEs. The data obtained showed that a significant dermal absorption and/or inhalation of ethanol occurred, and that the use of detergents produce urinary EtG concentrations both higher than the cut-offs normally used for clinical and forensic analyses (either 100 and 500 ng/mL, depending on the context). The concentrations of the ethanol metabolites in the keratin matrices were, respectively, below the cut-off of 7 pg/mg for EtG and below 0.5 ng/mg for FAAEs (0.35 ng/mg for ethyl palmitate). In conclusion, the regular use of alcohol-based hand sanitizers can affect the concentration of urinary EtG and lead to positive analytical results, particularly when specimens are obtained shortly after sustained use of ethanol-containing hand sanitizer. On the other hand, direct biomarkers of alcohol abuse in the keratin matrix are capable of distinguishing between ethanol consumption and incidental exposures.

Effects of various sample pretreatment procedures on ethyl glucuronide quantification in hair samples: Comparison of positivity rates and appraisal of cut-off values.

The quantification of ethylglucuronide (EtG) in hair is nowadays recognized as the approach with the highest diagnostic performance to evaluate harmful drinking. A widely accepted cut-off of 30pg/mg has been selected after several accurate compared studies. While most of the studies that were used to establish the appropriate cut-off value prescribed to cut hair into small segments before their extraction, hair milling has subsequently been identified as the most efficient pretreatment procedure and was therefore recommended in the last Consensus document issued by the Society of Hair Testing. In this study, we initially compared the results obtained with the two sample preparations, namely cutting and milling, both being applied to the same specimens (n=781). Among these, 205 samples produced measurable EtG values with both methods, with differences ranging from -41.7% up to +415% (the mean increase in EtG concentration, switching from cutting to milling, was +62.1% and the median was +42.3%). Among the aforementioned 205 samples, 29 specimens (3.7% of the total 781 samples) produced significantly different outcome, being classified as negative (i.e., below 30pg/mg) if the cutting procedure is used, but largely positive (above 40pg/mg) when milling is used. Subsequently, the positivity rates obtained on a large population dataset (>27,000 samples) with the two procedures, were retrospectively compared using variable cut-offs values. The percentage of head hair samples with EtG concentration exceeding 30pg/mg upon application of the milling procedure shows a 45% increase (from 10.9% to 15.8%) with respect to cutting procedure, whereas the fraction of hair samples with EtG exceeding 40pg/mg (10.5%) overlaps the percentage of positive samples obtained after cutting pretreatment and applying a cut-off of 30pg/mg. On the basis of these results, it would be worth considering the application of cut-off values linked with the pretreatment procedure, taking into account the results of forthcoming inter-laboratory calibrations.

European guidelines for workplace drug and alcohol testing in hair.

Guidelines for Legally Defensible Workplace Drug Testing have been prepared and updated by the European Workplace Drug Testing Society (EWDTS). They are based on the 2010 version published by Pascal Kintz and Ronald Agius (Guidelines for European workplace drug and alcohol testing in hair. Drug Test. Anal. 2010, 2, 367) and in concordance with the Society of Hair Testing guidelines (Society of Hair Testing guidelines for drug testing in hair. Forensic Sci. Int. 2012, 218, 20-24). The European Guidelines are designed to establish best practice procedures whilst allowing individual countries to operate within the requirements of national customs and legislation. The EWDTS recommends that all European laboratories that undertake legally defensible workplace drug testing use these guidelines as a template for accreditation. Copyright © 2016 John Wiley & Sons, Ltd.

Enhanced solubility and antibacterial activity of lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazoles by complexation with ß-cyclodextrins.

