A method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the direct quantitation of endogenous steroid sulfates has been developed to be able to evaluate these metabolites as biomarkers to detect the misuse of endogenous androgenic anabolic steroids in sports. For sample preparation, a mixed-mode solid-phase extraction was optimized to eliminate the glucuronide fraction in the washing step thus obtaining only the sulfate fraction. Chromatographic separation was optimized to achieve adequate resolution between isomers. The electrospray ionization and the product ion mass spectra of the sulfates were studied in order to obtain the most specific and selective transitions. The method was validated for quantitative purposes for 11 steroid sulfates obtaining satisfactory values for linearity, accuracy, and intra- and inter-day precision (relative standard deviation better than 16.2%). Limits of quantitation ranged between 0.5 and 2 ng/mL. Extraction recoveries for sulfate metabolites were between 90 and 94%. Matrix effect ranged from 90 to 110% showing the absence of significant ion suppression/enhancement. Samples were found to be stable after 2 freeze/thaw cycles. The applicability of the method was checked by the analysis of 75 urine samples from healthy volunteers (54 males, 37 Caucasian and 17 Asian, and 21 Caucasian females) to evaluate the concentration levels of endogenous sulfate metabolites in basal conditions.

The detection of testosterone (T) misuse is performed using the steroid profile that includes concentrations of T and related metabolites excreted free and glucuronoconjugated, and the ratios between them. In this work, the usefulness of 14 endogenous steroid sulfates to improve the detection capabilities of oral T administration has been evaluated. Quantitation of the sulfate metabolites was performed using solid-phase extraction and analysis by liquid chromatography-tandem mass spectrometry. Urine samples were collected up to 144 hours after a single oral dose of T undecanoate (120 mg) to five Caucasian male volunteers. Detection times (DTs) of each marker were estimated using reference limits based on a population study and also monitoring the individual threshold for each volunteer. High inter-individual variability was observed for sulfate metabolites and, therefore, better DTs were obtained using individual thresholds. Using individual threshold limits, epiandrosterone sulfate (epiA-S) improved the DT with respect to testosterone/epitestosterone (T/E) ratio in all volunteers. Androsterone, etiocholanolone, and two androstanediol sulfates also improved DTs for some volunteers. Principal component analysis was used to characterize the sample cohort, obtaining 13 ratios useful for discrimination. These ratios as well as the ratio epiA-S/ dehydroepiandrosterone sulfate were further examined. The most promising results were obtained using ratios between sulfates of epiA, androsterone, or androstanediol 1 and E, and also sulfates of epiA or androstanediol 1, and dehydroandrosterone.These selected ratios prolonged the DT of oral T administration up to 144 hours, which corresponded to a significantly higher retrospectivity compared to those obtained using concentrations or the conventional T/E ratio.

27Liquid chromatography-electrospray-tandem mass spectrometry MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-

A novel LC-MS/MS method has been developed for the determination of 13 aminoglycoside antibiotics in meat products. Among the chromatographic columns tested, the mixed-mode Obelisc R provided the best performance. Electrospray has been used for the coupling of the LC and the effect of temperature on the ionization has been evaluated. The mass spectra of AGs have been studied in order to select the most adequate precursor and product ions for quantitation and confirmation in SRM mode, showing that the single charged [M+H](+) provided better precisions than the double charged [M+2H](2+). Accurate mass measurements have been performed in order to confirm the molecular composition of the product ions, allowing the establishment of a new mechanism for some product ions of STR and DHSTR. A sample treatment based on an extraction and a SPE clean-up has been applied to a wide variety of meat products such as frankfurters; sausages; and minced meat of pork, veal, and chicken. Method limits of quantitation in the low microgram per kilogram level (1-50 μg kg(-1)), precisions %RSD below 15 % and accuracies expressed as relative errors below 23 % have been obtained, making the proposed method suitable for routine analysis.

