RESUMEN

Sulfate metabolites of endogenous anabolic androgenic steroids (EAAS) have been shown to prolong the detection times compared with the conventional urinary markers of the steroid profile for oral and intramuscular administrations of testosterone (T). In this work, the sensitivity of sulfate EAAS markers for the detection of T gel administration has been evaluated in six Caucasian and six Asian male volunteers. Fourteen sulfate metabolites were measured in basal and post-administration samples after multiple doses of T gel (100 mg/day, three consecutive days), and the detection times based on individual thresholds for each volunteer were evaluated. Sulfate concentrations did not show adequate sensitivity, but the results of sulfate ratios were much more promising. Androsterone sulfate/testosterone sulfate (A-S/T-S), epiandrosterone sulfate/epitestosterone sulfate (epiA-S/E-S), epiA-S/T-S, and etiocholanolone sulfate/epitestosterone sulfate (Etio-S/E-S) provided the most consistent detectability for all volunteers and populations, with detection times ranging from 60 to 96 h since the first dose. Additional ratios improved detectability to up to 7 days, but only in particular volunteers. In general, sensitivity was similar to or better than the conventional testosterone/epitestosterone ratio (T/E) of the steroid profile, which further reinforces the conclusion that sulfate EAAS metabolites can be a good complement for the current steroid profile.

RESUMEN

In this work, the collision cross section (CCS) value of 103 steroids (including unconjugated metabolites and phase II metabolites conjugated with sulfate and glucuronide groups) was determined by liquid chromatography coupled to traveling wave ion mobility spectrometry (LC-TWIMS). A time of flight (QTOF) mass analyzer was used to perform the analytes determination at high-resolution mass spectrometry. An electrospray ionization source (ESI) was used to generate [M+H]+, [M + NH4]+ and/or [M - H]- ions. High reproducibility was observed for the CCS determination in both urine and standard solutions, obtaining RSD lower than 0.3% and 0.5% in all cases respectively. CCS determination in matrix was in accordance with the CCS measured in standards solution showing deviations below 2%. In general, CCS values were directly correlated with the ion mass and allowed differentiating between glucuronides, sulfates and free steroids although differences among steroids of the same group were less significant. However, more specific information was obtained for phase II metabolites observing differences in the CCS value of isomeric pairs concerning the conjugation position or the α/ß configuration, which could be useful in the structural elucidation of new steroid metabolites in the anti-doping field. Finally, the potential of IMS reducing interferences from the sample matrix was also tested for the analysis of a glucuronide metabolite of bolasterone (5ß-androstan-7α,17α-dimethyl-3α,17ß-diol-3-glucuronide) in urine samples.

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In this work, desorption electrospray ionization and paper spray ionization both with high-resolution mass spectrometry (DESI-HRMS and PSI-HRMS) were explored for the fast and direct analysis of stimulants and diuretics in urine samples. The analysis was performed at a resolution of 70 000 FWHM (m/z 200) using a quadrupole-Orbitrap mass spectrometer in full scan acquisition mode, detecting stimulants and diuretics in positive and negative ion modes, respectively. The most critical parameters affecting the desorption and ionization efficiencies of compounds were optimized, paying particular attention to the optimization of the spray solvent for PSI-HRMS analysis and to the selection of the DESI sample substrate. For stimulants, the PSI-HRMS method performed better than DESI-HRMS, allowing the direct analysis of raw urine samples with better signal-to-noise ratios than DESI. However, results obtained for diuretics were not as satisfactory as we expected. The PSI-HRMS method was applied to the screening of 52 stimulants for doping control purposes, providing satisfactory detectability for most of them at the Minimum Reporting Level (MRL) in less than 2 minutes for each single analysis. Despite the advantages offered by the PSI-HRMS method, in this study is also included a discussion on the limitations observed because of the presence of interference for some compounds.

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This work describes the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for the determination of 23 primary aromatic amines (PAAs) that can potentially migrate from food contact materials. The chromatographic separation was performed in a pentafluorophenylpropyl (PFPP) column achieving the separation of all PAAs in less than 6.5 min using water to acetonitrile (0.1% acetic acid in both solvents) as mobile phase and a gradient elution. The feasibility of atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) was evaluated as alternative to electrospray ionization (ESI) for the analysis of PAAs. Results showed that for most of the compounds, better responses were obtained with APCI, which shows the advantage of being less susceptible to matrix effects. Tandem mass spectrometry (MS/MS) fragmentation studies of [M + H]+ allowed for the selection of the two most characteristic and abundant product ions of the 23 PAAs which led to the development of a selective and sensitive UHPLC-APCI-MS/MS method with limits of detection ranging from 0.2 to 2 µg kg-1. Moreover, intra-day and inter-day precisions of the method in terms of relative standard deviation (RSD%) were lower than 10% and 15%, while trueness as relative error was <15% for most of the compounds. The UHPLC-APCI-MS/MS method was applied to the analysis of twenty black Nylon kitchenware samples that were submitted to migration tests using food simulant B (3% acetic acid, w/v), and the presence of PAAs were detected in eighteen samples at concentrations above the legislated limit (2 µg kg-1 of food or food simulants).

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A gas chromatography-atmospheric pressure photoionization-high-resolution mass spectrometry (GC-APPI-HRMS) method was developed for the determination of eight phenylalkylamine stimulants in urine samples. Spiked urine samples were hydrolyzed, processed by solid-phase extraction, and derivatized before analysis. Two derivatization reactions were studied: the formation of trimethylsilyl (TMS) derivatives with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and trimethylsilyl/trifluoroacetyl (TMS/TFA) derivatives with MSTFA and N-methyl-bis (trifluoroacetamide) (MBTFA) as derivatization reagents. Gas chromatography of both derivatives was performed with a 100% dimethylsiloxane column and a good separation of all isomeric compounds was achieved. To maximize the signal of the protonated molecule [M+H]+, the APPI most critical parameters were optimized. Three solvents were tested as dopant agents, with acetone yielding the lower in-source collision-induced dissociation (CID) fragmentation. The acquisition was performed in full scan and product ion scan (parallel reaction monitoring, PRM) using a quadrupole-Orbitrap mass analyzer (35,000 FWHM at m/z 200) in positive ion detection mode. At the optimal working conditions, the full scan method was evaluated for the fulfillment of identification requirements in doping analysis. Selectivity, limits of detection, matrix effect, and precision were estimated to validate the method for confirmation purposes and its applicability was tested by the analysis of spiked samples as well as by the analysis of samples obtained after the administration of some of the compounds to healthy volunteers. Results were compared with those obtained by GC-electron ionization-MS, demonstrating that the GC-APPI-HRMS method improved selectivity and sensibility, achieving lower limits of detection and satisfactory reproducibility.

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