Higher Creatinine Concentrations in Ethyl Glucuronide-Positive Urine Specimens Collected from Subjects in a Controlled Alcohol Abstinence Program: Is Serum Creatinine a Good Marker of Renal Function in Drinkers?

AIMS: The aim of this study was to examine urine creatinine concentrations in drivers submitted to controlled alcohol abstinence programs. METHODS: Urine samples (n = 32,210) were screened for ethyl glucuronide (EtG) by immunoassay during a 2-year period. Non-negatives underwent EtG and ethyl sulfate (EtS) confirmation by coupled-column Liquid Chromatography-Tandem Mass Spectrometry. Urine samples were tested for dilution by the analysis of creatinine content with <0.2 g/l indicating a dilute specimen. RESULTS: The mean urine creatinine was significantly higher in EtG positives compared to negatives (1.47 ± 0.98 vs. 1.17 ± 0.79 g/l). The difference between positives and negatives was consistent within genders and age groups (<45; ≥45). The higher urinary creatinine in EtG positives is explained by a late antidiuretic effect of alcohol. CONCLUSION: Attempts to dilute urine specimens by drinking water or other liquids before voiding are less effective for EtG/EtS compared with illicit drugs excreted in urine. If the temporary decrease in serum creatinine as a consequence of the late antidiuretic effect of alcohol is confirmed by controlled studies, serum creatinine as an indicator of kidney function should be reconsidered in drinkers.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part A.

The (75)Se internal bioavailability was investigated in microalgae, mussels and rats as biological experimental models. The (75)Se accumulation from freshwater to microalgae [Scenedesmus obliquus (Turpin) Kützing], from freshwater to mussels (Unio mancus Lamark) and, finally, per os to rats (Rattus norvegicus Berkenhout) was followed using (75)Se-labelled selenite looking at (75)Se uptake, retention, intracellular distribution and binding with cellular biocomplexes. After exposure to 10, 50 and 500 µg Se L(-1), the microalgae showed an inhibitory effect on population growth only at the highest concentration. Mussels exposed to 105 µg Se L(-1) showed an accumulation of the element with time in all tissues. Intracellularly, Se was present in all subcellular fractions, especially in the cytosol. Rats were treated via oral administration with 5 µg Se rat(-1). After 24 h, liver and kidney showed the highest Se concentration.

Uptake from water, internal distribution and bioaccumulation of selenium in Scenedesmus obliquus, Unio mancus and Rattus norvegicus: part B.

(75)Se-selenite transfer was investigated in a phytoplankton-mussel-rat food chain model consisting of Scenedesmus obliquus (Turpin) Kützing, Unio mancus Lamark and Rattus norvegicus Berkenhout. (75)Se-metabolized forms were investigated in order to identify potential critical steps in the food chain, as well as its relative bioavailability looking also at intracellular, cellular and organ partitioning. Tissue and intracellular distribution of (75)Se in mussels fed with (75)Se-S. obliquus was different compared to those exposed only to inorganic (75)Se-selenite. The intracellular distribution of (75)Se in the hepatopancreas and mantle of mussels fed (75)Se-microalgae was similar to hepatic and renal distributions in rats, suggesting that their stomach dissociated larger (75)Se-containing molecules. The (75)Se partitioned from water (culture medium) to microalgae showing a bioconcentration factor of 435. The bottleneck in the trophic transfer of (75)Se occurred between S. obliquus-U. mancus. From microalgae to mussels and subsequently to rats no bioaccumulation was verified.

Determination of cocaine and metabolites in hair by column-switching LC-MS-MS analysis.

