Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. II: Confirmatory analysis.

For doping control, analyses of samples are generally achieved in two steps: a rapid screening and, in the case of a positive result, a confirmatory analysis. A two-step methodology based on ultra-high-pressure liquid chromatography coupled to a quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was developed to screen and confirm 103 doping agents from various classes (e.g., beta-blockers, stimulants, diuretics, and narcotics). The screening method was presented in a previous article as part I (i.e., Fast analysis of doping agents in urine by ultra-high-pressure liquid chromatography-quadrupole time-of-flight mass spectrometry. Part I: screening analysis). For the confirmatory method, basic, neutral and acidic compounds were extracted by a dedicated solid-phase extraction (SPE) in a 96-well plate format and detected by MS in the tandem mode to obtain precursor and characteristic product ions. The mass accuracy and the elemental composition of precursor and product ions were used for compound identification. After validation including matrix effect determination, the method was considered reliable to confirm suspect results without ambiguity according to the positivity criteria established by the World Anti-Doping Agency (WADA). Moreover, an isocratic method was developed to separate ephedrine from its isomer pseudoephedrine and cathine from phenylpropanolamine in a single run, what allowed their direct quantification in urine.

Determination of nicotine and nicotine metabolites in urine by hydrophilic interaction chromatography-tandem mass spectrometry: Potential use of smokeless tobacco products by ice hockey players.

Consumption of nicotine in the form of smokeless tobacco (snus, snuff, chewing tobacco) or nicotine-containing medication (gum, patch) may benefit sport practice. Indeed, use of snus seems to be a growing trend and investigating nicotine consumption amongst professional athletes is of major interest to sport authorities. Thus, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the detection and quantification of nicotine and its principal metabolites cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide in urine was developed. Sample preparation was performed by liquid-liquid extraction followed by hydrophilic interaction chromatography-tandem mass spectrometry (HILIC-MS/MS) operated in electrospray positive ionization (ESI) mode with selective reaction monitoring (SRM) data acquisition. The method was validated and calibration curves were linear over the selected concentration ranges of 10-10,000 ng/mL for nicotine, cotinine, trans-3-hydroxycotinine and 10-5000 ng/mL for nicotine-N'-oxide and cotinine-N-oxide, with calculated coefficients of determination (R(2)) greater than 0.95. The total extraction efficiency (%) was concentration dependent and ranged between 70.4 and 100.4%. The lower limit of quantification (LLOQ) for all analytes was 10 ng/mL. Repeatability and intermediate precision were ?9.4 and ?9.9%, respectively. In order to measure the prevalence of nicotine exposure during the 2009 Ice Hockey World Championships, 72 samples were collected and analyzed after the minimum of 3 months storage period and complete removal of identification means as required by the 2009 International Standards for Laboratories (ISL). Nicotine and/or metabolites were detected in every urine sample, while concentration measurements indicated an exposure within the last 3 days for eight specimens out of ten. Concentrations of nicotine, cotinine, trans-3-hydroxycotinine, nicotine-N'-oxide and cotinine-N-oxide were found to range between 11 and 19,750, 13 and 10,475, 10 and 8217, 11 and 3396, and 13 and 1640 ng/mL, respectively. When proposing conservative concentration limits for nicotine consumption prior and/or during the games (50 ng/mL for nicotine, cotinine and trans-3-hydroxycotinine and 25 ng/mL for nicotine-N'-oxide and cotinine-N-oxide), about half of the hockey players were qualified as consumers. These findings significantly support the likelihood of extensive smokeless nicotine consumption. However, since such conclusions can only be hypothesized, the potential use of smokeless tobacco as a doping agent in ice hockey requires further investigation.

Determination of chlorzoxazone and 6-hydroxychlorzoxazone in plasma by gas chromatography--mass spectrometry.

A gas chromatographic-mass spectrometric method is presented which allows the determination of chlorzoxazone and 6-hydroxychlorzoxazone after derivatization with the reagent N-tert.-butyldimethylsilyl-N-methyltrifluoroacetamide. No interference was observed from endogenous compounds following the extraction of plasma samples from six different human subjects. The standard curves were linear over a working range of 20 to 4000 ng/ml and of 20 to 1000 ng/ml for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. Recoveries ranged from 65 to 97% for the two compounds and intra- and inter-day coefficients of variation were always less than 9%. The limit of quantitation of the method was found to be 5 ng/ml for the two compounds, hence allowing its use for single low dose pharmacokinetics.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

Accuracy profile validation of a new method for carbon monoxide measurement in the human blood using headspace-gas chromatography-mass spectrometry (HS-GC-MS).