Some lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazole antibacterial agents have been evaluated for their activity in the presence of cyclodextrins (CDs) containing aqueous solutions where CDs are adopted as solubilizing excipients for improving the poor water solubility of these compounds. For such purpose both the natural ß-CD and one of FDA/EMA approved CDs for parenteral use (i.e. HP-ß-CD) have been employed. The solubility rank order observed was accounted for by thermal analysis (Differential Scanning Calorimetry) and FT-IR spectroscopy. The most promising compound was subjected to further NMR spectroscopic studies and molecular modelling simulations to verify the interactions between the guest molecule and the CD cavity. The assessment of the antibacterial activity of such compounds against selected Gram positive and Gram negative bacterial strains clearly showed that their antimicrobial effectiveness may, quite in all instances, be positively affected by complexation with ß-CD and HP-ß-CD. These results, which are in some ways in contrast with those already reported in the literature, are herein discussed on the basis of plausible mechanisms. Moreover, this investigation also reveals that the described methodology of complexing both lipophilic and hydrophilic antimicrobial agents with CDs may be an useful approach to enhance their effectiveness as well as a promising strategy to overcome even the microbial resistance problem.

Cut-off proposal for the detection of ketamine in hair.

Ketamine is a powerful anesthetic drug used in both human and veterinary surgery, but it is also commonly misused because of its psychotropic properties. Since the abuse of this drug has been reported in many countries worldwide, its determination in hair samples is offered as a specialist test by hundreds of laboratories. However, unlike other common drugs of abuse, a cut-off level for ketamine in hair has not been fixed yet. Therefore, aim of this study is to propose a concentration value for ketamine in hair analysis, in order to discriminate between chronic and occasional use, and between active use and external contamination. After considering the chemical properties of this molecule, and the experimental data collected in our laboratory or reported in several other published studies, we propose a cut-off level of 0.5ng/mg, as indicative of repeated exposure to ketamine. Additionally, we suggest that the detection of the metabolite norketamine should be mandatory to prove active intake and exclude false positive result from external contamination. Thus, a reasonable cut-off value for norketamine could be fixed at 0.1ng/mg, while the minimal concentration ratio norketamine/ketamine may be positively established at 0.05.

Hair analysis as a tool to evaluate the prevalence of synthetic cannabinoids in different populations of drug consumers.

Among the new psychoactive products, herbal mixtures containing synthetic cannabimimetics are likely the most abused worldwide. In this study, a specific ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the detection of 23 synthetic cannabinoids in hair samples was developed in order to (1) expand the number of screened compounds, coherent with new substances emerging in the European territory, (2) evaluate their consumption on a large period of examination, and (3) evaluate the diffusion of cannabimimetics among different populations of drug consumers. The method employs digestion of hair sample with NaOH followed by extraction with n-hexane/ethylacetate, and injection into the UHPLC-MS/MS system. After validation, the method was applied to the analysis of 344 hair samples previously tested in our laboratory for the most common drugs. Overall, 15 samples were found positive for at least one synthetic cannabinoid. Coherent with previously published results, the present data show that young males, former or still active Cannabis consumers, represent the population most often involved in synthetic cannabimimetics consumption. Several cases of poly-abuse were also determined. The drug most frequently detected was JWH-073 (11 samples) generally at low concentration (mean 7.69 ± 14.4 pg/mg, median 1.9 pg/mg, range 1.6-50.5 pg/mg), followed by JWH-122 (8 samples, mean concentration: 544 ± 968 pg/mg, median 28.4 pg/mg, range 7.4-2800 pg/mg). Other detected drugs included JWH-250, JWH-081, JWH-018, JWH-210, JWH-019, and AM-1220. For several positive samples, the synthetic cannabinoid concentration was lower than 50 pg/mg, underlining the need for established cut-off values for discrimination between chronic consumption and occasional use (or external contamination).

Determination of ethyl glucuronide levels in hair for the assessment of alcohol abstinence.