A method for rapid, selective, and robust determination of cocaine (CO) and metabolites in 5-mg hair samples was developed and fully validated using a column-switching liquid chromatography-tandem mass spectrometry system (LC-MS-MS). Hair samples were decontaminated, segmented, incubated overnight in diluted HCl, and centrifuged, and the diluted (1:10 with distilled water) extracts were analyzed in positive ionization mode monitoring two reactions per analyte. Quantifier transitions were: m/z 304.2→182.2 for CO, m/z 290.1→168.1 for benzoylecgonine (BE), and m/z 318.2→196.2 for cocaethylene (CE). The lower limit of quantification (LLOQ) was set at 0.05 ng/mg for CO and CE, and 0.012 ng/mg for BE. Imprecision and inaccuracy at LLOQ were lower than 20 % for all analytes. Linearity ranged between 0.05 and 50.0 ng/mg for CO and CE and 0.012 and 12.50 ng/mg for BE. Selectivity, matrix effect, process efficiency, recovery, carryover, cross talk, and autosampler stability were also evaluated during validation. Eighteen real hair samples and five samples from a commercial proficiency testing program were comparatively examined with the proposed multidimensional chromatography coupled with tandem mass spectrometry procedure and our reference gas chromatography coupled to mass spectrometry (GC-MS) method. Compared with our reference GC-MS method, column-switching technique and the high sensitivity of the tandem mass spectrometry detection system allowed to significantly reduce sample amount (×10) with increased sensitivity (×2) and sample throughput (×4), to simplify sample preparation, and to avoid that interfering compounds and ions impaired the ionization and detection of the analytes and deteriorate the performance of the ion source.

Synthetic cannabinoids in hair-Prevalence of use in abstinence control programs for driver's license regranting in Germany.

Although the use, structural variety, and prevalence of synthetic cannabinoids (SCs) have steadily increased on the drug market, they are rarely analyzed in abstinence control programs for driver's license regranting. The aim of this study was to determine the SC prevalence in these programs by analyzing hair samples collected between March 2020 and March 2021 from various regions in Germany, mainly Bavaria (40%). Specimens were analyzed quantitatively for drugs of abuse and qualitatively for 107 SCs. Hair samples were screened by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and to search for unknown SC analogs, positive samples were additionally screened by liquid chromatography-high resolution time of flight mass spectrometry (LC-qTOF/MS). The analysis of 5097 hair samples resulted in 181 SC detections (3.6%), showing a wide range of 44 SCs, with up to 13 different compounds found in a single sample. The most prevalent compounds were 5F-MDMB-PICA and MDMB-4en-PINACA; furthermore, 10 new substances not initially covered by LC-MS/MS analysis were detected by LC-qTOF/MS. The SC positivity rate was comparable to cocaine (5.4%) and amphetamine (2.6%). Only in 35 cases (0.7%), SC analysis was requested by the clients, highlighting the insufficient coverage of SC consumption in the studied collective. In summary, hair sample analysis proved to be a valuable tool to monitor the use of SCs. In order to keep pace with newly emerging SC analogs, an up-to-date analytical method is essential. Prospectively, SCs should be more routinely screened in hair analysis for abstinence control to avoid cannabis substitution by SCs.

Evidence for gender differences in the Amphetamine/Methamphetamine ratio in the hair of subjects undergoing Fitness-to-Drive testing.

BACKGROUND AND AIMS: Retrospective analysis of hair testing data provides insights in drugs abuse patterns and improves results interpretation. Cases from subjects undergoing driving fitness assessment (2010-2020) were examined to evidence patterns in methamphetamine (MA) abuse. MATERIALS AND METHODS: All cases with positive MA (≥0.025 ng/mg) were included (n = 585). Data available were gender, age, MA and A (amphetamine) in hair (h), hair color/treatment, length of proximal hair. Cases with Ah/MAh ≤ 0.35 (n = 469) were arbitrarily selected to remove as many combined A, MA users. ANOVA was performed to detect Ah/MAh predictors. RESULTS: No predictors affected Ah/MAh. A bimodal frequency distribution was observed. We clustered cases in two groups (1, Ah/MAh 0.025-0.070; 2, Ah/MAh 0.071-0.120) and performed logistic regression. Only gender exhibited significant difference across groups (p = 0.0080). Odds ratio for females falling into group 2 was 2.86 times higher (CI97.5 1.34-6.44). CONCLUSION: Literature data support the hypothesis that the two Ah/MAh groups represent different phenotypes of the CYP2D6-mediated MA N-demethylation. Whether gender plays a role in such difference could not be confirmed. However, these results provide further suggestion of an association of gender and pharmacogenomics with MA disposition, requiring these factors to be considered in future research.

[Assessment of fitness to drive in correlation with narcotic and psychotropic drug use. Epidemiologic study in Verona]. / La valutazione dell'idoneità alla guida in relazione all'uso di sostanze stupefacenti e psicotrope. Studio epidemiologico della casistica veronese.