The aim of our study was to provide an innovative headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable for the routine determination of blood CO concentration in forensic toxicology laboratories. The main drawback of the GC/MS methods discussed in literature for CO measurement is the absence of a specific CO internal standard necessary for performing quantification. Even if stable isotope of CO is commercially available in the gaseous state, it is essential to develop a safer method to limit the manipulation of gaseous CO and to precisely control the injected amount of CO for spiking and calibration. To avoid the manipulation of a stable isotope-labeled gas, we have chosen to generate in a vial in situ, an internal labeled standard gas ((13)CO) formed by the reaction of labeled formic acid formic acid (H(13)COOH) with sulfuric acid. As sulfuric acid can also be employed to liberate the CO reagent from whole blood, the procedure allows for the liberation of CO simultaneously with the generation of (13)CO. This method allows for precise measurement of blood CO concentrations from a small amount of blood (10 μL). Finally, this method was applied to measure the CO concentration of intoxicated human blood samples from autopsies.

Use of accuracy profile for the validation of a gas chromatography-negative chemical ionization tandem mass spectrometry method: quantification of ethyl glucuronide in hair

Introduction: Ethylglucuronide (EtG) is a direct and specific metabolite of ethanol. Its determination in hair is of increasing interest for detecting and monitoring alcohol abuse. The quantification of EtG in hair requires analytical methods showing highest sensitivity and specificity. We present a fully validated method based on gas chromatography-negative chemical ionization tandem mass spectrometry (GC-NCI-MS/MS). The method was validated using French Society of Pharmaceutical Sciences and Techniques (SFSTP) guidelines which are based on the determination of the total measurement error and accuracy profiles. Methods: Washed and powdered hair is extracted in water using an ultrasonic incubation. After purification by Oasis MAX solid phase extraction, the derivatized EtG is detected and quantified by GC-NCI-MS/MS method in the selected reaction monitoring mode. The transitions m/z 347 / 163 and m/z 347 / 119 were used for the quantification and identification of EtG. Four quality controls (QC) prepared with hair samples taken post mortem from 2 subjects with a known history of alcoholism were used. A proficiency test with 7 participating laboratories was first run to validate the EtG concentration of each QC sample. Considering the results of this test, these samples were then used as internal controls for validation of the method. Results: The mean EtG concentrations measured in the 4 QC were 259.4, 130.4, 40.8, and 8.4 pg/mg hair. Method validation has shown linearity between 8.4 and 259.4 pg/mg hair (r2 > 0.999). The lower limit of quantification was set up at 8.4 pg/mg. Repeatability and intermediate precision were found less than 13.2% for all concentrations tested. Conclusion: The method proved to be suitable for routine analysis of EtG in hair. GC-NCI-MS/MS method was then successfully applied to the analysis of EtG in hair samples collected from different alcohol consumers.

Urinary analysis of 16(5alpha)-androsten-3alpha-ol by gas chromatography/combustion/isotope ratio mass spectrometry: implications in anti-doping analysis.

We present a method for the analysis of urinary 16(5alpha)-androsten-3alpha-ol together with 5beta-pregnane-3alpha,20alpha-diol and four testosterone metabolites: androsterone (Andro), etiocholanolone (Etio), 5alpha-androstane-3alpha,17beta-diol (5alphaA), 5beta-androstane-3alpha,17beta-diol (5betaA) by means of gas chromatography/combustion/isotopic ratio mass spectrometry (GC/C/IRMS). The within-assay and between-assay precision S.D.s of the investigated steroids were lower than 0.3 and 0.6 per thousand, respectively. A comparative study on a population composed of 20 subjects has shown that the differences of the intra-individual delta(13)C-values for 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol are less than 0.9 per thousand. Thereafter, the method has been applied in the frame of an excretion study following oral ingestion of 50 mg DHEA initially and oral ingestion of 50mg pregnenolone 48 h later. Our findings show that administration of DHEA does not affect the isotopic ratio values of 16(5alpha)-androsten-3alpha-ol and 5beta-pregnane-3alpha,20alpha-diol, whereas the isotopic ratio values of 5beta-pregnane-3alpha,20alpha-diol vary by more 5 per thousand upon ingestion of pregnenolone. We have observed delta(13)C-value changes lower than 1 per thousand for 16(5alpha)-androsten-3alpha-ol, though pregnenolone is a precursor of the 16-ene steroids. In contrast to 5beta-pregnane-3alpha,20alpha-diol, the 16-ene steroid may be used as an endogenous reference compound when pregnenolone is administered.

