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Prednisolone (PRED) and prednisone (PSONE) are prohibited in sports competitions when administered by systemic routes, and they are allowed by other routes for therapeutic purposes. There is no restriction of use in out-of-competition periods. The present study aimed to evaluate the urinary excretion of PRED, PSONE, and their most important metabolites after systemic and nonsystemic treatments in order to verify the suitability of the current reporting level of 30 ng/ml used to distinguish allowed and prohibited administrations and to establish washout periods for oral treatments performed in out-of-competition periods. PRED was studied after dermatological administration (5 mg/day for 5 days, n = 6 males) and oral administration (5 mg, n = 6 males; 10 mg, n = 2 males). PSONE was studied after oral administration (10 mg, n = 2 males; 30 mg, n = 1 male and 1 female). Concentrations in urine were measured using an LC-MS/MS method. Concentrations after dermatological treatment were low for all metabolites. After oral administration, concentrations were very high during the first 24 h after administration ranging from 1.6 to 2261 ng/ml and from 4.6 to 908 ng/ml for PRED and PSONE, respectively. Concentrations of most of the metabolites measured were lower than 30 ng/ml from 24 h after all oral administrations. New reporting levels are proposed for PRED and PSONE considering data of our study and other information published after nonsystemic administrations of the compounds. Washout periods of at least 24 h are recommended to ensure no false positives when oral treatments need to be performed in out-of-competition periods.

Betamethasone (BET) is prohibited in sports competitions when administered by systemic routes, and it is allowed by other routes for therapeutic purposes. In out-of-competition periods, there is no restriction of use. The present work aimed to assess the urinary excretion of BET and its metabolites after allowed and prohibited administrations to verify the suitability of the current reporting level of 30 ng/ml used to distinguish allowed and prohibited administrations and to establish washout periods for oral and intramuscular (IM) administrations when out-of-competition treatments are needed. BET was administered to healthy volunteers by different routes: topical (10 mg/day for 5 days, n = 6 males), intranasal (320 μg/day for 3 days, n = 4 males and 4 females), oral (0.5 mg, n = 8 males) or IM (6 mg, n = 6 males, or 12 mg, n = 4 males and 4 females). Urine and plasma samples collected before and after administration were analysed using liquid chromatography-tandem mass spectrometry. Among all studied metabolites, the parent drug was selected as the best discriminatory marker. After topical administration, BET concentrations were lower than 6.6 ng/ml. However, after intranasal treatment, some samples at concentrations close to or higher than 30 ng/ml were detected, suggesting the need to revise the current reporting level. Urinary concentrations after oral and intranasal administrations were similar, and after IM administration, concentrations were much higher. Taking into account all information, a urinary reporting level of 60 ng/ml is proposed. Washout periods of at least 48 and 96 h are recommended after oral and IM administrations, respectively.

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 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.

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.

In this work an LC-MS/MS method has been developed for the simultaneous analysis of kasugamycin (KAS) and streptomycin (STR) in vegetable samples. The use of a mixed-mode HPLC column and a ternary mobile phase acetonitrile:water:0.5% aqueous formic acid in gradient elution mode are proposed to provide enough retention and resolution for these highly polar compounds. Heated-electrospray ionization (H-ESI) has been used to ionize KAS and STR when coupling liquid chromatography to mass spectrometry. In contrast to what happens with most aminoglycosides, KAS only showed singly charged ions in the full-scan mass spectrum. The [M + H]+ of both KAS and STR are proposed as precursor ions for tandem mass spectrometry since more stable product ions and better ion ratio precisions are obtained. Fragmentation of KAS has been studied, showing that the cleavage of the glycosidic bonds provided the main product ions and the most abundant and selective ones are proposed for quantitation and confirmation purposes in MRM mode. The LC-MS/MS method developed has been applied to the analysis of vegetables. A simple clean-up procedure using hydrophilic–lipophilic-balanced cartridges was applied to several vegetable extracts from tomato, zucchini, chard and lettuce, obtaining recoveries >80% for both compounds. The high matrix suppression observed for KAS in all matrices was reduced by a 1 : 5 sample dilution with acetonitrile, providing a 100-fold improvement in sensitivity for this compound. Method quality parameters have been established for tomato matrices, obtaining method limits of quantitation in the low µg kg−1 level (5–10 µg kg−1) for both compounds, precisions expressed in %RSD better than 7% and accuracies expressed as relative error better than 8% at the 100 µg kg−1 level, making this method suitable for routine analysis.

Liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS) was applied to the analysis and authentication of fruit-based products and fruit-based pharmaceutical preparations. A Kinetex C18 reversed-phase column under gradient elution with 0.1 % formic acid aqueous solution and methanol mobile phases was used for the simultaneous determination of 26 polyphenols, allowing an acceptable separation in less than 22 min. Instrumental quality parameters such as limits of detection (LOD, values between 12 and 14 μg/L for 19 of the 26 analyzed polyphenols), linearity (r (2) 0.991), run-to-run and day-to-day precisions (relative standard deviation (RSD) values lower than 9.9 and 13.5 %, respectively), and accuracy (relative errors lower than 8 %) were established. A simple extraction method, consisting of a sample sonication with acetone/water/hydrochloric acid (70:29.9:0.1 v/v/v) and centrifugation, was proposed. Two calibration procedures, external calibration using standards prepared in water and standard addition, were evaluated for polyphenol quantification in several grape and cranberry fruits and processed fruit products. For a 95 % confidence level, no statistical differences were observed between the two calibration methods (p values between 0.06 and 0.95), denoting that external calibration was suitable enough for the quantitative analysis of polyphenols in fruit-based products. The proposed LC-ESI-MS/MS method was then applied to the analysis of polyphenols in 23 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic concentration data was then analyzed by principal component analysis (PCA) to extract information of the most significant profile data contributing to authentication of natural extracts according to their fruit of origin.