Driving under the influence of drugs is a serious problem for road traffic safety. According to the Italian Road Traffic Code, the driving licence must not be issued to anyone who abuses, is addicted to, or suffers for dependence to illicit or psychotropic drugs. The diagnosis of such clinical conditions is performed by Provincial Medical Commissions of the Public Health Service also on the basis of drugs of abuse testing results on urine and/or hair samples. This study aimed at examining test results obtained by the Forensic Toxicology laboratory of the Department of Public Health & Community Medicine, University of Verona, upon request of the local Medical Commission, over the period 2003-2008 with the purposes of (i) defining trends in drug abuse in the examined population (ii) identifying specific risk factors for testing positive and for relapse, (iii) selecting the most effective and efficient analytical strategy to detect illicit drugs use. During the study period, cocaine was the most frequently detected illicit drug. The comparison of results from urine and hair testing confirmed the complementary features of these two biological substrates and the importance to have both data in order to increase the sensitivity in detecting illicit drug use. Moreover, this study showed that testing for driving fitness is an effective deterrent to illicit drug use, as only about one quarter of subjects testing positive at the first testing are still positive at the second testing.

LC-QTOF-MS Presumptive Identification of Synthetic Cannabinoids without Reference Chromatographic Retention/Mass Spectral Information. II. Evaluation of a Computational Approach for Predicting and Identifying Unknown High-Resolution Product Ion Mass Spectra.

Despite liquid chromatography-high-resolution tandem mass spectrometry (MS2) enables untargeted acquisition, data processing in toxicological screenings is almost invariably performed in targeted mode. We developed a computational approach based on open source chemometrics software that, starting from a suspected synthetic cannabinoid (SC) determined formula, searches for isomers in different new psychoactive substances web databases, predicts retention time (RT) and high-resolution MS2 spectrum, and compares them with the unknown providing a rank-ordered candidates list. R was applied on 105 SC measured data to develop and validate a multiple linear regression quantitative structure-activity relationship model predicting RT. Competitive Fragmentation Modeling for Metabolite Identification (CFM-ID) freeware was used to predict/compare spectra with Jaccard similarity index. Data-dependent acquisition was performed with an Agilent Infinity 1290 LC-6550 iFunnel Q-TOF MS with ZORBAX Eclipse-Plus C18 (100 × 2.1 mm2/1.8 µm) in water/acetonitrile/ammonium formate gradient. Ability of the combined RT/MS2 prediction to identify unknowns was evaluated on SC standards (with leave-one-out from the RT model) and on unexpected SC encountered in real cases. RT prediction reduced the number of isomers retrieved from a group of new psychoactive substances web databases to one-third (2,792 ± 3,358→845 ± 983) and differentiated between SC isomers when spectra were not selective (4F-MDMB-BUTINACA, 4F-MDMB-BUTINACA 2'-indazole isomer) or unavailable (4CN-Cumyl-B7AICA, 4CN-Cumyl-BUTINACA). When comparing 30/40 eV measured spectra of 99 SC against RT-selected, CFM-ID predicted spectra of isomers, the right candidate ranked 1st on median and 4th on average; 54% and 88% of times the right match ranked 1st or within the first 5 matches, respectively. To our knowledge, this is the first case of extensive chemometrics application to toxicological screening. In most cases, presumptive identification (being based on computation, it requires further information for confirmation) of unexpected SC was achieved without reference measured information. This method is currently the closest possible to true unbiased/untargeted screening. The bottleneck of the method is the processing time required to predict mass spectra (ca. 30-35 s/compound using a 64-bit 2.50-GHz Intel® Core™ i5-7200U CPU). However, strategies can be implemented to reduce prediction processing time.

LC-QTOF-MS Presumptive Identification of Synthetic Cannabinoids without Reference Chromatographic Retention/Mass Spectral Information. I. Reversed-Phase Retention Time QSPR Prediction as an Aid to Identification of New/Unknown Compounds.