This study examined the potential of a highly sensitive LC-MS/MS method for the determination of EtG in head hair (i) to ascertain alcohol abstinence, (ii) to estimate the basal level of EtG (sub-ppb concentrations) in head hair in a population of alcohol abstainers and (iii) to suggest a revision of cut-off values for assessing alcohol abstinence. An UHPLC-MS/MS protocol previously developed was modified and validated again to detect low EtG levels in head hair samples from a population of 44 certain abstainers and teetotalers. Basal level of EtG in hair was determined by a standard addition quantification method. The validated UHPLC-MS/MS method allowed detecting and quantifying 0.5 and 1.0 pg/mg of EtG in hair, respectively. EtG concentrations lower than 1.0 pg/mg were determined for 95% of abstainers; 30% of them had non-detectable (<0.5 pg/mg) EtG values. Two samples evidenced EtG concentrations higher than 1.0 pg/mg that were subsequently explained by unintentional ethanol exposure. The method's feature of high analytical sensitivity makes it particularly suitable for alcohol abstinence ascertainment and, in the same time, allows to tentatively estimate basal EtG concentrations in hair around 0.8±0.4 pg/mg. This finding opens a discussion on the possible origin of basal EtG concentration and potential sources of bias in the evaluation of alcohol abstinence. Cut-off value in the range of 1.0-2.0 pg/mg can be reliably proposed to support alcohol abstinence.

Determination of pharmaceutical and illicit drugs in oral fluid by ultra-high performance liquid chromatography-tandem mass spectrometry.

A simple and extremely fast procedure for the quantitative determination in oral fluid samples of 44 substances, including the most common drugs of abuse and several pharmaceutical drugs, was developed and fully validated. Preliminary sample treatment was limited to protein precipitation. The resulting acetonitrile solution was directly injected into an ultra-high performance liquid chromatograph (UHPLC) equipped with a C18 column (100mm×2.1mm, 1.7µm). The mobile phase eluted with linear gradient (water/formic acid 5mM: acetonitrile/formic acid 5mM; v:v) from 98:2 to 0:100 in 5.0min, followed by isocratic elution at 100% B for 1.0min. The flow rate was 0.6mL/min and the total run time was 9.0min including re-equilibration at the initial conditions. The analytes were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The method proved to be simple, accurate, rapid and highly sensitive, allowing the simultaneous detection of all compounds. The ease of sample treatment, together with the wide range of detectable substances, all with remarkable analytical sensitivity, make this procedure ideal for the screening of large populations in several forensic and clinical contexts, whenever oral fluid sampling has to be preferred to blood sampling, as for example in short retrospective investigations.

Simultaneous determination in hair of multiclass drugs of abuse (including THC) by ultra-high performance liquid chromatography-tandem mass spectrometry.

A simple procedure for the quantitative determination in hair samples of 13 common drugs of abuse or metabolites (morphine, 6-acetylmorphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, benzoylecgonine, cocaine, buprenorphine, methadone and Δ(9)-tetrahydrocannabinol) has been developed and fully validated. The analytes were extracted from the matrix by a simple overnight incubation with methanol at 55 °C. An aliquot of the extract was directly injected into an ultra-high performance liquid chromatography system equipped with Waters Acquity UHPLC BEH C18 column (100 mm × 2.1mm, 1.7 µm). The mobile phase eluted with a linear gradient (water/formic acid 5 mM:acetonitrile; v:v) from 98:2 to 0:100 in 4.5 min, followed by isocratic elution at 100% B for 1.0 min. The flow rate was 0.6 mL/min and the total run time was 8.0 min including re-equilibration at the initial conditions. The compounds were revealed by a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method proved linear in the interval from the limit of quantification to 5.0 ng/mg (1.0 ng/mg for Δ9-tetrahydrocannabinol) with correlation coefficient values ranging from 0.9970 to 0.9997. Quantitation limits were below the cut-off values recommended by the Society of Hair Testing and ranged from 0.02 to 0.08 ng/mg. Application of the present UHPLC-MS/MS procedure and instrumentation to hair analysis allows high sample-throughput, together with excellent sensitivity and selectivity, in workplace drug-screening controls and forensic investigations. These qualities, combined with minimal sample workup, make the cost of this screening affordable for most private and public administrations.

Distribution of chloralose in a fatal intoxication.