Surface-sampling and analysis of TATP by swabbing and gas chromatography/mass spectrometry

The method of sample recovery for trace detection and identification of explosives plays a critical role in several criminal investigations. After bombing, there can be difficulties in sending big objects to a laboratory for analysis. Traces can also be searched for on large surfaces, on hands of suspects or on surfaces where the explosive was placed during preparatory phases (e.g. places where an IED was assembled, vehicles used for transportation, etc.). In this work, triacetone triperoxide (TATP) was synthesized from commercial precursors following reported methods. Several portions of about 6 mg of TATP were then spread on different surfaces (e.g. floors, tables, etc.) or used in handling tests. Three different swabbing systems were used: a commercial swab, pre-wetted with propan-2-ol (isopropanol) and water (7:3), dry paper swabs, and cotton swabs wetted with propan-2-ol. Paper and commercial swabs were also used to sample a metal plate, where a small charge of about 4 g of TATP was detonated. Swabs were sealed in small glass jars with screw caps and Parafilm® M and sent to the laboratory for analysis. Swabs were extracted and analysed several weeks later by gas chromatography/mass spectrometry. All the three systems gave positive results, but wetted swabs collected higher amounts of TATP. The developed procedure showed its suitability for use in real cases, allowing TATP detection in several simulations, including a situation in which people wash their hands after handling the explosive.

Analysis and quantification of vitamin D metabolites in serum by ultra-performance liquid chromatography coupled to tandem mass spectrometry and high-resolution mass spectrometry: a method comparison and validation.

RATIONALE: The aim of the work was to develop and validate a method for the quantification of vitamin D metabolites in serum using ultra-high-pressure liquid chromatography coupled to mass spectrometry (LC/MS), and to validate a high-resolution mass spectrometry (LC/HRMS) approach against a tandem mass spectrometry (LC/MS/MS) approach using a large clinical sample set. METHODS: A fast, accurate and reliable method for the quantification of the vitamin D metabolites, 25-hydroxyvitamin D2 (25OH-D2) and 25-hydroxyvitamin D3 (25OH-D3), in human serum was developed and validated. The C3 epimer of 25OH-D3 (3-epi-25OH-D3) was also separated from 25OH-D3. The samples were rapidly prepared via a protein precipitation step followed by solid-phase extraction (SPE) using an HLB μelution plate. Quantification was performed using both LC/MS/MS and LC/HRMS systems. RESULTS: Recovery, matrix effect, inter- and intra-day reproducibility were assessed. Lower limits of quantification (LLOQs) were determined for both 25OH-D2 and 25OH-D3 for the LC/MS/MS approach (6.2 and 3.4 µg/L, respectively) and the LC/HRMS approach (2.1 and 1.7 µg/L, respectively). A Passing & Bablok fit was determined between both approaches for 25OH-D3 on 662 clinical samples (1.11 + 1.06x). It was also shown that results can be affected by the inclusion of the isomer 3-epi-25OH-D3. CONCLUSIONS: Quantification of the relevant vitamin D metabolites was successfully developed and validated here. It was shown that LC/HRMS is an accurate, powerful and easy to use approach for quantification within clinical laboratories. Finally, the results here suggest that it is important to separate 3-epi-25OH-D3 from 25OH-D3. Copyright © 2012 John Wiley & Sons, Ltd.

Confirmation of natural gas explosion from methane quantification by headspace gas chromatography-mass spectrometry (HS-GC-MS) in postmortem samples: a case report.

A new analytical approach for measuring methane in tissues is presented. For the first time, the use of in situ-produced, stably labelled CDH(3) provides a reliable and precise methane quantification. This method was applied to postmortem samples obtained from two victims to help determine the explosion origin. There was evidence of methane in the adipose tissue (82 nmol/g) and cardiac blood (1.3 nmol/g) of one victim, which corresponded to a lethal methane outburst. These results are discussed in the context of the available literature to define an analysis protocol for application in the event of a gas explosion.

Adverse effects of industrial multiwalled carbon nanotubes on human pulmonary cells

The aim of this study was to evaluate adverse effects of multiwalled carbon nanotubes (MWCNT), produced for industrial purposes, on the human epithelial cell line A549. MWCNT were dispersed in dipalmitoyl lecithin (DPL), a component of pulmonary surfactant, and the effects of dispersion in DPL were compared to those in two other media: ethanol (EtOH) and phosphate-buffered saline (PBS). Effects of MWCNT were also compared to those of two asbestos fibers (chrysotile and crocidolite) and carbon black (CB) nanoparticles, not only in A549 cells but also in mesothelial cells (MeT5A human cell line), used as an asbestos-sensitive cell type. MWCNT formed agglomerates on top of both cell lines (surface area 15-35 μm2) that were significantly larger and more numerous in PBS than in EtOH and DPL. Whatever the dispersion media, incubation with 100 μg/ml MWCNT induced a similar decrease in metabolic activity without changing cell membrane permeability or apoptosis. Neither MWCNT cellular internalization nor oxidative stress was observed. In contrast, asbestos fibers penetrated into the cells, decreased metabolic activity but not cell membrane permeability, and increased apoptosis, without decreasing cell number. CB was internalized without any adverse effects. In conclusion, this study demonstrates that MWCNT produced for industrial purposes exert adverse effects without being internalized by human epithelial and mesothelial pulmonary cell lines. [Authors]