The application of Quantitative Structure-Property Relationship (QSPR) modeling to the prediction of reversed-phase liquid chromatography retention behavior of synthetic cannabinoids (SC), and its use in aiding the untargeted identification of unknown SC are described in this paper. 1D, 2D molecular descriptors and fingerprints of 105 SC were calculated with PaDEL-Descriptor, selected with Boruta algorithm in R environment, and used to build-up a multiple linear regression model able to predict retention times, relative to JWH-018 N-pentanoic acid-d5 as internal standard, under the following conditions: Agilent ZORBAX Eclipse Plus C18 (100 mm × 2.1 mm I.D., 1.8 µm) column with Phenomenex SecurityGuard Ultra cartridge (C18, 10 mm × 2.1 mm I.D., < 2 µm) kept at 50°C; gradient elution with 5-mM ammonium formate buffer (pH 4 with formic acid) and acetonitrile with 0.01% formic acid, flow rate 0.5 mL/min. The model was validated by repeated k-fold cross-validation using two-thirds of the compounds as training set and one-third as test set (Q2 0.8593; root mean squared error, 0.087, ca. 0.56 min; mean absolute error, 0.060) and by predicting relative Retention Times (rRT) of 5 SC left completely out of the modeling study. Application of the model in routine work showed its capacity to discriminate isomers, to identify unexpected SC in combination with mass spectral information, and to reduce the length of the list of candidate isomers to ca. one-third, thus reducing significantly the time required for predicting high-resolution product ion spectra to be compared to the unknown using a computational Mass Spectrometry (MS) search/identification approach.

Ethyl glucuronide in hair compared with traditional alcohol biomarkers--a pilot study of heavy drinkers referred to an alcohol detoxification unit.

BACKGROUND: Traditional biomarkers for heavy alcohol use include serum carbohydrate-deficient transferrin (CDT), the enzymes aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as gamma-glutamyl transferase (GGT). Measurement of the nonoxidative ethanol metabolite, ethyl glucuronide (EtG) in hair, has been proposed as a new marker with superior qualities. The aim of this study was to investigate the sensitivity of EtG in hair to detect heavy alcohol use compared with CDT, AST, ALT, and GGT. We also wanted to study the quantitative relation between alcohol intake and the different biomarkers. METHODS: Sixteen patients with a history of heavy alcohol use over the previous 3 months were recruited directly after admission to a withdrawal clinic. They were thoroughly interviewed about their drinking pattern as well as relevant diseases and use of medicines or drugs. Serum was sampled and analyzed for %CDT, AST, ALT, and GGT. Hair samples were collected and analyzed for EtG. RESULTS: The mean estimated daily intake (EDI) over the previous 3 months was 206 +/- 136 g pure alcohol. All patients fulfilled the criteria for heavy alcohol use. The sensitivity to detect heavy alcohol use was 64% for %CDT, 67% for AST, 67% for ALT, 93% for GGT, and 94% for EtG. There was no correlation between the quantitative values of EDI and %CDT, AST, ALT, and GGT. There was a positive, statistically significant correlation between EDI and the level of EtG in hair. CONCLUSIONS: In this study, EtG in hair and GGT showed the best sensitivity to detect heavy alcohol use and there was a positive correlation between EDI and the concentrations of EtG in hair. Before giving recommendations for clinical practice, further studies should be carried out on larger materials and populations with a wider range of alcohol intake.

Serum/whole blood concentration ratio for ethylglucuronide and ethyl sulfate.

Serum/blood (S/B) concentration ratios for ethyl glucuronide (EtG) and ethyl sulfate (EtS) are missing from the literature, and the aim of this study was to determine these ratios in samples from patients at admission to an alcohol rehabilitation clinic. Two blood samples were collected simultaneously, and EtG and EtS were analyzed in whole blood and serum, respectively, using a liquid chromatography-mass spectrometry method. Separate calibration standards were prepared in both whole blood and serum for the calculation of whole blood and serum concentrations, respectively. Thirteen pairs of serum and whole blood were analyzed. The median S/B value for EtG was 1.69, and the range was 1.33-1.90. For EtS, the median S/B ratio was 1.30, and the range was 1.08-1.47. The S/B ratio was significantly lower for EtS than for EtG (p < 0.001). The higher concentrations of EtG and EtS in serum than in whole blood have to be considered when whole blood results obtained from forensic toxicology are compared to serum or plasma results from clinical laboratories.

Implementation and performance evaluation of a database of chemical formulas for the screening of pharmaco/toxicologically relevant compounds in biological samples using electrospray ionization-time-of-flight mass spectrometry.