Nowadays most LC-MS methods rely on tandem mass spectrometry not only for quantitation and confirmation of compounds by multiple reaction monitoring (MRM), but also for the identification of unknowns from their product ion spectra. However, gas-phase reactions between charged and neutral species inside the mass analyzer can occur, yielding product ions at m/z values higher than that of the precursor ion, or at m/z values difficult to explain by logical losses, which complicate mass spectral interpretation. In this work, the formation of adduct ions in the mass analyzer was studied using several mass spectrometers with different mass analyzers (ion trap, triple quadrupole, and quadrupole-Orbitrap). Heterocyclic amines (AαC, MeAαC, Trp-P-1, and Trp-P-2), photo-initiators (BP and THBP), and pharmaceuticals (phenacetin and levamisole) were selected as model compounds and infused in LCQ Classic, TSQ Quantum Ultra AM, and Q-Exactive Orbitrap (ThermoFisher Scientific) mass spectrometers using electrospray as ionization method. The generation of ion-molecule adducts depended on the compound and also on the instrument employed. Adducts with neutral organic solvents (methanol and acetonitrile) were only observed in the ion trap instrument (LCQ Classic), because of the ionization source on-axis configuration and the lack of gas-phase barriers, which allowed inertial entrance of the neutrals into the analyzer. Adduct formation (only with water) in the triple quadrupole instruments was less abundant than in the ion trap and quadrupole-Orbitrap mass spectrometers, because of the lower residence time of the reactive product ions in the mass analyzer. The moisture level of the CID and/or damper gas had a great effect in beam-like mass analyzers such as triple quadrupole, but not in trap-like mass analyzers, probably because of the long residence time that allowed adduct formation even with very low concentrations of water inside the mass spectrometer.

Fifty samples of finished drinking waters (FDWs) from Spain covering 12 million inhabitants were tested for 53 pharmaceuticals pertaining to 12 different Anatomical Therapeutic Chemical (ATC) classification system codes. The studied compounds are a combination of most commonly consumed pharmaceuticals with other barely reported in the literature. Five compounds, azithromycin, clarithromycin, erythromycin, sulfamethoxazole, and ibuprofen were tentatively identified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in some samples (2 to 15 %), but only ibuprofen and azithromycin could be confirmed when analyzed by liquid chromatography-high-resolution mass spectrometry (LC-HRMS) with a quadrupole-Orbitrap instrument. Concentration levels of ibuprofen in the positive samples ranged from 12 to 17 ng/L (n = 6) while for azithromycin values from 5 to 9.5 ng/L (n = 3) were found. Ibuprofen fragmentation behaviour in different mass spectrometry instrument configurations (triple quadrupole, quadrupole-ion trap, and quadrupole-Orbitrap) was evaluated.

UHPLC-APPI-MS/MS was used for authentication of fruit-based products and pharmaceutical preparations.

The introduction of alternative markers to the steroid profile can be an effective approach to improving the screening capabilities for the detection of testosterone (T) misuse. In this work, endogenous steroid sulfates were evaluated as potential markers to detect intramuscular (IM) T administration. Fourteen sulfate metabolites were quantified using mixed‐mode solid‐phase extraction and analysis by liquid chromatography–tandem mass spectrometry (LC–MS/MS). Urine samples after a single IM injection (100 mg) of T cypionate to six Caucasian and six Asian healthy male volunteers were analyzed. Principal component analysis (PCA) was used to characterize the sample cohort and to obtain the most useful markers for discrimination between pre‐ and post‐administration samples. For Caucasian volunteers, a separation between pre‐ and post‐administration samples was observed in PCA, whereas for Asian volunteers no separation was obtained. Seventeen ratios between sulfate metabolites were selected and further considered. Detection times (DTs) of each marker were evaluated using individual thresholds for each volunteer. The best results were obtained using ratios involving T and epitestosterone (E) sulfates in the denominator. The best marker was the ratio androsterone sulfate/testosterone sulfate (A‐S/T‐S) which prolonged the DT 1.2–2.1 times in respect to those obtained using T/E ratio in all Caucasian volunteers and 1.3–1.5 times in two Asian volunteers. Other ratios between A‐S or etiocholanolone sulfate and E‐S, and sulfates of etiocholanolone, dehydroandrosterone or epiandrosterone, and T‐S were also found adequate. These ratios improve the DT after IM T administration and their incorporation to complement the current steroid profile is recommended.