Chloralose (alpha-chloralose) is a poisonous substance currently used as a rodenticide or avicide. It has primarily been used in Europe since 1893 as a human and veterinary hypnotic agent. Chloralose is a central nervous system depressant also acting as a stimulant on spinal reflexes. In the present case, a 24-year-old man was found dead in his bedroom near vomit residues. Several items were seized from the scene, including an empty bottle of Murex 50 g (α-chloralose), sold in Italy as rodenticide. Postmortem examination revealed no evidence of natural disease or trauma. Heart blood, urine, gastric contents, vitreous humour, brain, bile and liver were collected and submitted for toxicological analysis. Several extraction procedures and a specific liquid chromatography-tandem mass spectrometry protocol were purposely developed and validated. Chloralose was found in blood at a concentration of 65.1 mg/L and high levels were also detected in the gastric contents, confirming its ingestion shortly before the man's death. The distribution of chloralose in the body was evaluated by analyzing urine, vitreous humour, brain, bile and liver specimens. Quantitation of chloralose in several body fluids and tissues adds new data about the distribution of this chemical in the human body after massive ingestion.

Simultaneous analysis of several synthetic cannabinoids, THC, CBD and CBN, in hair by ultra-high performance liquid chromatography tandem mass spectrometry. Method validation and application to real samples.

A simple procedure for the quantitative detection of JWH-018, JWH-073, JWH 200, JWH-250, HU-210, Δ(9)-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabinol (CBN) in hair has been developed and fully validated. After digestion with NaOH and liquid-liquid extraction, the separation was performed with an ultra-high performance liquid chromatography system coupled to a triple quadrupole mass spectrometer operating in the selected reaction monitoring mode. The absence of matrix interferents, together with excellent repeatability of both retention times and relative abundances of diagnostic transitions, allowed the correct identification of all analytes tested. The method was linear in two different intervals at low and high concentration, with correlation coefficient values between 0.9933 and 0.9991. Quantitation limits ranged from 0.07 pg/mg for JWH-200 up to 18 pg/mg for CBD The present method for the determination of several cannabinoids in hair proved to be simple, fast, specific and sensitive. The method was successfully applied to the analysis of 179 real samples collected from proven consumers of Cannabis, among which 14 were found positive to at least one synthetic cannabinoid.

Rapid determination of anti-estrogens by gas chromatography/mass spectrometry in urine: Method validation and application to real samples.

A fast screening protocol was developed for the simultaneous determination of nine anti-estrogenic agents (aminoglutethimide, anastrozole, clomiphene, drostanolone, formestane, letrozole, mesterolone, tamoxifen, testolactone) plus five of their metabolites in human urine. After an enzymatic hydrolysis, these compounds can be extracted simultaneously from urine with a simple liquid-liquid extraction at alkaline conditions. The analytes were subsequently analyzed by fast-gas chromatography/mass spectrometry (fast-GC/MS) after derivatization. The use of a short column, high-flow carrier gas velocity and fast temperature ramping produced an efficient separation of all analytes in about 4 min, allowing a processing rate of 10 samples/h. The present analytical method was validated according to UNI EN ISO/IEC 17025 guidelines for qualitative methods. The range of investigated parameters included the limit of detection, selectivity, linearity, repeatability, robustness and extraction efficiency. High MS-sampling rate, using a benchtop quadrupole mass analyzer, resulted in accurate peak shape definition under both scan and selected ion monitoring modes, and high sensitivity in the latter mode. Therefore, the performances of the method are comparable to the ones obtainable from traditional GC/MS analysis. The method was successfully tested on real samples arising from clinical treatments of hospitalized patients and could profitably be used for clinical studies on anti-estrogenic drug administration.

A fatal case of simultaneous ingestion of mirtazapine, escitalopram, and valproic acid.