Electrospray ionization (ESI)-time-of-flight (TOF) MS enables searching a wide number of pharmaco/toxicologically relevant compounds (PTRC) in biosamples. However, the number of identifiable PTRC depends on extension of reference database of chemical formulas/compound names. Previous approaches proposed in-house or commercial databases with limitations either in PTRC number or content (e.g., few metabolites, presence of non-PTRC). In the frame of development of a ESI-TOF PTRC screening procedure, a subset of PubChem Compound as reference database is proposed. Features of this database (approximately 50,500 compounds) are illustrated, and its performance evaluated through analysis by capillary electrophoresis (CE)-ESI-TOF of hair/blood/urine collected from subjects under treatment with known drugs or by comparison with reference standards. The database is rich in parent compounds of pharmaceutical and illicit drugs, pesticides, and poisons and contains many metabolites (including about 6000 phase I metabolites and 180 glucuronides) and related substances (e.g., impurities, esters). The average number of hits with identical chemical formula is 1.82 +/- 2.27 (median = 1, range 1-39). Minor deficiencies, redundancies, and errors have been detected that do not limit the potential of the database in identifying unknown PTRC. The database allows a much broader search for PTRC than other commercial/in-house databases of chemical formulas/compound names previously proposed. However, the probability that a search retrieves different PTRC having identical chemical formula is higher than with smaller databases, and additional information (anamnestic/circumstantial data, concomitant presence of parent drug and metabolite, selective sample preparation, liquid chromatographic retention, and CE migration behavior) must be used in order to focus the search more tightly.

A direct screening procedure for diuretics in human urine by liquid chromatography-tandem mass spectrometry with information dependent acquisition.

BACKGROUND: Diuretics are a class of compounds largely used for either therapeutic (edemas, hypertension, etc.) or illegal (doping) purposes. Probably owing to the substantial variety of their chemical structures, which makes them hardly extractable from a biological matrix in a single procedure, a quite short list of screening methods can be retrieved in the literature. METHODS: This work presents a screening procedure for 24 diuretics based on the direct injection of urine (after 50 folds dilution) in a LC-ESI-MS/MS system (Applied Biosytems 4000 QTrap). Two information dependent acquisitions (IDA), one in positive, one in negative ionization, allowed the acquisition of one selected reaction monitoring transition for each compound, which, when a significant peak was found, triggered the acquisition of the enhanced product ion (EPI) spectrum. RESULTS AND CONCLUSIONS: EPI spectra were stored in a library and the procedure was able to recognize by library matching various diuretics in real positive samples. The limits of detection were comprised between 0.002 and 0.25 mg/l and ion suppression was not found to significantly influence the analysis.

Ethyl glucuronide and ethyl sulphate determination in serum by liquid chromatography-electrospray tandem mass spectrometry.

BACKGROUND: Ethyl glucuronide (EtG) and ethyl sulphate (EtS) are two ethanol metabolites that can be detected in serum up to 8 h after ethanol elimination. Their presence is therefore indicative of recent ethanol consumption in case of delayed sampling after an event (e.g. car crash). METHODS: A LC-ESI-MS-MS method for the determination of EtG and EtS in serum was developed and validated. Two product ions together with the parent ions were monitored for identification. Pentadeuterated-EtG was used as internal standard. RESULTS: Excellent linearity for EtG (from 0.22 to 45 micromol/l) and EtS (from 0.40 to 80 micromol/l) was observed (r(2)>or=0.9998). LOD and LLOQ were 0.04 and 0.20 micromol/l for EtG and 0.08 and 0.40 micromol/l for EtS, respectively. Accuracy (bias) and precision (relative standard deviation), studied at four different quality control levels, were always better than 7%. Matrix effects were found to be negligible. The method was applied to several samples obtained from known alcoholics and social drinkers. CONCLUSIONS: A sensitive and specific determination of EtG and EtS in serum samples was achieved despite a simple and fast sample preparation. To our knowledge, this is the first fully validated method for the simultaneous determination of the two alcohol metabolites in serum.

LC-MS-MS analysis of 2,4-dinitrophenol and its phase I and II metabolites in a case of fatal poisoning.