Mirtazapine, escitalopram, and valproic acid are newer antidepressant drugs than traditional tricyclic antidepressants and are supposed to be less toxic. Nevertheless, intoxication cases due to their overdosage have been repeatedly reported. In the case presently reported, a 64-year-old woman with a previous history of chronic depression was found dead in her apartment. Several packages of pharmaceutical drugs were found, including mirtazapine, escitalopram, and valproic acid. During the autopsy, no evidence of natural disease or trauma was found to account for this death. In order to determine whether massive drug assumption might have determined a lethal intoxication, heart blood, urine, and gastric content were collected and submitted to toxicological analysis. Specific liquid chromatography-tandem mass spectrometry protocols were purposely developed and validated. Blood concentrations of mirtazapine, escitalopram, and valproic acid were 20.3, 65.5, and 417 mg/L, respectively, whereas urine concentrations were 17.0, 94.5, and 423 mg/L, respectively. High concentrations of these drugs were also detected in the gastric content, confirming their ingestion shortly before death. The agreement between authoptic examination by forensic pathologists and toxicological findings are consistent with the suicidal hypothesis, where the death arose by drug intoxication due to simultaneous high-dosage ingestion of mirtazapine, escitalopram, and valproic acid.

A study of distribution of ethyl glucuronide in different keratin matrices.

Ethyl glucuronide (EtG) is a direct metabolite of ethanol, frequently used as a biomarker of alcohol abuse. To this purpose, EtG is preferentially determined in hair samples, using a cut-off value of 30pg/mg to discriminate between social and heavy drinkers, as recently fixed by an international consensus conference. Although this cut-off value is assumed for head hair, alternative matrices, such as pubic, axillary and chest hair, are often analyzed when head hair is not available. Previous studies suggested that determination of EtG in various keratin matrices may lead to different results; growth cycle and rate, urine contamination, distribution of sebum glands and other environmental factors are likely to contribute to these differences. We analyzed more than 2700 samples (head, pubic, chest and axillary hair) to evaluate the inter- and intra-individual distribution of the EtG concentration in the different keratin matrices. The data were interpreted on a statistical basis, on the assumption that large population data-sets will level off the average alcohol consumption of each group. From both inter- and intra-individual distribution data, significant differences were observed in EtG concentrations recorded in head, axillary and pubic hair samples. It is concluded that pubic hair cannot be utilized alternatively to head hair to prove chronic alcohol abuse, nor is axillary hair, since positive and negative biases respectively affect these determinations. In contrast, for chest hair, EtG distributions similar to head hair were found, although the large discrepancy between the examined population dimensions presently prevents any definitive conclusion. Thus, chest hair represents a promising alternative to head hair for EtG determinations, deserving further investigation on samples collected from the same individuals, in order to establish a clear correlation between their respective EtG concentrations.

Validation of a GC/MS method for the detection of two quinolinone-derived selective androgen receptor modulators in doping control analysis.

Selective androgen receptor modulators (SARMs) represent an emerging class of drugs likely to be abused in sport. For clinical applications, these substances provide a promising alternative to testosterone-replacement therapies and their advantages include oral bioavailability, androgen receptor specificity, tissue selectivity, and the absence of steroid-related side effects. Although not yet commercially available, since January 2008 SARMs have been included on the prohibited list issued yearly by the World Anti-Doping Agency (WADA), so control laboratories need to update their procedures to detect either the parent drugs or their metabolites. Within this context, two quinolinone SARM models were synthesized and automatically characterized to update the existing routine screening procedures. The conditions for the new target analytes are compatible with the existing laboratory protocols used for both in-competition and out-of-competition controls and can be included in them. Validation parameters according to ISO 17025 and WADA guidelines were successfully determined. For analytical determinations, spiked urine samples were hydrolyzed and extracted at pH 9.6 with 10 mL of tert-butyl methyl ether. Then, the analytes were subsequently converted into trimethylsilyl derivatives and detected by gas chromatography-mass spectrometry. The absence of interferents, together with excellent repeatability of both retention times and the relative abundances of diagnostic ions, allowed proper identification of all SARM analytes. The analytes' quantification was linear up to 500 ng/mL and precision criteria were satisfied (coefficient of variation less than 25% at 10 ng/mL). The limits of detection were 1 ng/mL for both SARMs, whereas recovery values were between 95.5 and 99.3%. The validated method can be efficiently used for urine screening of the 2-quinolinone-derived SARMs tested.