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis of biological fluids (blood, urine, gastric content, and bile) collected at autopsy in a case of suspected 2,4-dinitrophenol (DNP) fatal poisoning allowed the determination of DNP and its known metabolites (2-amino-4-nitrophenol and nitro-4-aminophenol). The tentative identification of three conjugated metabolites (DNP glucuronide, DNP sulfate, and 2-amino-4-nitrophenol glucuronide) could be made on the basis of their pseudomolecular ion, isotopic and fragmentation patterns, and retention characteristics. Another DNP metabolite reported in the literature, 2,4-diaminophenol, was not detected in the samples. Postmortem blood concentrations were 48.4 mg/L for DNP and 1.2 mg/L for 2-amino-4-nitrophenol. Gas chromatography-MS screening and quantification in postmortem blood revealed the presence of toxic concentrations of citalopram and its desmethylated metabolite (0.58 and 0.40 mg/L, respectively) and therapeutic or lower than therapeutic levels of olanzapine (0.04 mg/L), desalkylflurazepam (0.02 mg/L), and nordazepam (0.01 mg/L). Based on LC-MS-MS results and on available literature data on DNP poisonings, it was concluded that DNP poisoning played a contributing role, together with citalopram, in the cause of death.

Determination of ethyl glucuronide in hair samples by liquid chromatography/electrospray tandem mass spectrometry.

A method for the determination of ethyl glucuronide (EtG) in hair samples, using liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS), was developed and validated. The treatment of hair samples was as follows: to 100 mg of washed (dichloromethane followed by methanol, 1 ml each) and cut (1-2 mm) material, 700 microl of water, 20 microl of internal standard solution (pentadeuterated EtG, D(5)-EtG, 500 microg/l) and 20 microl of methanol were added. Samples were incubated at 25 degrees C overnight and then ultrasonicated for 2 h. Finally, 8 microl of the centrifuged solution (13,000 rpm) were analyzed by LC/ESI-MS/MS in negative ion mode. The surviving ions of EtG and D(5)-EtG were monitored together with the following MRM transitions: m/z 221 --> 75, m/z 221 --> 85 (EtG) and m/z 226 --> 75, m/z 226 --> 85 (D(5)-EtG). The method exhibited a mean correlation coefficient better than 0.9998 over the dynamic range (3-2000 pg/mg). The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 3 and 2 pg/mg respectively. The intra- and interday precision and accuracy were studied at four different concentration levels (3, 5, 56 and 160 pg/mg) and were always better than 7% (n = 5). Matrix effects did not exceed 20%. The method was applied to several hair samples taken from autopsies of known alcoholics, from patients in withdrawal treatment, from social drinkers, from adult teetotalers and from children not exposed to ethanol, with EtG concentrations globally ranging from < or =2 to 4180 pg/mg.

Ethyl glucuronide in hair: Is it a reliable marker of chronic high levels of alcohol consumption?

AIMS: This study aims to investigate the relationship between ethanol daily intake (EDI) and the levels of ethyl glucuronide in hair. DESIGN: Ethyl glucuronide concentration was determined in hair samples from different classes of ethanol drinkers and results were compared with the reported information about drinking habits. SETTING: Pavia, Italy. PARTICIPANTS: Twenty-two known alcoholics, 21 volunteers self-reporting an EDI from 2 to 60 g, and seven teetotallers were involved in this study. MEASUREMENTS: Ethyl glucuronide determination in hair samples was performed by liquid chromatography-tandem mass spectrometry (limit of detection: 2 pg/mg, lower limit of quantification: 3 pg/mg). FINDINGS: Current known alcoholics (n = 21) had ethyl glucuronide hair concentration in the range 4.0-434.7 pg/mg (average: 62.8, median 37.4 pg/mg); ethyl glucuronide was not detected in hair samples from teetotallers (n = 7); all volunteers reporting an EDI of at least 30 g ('non-moderate drinkers' according to the US Department of Health and Human Services) tested positive for ethyl glucuronide (cut-off: 4 pg/mg). All volunteers declaring an ethanol daily intake higher than 40 g ('heavy drinkers' according to the World Health Organization, Regional Committee for Europe) tested positive for this compound (cut-off: 5 pg/mg). The application of a cut-off of either 4 pg/mg or 5 pg/mg resulted in one false positive, coming from a volunteer asserting an ethanol daily intake of 30 g. No false negatives were found. CONCLUSIONS: The concentration of ethyl glucuronide in hair appears to correlate with EDI.

The role of cocaine in heroin-related deaths. Hypothesis on the interaction between heroin and cocaine.

In recent years, there has been an increase in cocaine-related deaths at the Department of Legal Medicine and Public Health of Pavia, probably reflecting the rising trend in cocaine use in Western Europe. Deaths from cocaine alone have increased from 6 cases in 1979-1991 (1.5% of drug-of-abuse deaths) to 13 in 1992-2002 (3.2%) and comparing the same periods, heroin-related deaths (HRDs) involving cocaine more than doubled from 8 (1.9%) to 22 (5.4%). In an attempt to investigate the role of cocaine in HRDs, acute narcotic death cases testing positive for cocaine use (blood cocaine or metabolite concentration >0.01 mg/l, COC+) were examined. Only cases from 1997 to 2001 were considered as in this period all data were obtained using the same analytical procedures (free morphine and total morphine by DPC Coat-A-Count radioimmunoassay before and after enzymatic hydrolysis, cocaine and metabolites in blood by SPE, TMS derivatization and gas chromatography-mass spectrometry (GC-MS)). The median, minimum and maximum concentrations of free morphine in blood (FM) and total morphine in blood (TM), urine (UM) and bile (BM) in the COC+ group (n = 9) were compared with those calculated in the group of "pure" HRDs (no other drugs detected in blood, COC-, n = 30). Differences among the medians in the two groups were statistically evaluated using the two-tailed Mann-Whitney U-Test. Statistical analysis was also carried out including in both groups cases with a blood alcohol concentration (BAC) > 20 mg/L (COC+, n = 19; COC-, n = 76). For the COC+ group, median TM was lower (0.32 mg/l versus 0.90 mg/l, P = 0.0214), median FM was lower, but not statistically significant (0.08 mg/l versus 0.28 mg/l, P = 0.1064), FM/TM ratio was similar (0.33 and 0.35), UM was also similar (21.0 mg/l and 18.0 mg/l), and BM was higher (90.0 mg/l versus 49.0 mg/l, P = 0.0268). Similar comparison results were obtained by repeating statistical analyses after including in the two groups cases with positive BAC. The picture observed for HRD cases involving cocaine is very different from what was previously observed for HRD cases involving ethanol [A. Polettini, A. Groppi, M. Montagna, The role of alcohol abuse in the etiology of heroin related deaths: evidence for pharmacokinetic interactions between heroin and alcohol, J. Anal. Toxicol. 23 (1999) 570-576], and updated with more recent data; in the high-ethanol (HE, BAC > 1000 mg/l) group, TM was lower than in the low-ethanol (LE, BAC

Applications of liquid chromatography-mass spectrometry in doping control.

This paper reviews liquid chromatographic-mass spectrometric (LC-MS) procedures for the screening, identification and quantification of doping agents in urine and other biological samples and devoted to drug testing in sports. Reviewed methods published approximately within the last five years and cited in the PubMed database have been divided into groups using the same classification of the 2004 World Anti-Doping Agency (WADA) Prohibited List. Together with procedures specifically developed for anti-doping analysis, LC-MS applications used in other fields (e.g., therapeutic drug monitoring, clinical and forensic toxicology, and detection of drugs illicitly used in livestock production) have been included when considered as potentially extensible to doping control. Information on the reasons for potential abuse by athletes, on the requirements established by WADA for analysis, and on the WADA rules for the interpretation of analytical findings are provided for the different classes of drugs.

Identification of phase I and II metabolites of the new designer drug α-pyrrolidinohexiophenone (α-PHP) in human urine by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

Pyrrolidinophenones represent one emerging class of newly encountered drugs of abuse, also known as 'new psychoactive substances', with stimulating psychoactive effects. In this work, we report on the detection of the new designer drug α-pyrrolidinohexiophenone (α-PHP) and its phase I and II metabolites in a human urine sample of a drug abuser. Determination and structural elucidation of these metabolites have been achieved by liquid chromatography electrospray ionisation quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS). By tentative identification, the exact and approximate structures of 19 phase I metabolites and nine phase II glucuronides were elucidated. Major metabolic pathways revealed the reduction of the ß-keto moieties to their corresponding alcohols, didesalkylation of the pyrrolidine ring, hydroxylation and oxidation of the aliphatic side chain leading to n-hydroxy, aldehyde and carboxylate metabolites, and oxidation of the pyrrolidine ring to its lactam followed by ring cleavage and additional hydroxylation, reduction and oxidation steps and combinations thereof. The most abundant phase II metabolites were glucuronidated ß-keto-reduced alcohols. Besides the great number of metabolites detected in this sample, α-PHP is still one of the most abundant ions together with its ß-keto-reduced alcoholic dihydro metabolite. Monitoring of these metabolites in clinical and forensic toxicology may unambiguously prove the abuse of the new designer drug α-PHP.