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.

Ultrahigh pressure liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry (UHPLC-APPI-MS/MS) was applied to the analysis and authentication of fruit-based products and pharmaceutical preparations. Two sub-2 μm C18 reversed-phase columns, Syncronis (100 × 2.1 mm, 1.7 μm) and Hypersil Gold (50 × 2.1 mm, 1.9 μm), were proposed under gradient elution with 0.1% formic acid aqueous solution and methanol mobile phases for the determination of 29 polyphenols, allowing us to obtain polyphenolic profiles in less than 13.5 and 23.5 min, respectively. Several atmospheric pressure ionization (API) sources (H-ESI, APCI, and APPI) were compared. For dopant-assisted APPI, four organic solvents, toluene, acetone, chlorobenzene and anisole, were evaluated as dopants. Both H-ESI and acetone-assisted APPI were selected as the best ionization sources for the analysis of targeted polyphenols. Acceptable sensitivity (LOD values down to 0.5 μg kg−1 in the best of cases), linearity (r2 higher than 0.995) and good precision (RSD values lower than 15.1%) and trueness (relative errors lower than 10.2%) were obtained using both UHPLC-API-MS/MS methods. A simple extraction procedure, consisting of sample sonication with acetone/water/hydrochloric acid (70 : 29.9 : 0.1 v/v/v) and centrifugation, was used. The proposed UHPLC-ESI-MS/MS and UHPLC-APPI-MS/MS methods with both C18 reversed-phase columns were then applied to the analysis of 32 grape-based and cranberry-based natural products and pharmaceutical preparations. Polyphenolic profile data were then analyzed by principal component analysis (PCA) to extract information on the most significant data contributing to the classification of natural extracts according to the type of fruit.

Recreational drugs (illicit drugs, human and veterinary medicines, legal highs, etc.) often contain lacing agents and adulterants which are not related to the main active ingredient. Serious side effects and even the death of the consumer have been related to the consumption of mixtures of psychoactive substances and/or adulterants, so it is important to know the actual composition of recreational drugs. In this work, a method based on flow injection analysis (FIA) coupled with high-resolution mass spectrometry (HRMS) is proposed for the fast identification of psychoactive substances in recreational drugs and legal highs. The FIA and HRMS working conditions were optimized in order to detect a wide range of psychoactive compounds. As most of the psychoactive substances are acid–base compounds, methanol–0.1 % aqueous formic acid (1:1 v/v) as a carrier solvent and electrospray in both positive ion mode and negative ion mode were used. Two data acquisition modes, full scan at high mass resolution (HRMS) and data-dependent tandem mass spectrometry (ddMS/HRMS) with a quadrupole–Orbitrap mass analyzer were used, resulting in sufficient selectivity for identification of the components of the samples. A custom-made database containing over 450 substances, including psychoactive compounds and common adulterants, was built to perform a high-throughput target and suspect screening. Moreover, online accurate mass databases and mass fragmenter software were used to identify unknowns. Some examples, selected among the analyzed samples of recreational drugs and legal highs using the FIA–HRMS(ddMS/HRMS) method developed, are discussed to illustrate the screening strategy used in this study. The results showed that many of the analyzed samples were adulterated, and in some cases the sample composition did not match that of the supposed marketed substance.

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.

In this work, the mass spectrometry behaviour of the veterinary drug family of phenicols, including chloramphenicol (CAP) and its related compounds thiamphenicol (TAP), florfenicol (FF) and FF amine (FFA), was studied. Several atmospheric pressure ionization sources, electrospray (ESI), atmospheric pressure chemical ionization and atmospheric pressure photoionization were compared. In all atmospheric pressure ionization sources, CAP, TAP and FF were ionized in both positive and negative modes; while for the metabolite FFA, only positive ionization was possible. In general, in positive mode, [M + H](+) dominated the mass spectrum for FFA, while the other compounds, CAP, TAP and FF, with lower proton affinity showed intense adducts with species present in the mobile phase. In negative mode, ESI and atmospheric pressure photoionization showed the deprotonated molecule [M-H](-), while atmospheric pressure chemical ionization provided the radical molecular ion by electron capture. All these ions were characterized by tandem mass spectrometry using the combined information obtained by multistage mass spectrometry and high-resolution mass spectrometry in a quadrupole-Orbitrap instrument. In general, the fragmentation occurred via cyclization and losses or fragmentation of the N-(alkyl)acetamide group, and common fragmentation pathways were established for this family of compounds. A new chemical structure for the product ion at m/z 257 for CAP, on the basis of the MS(3) and MS(4) spectra is proposed. Thermally assisted ESI and selected reaction monitoring are proposed for the determination of these compounds by ultra high-performance liquid chromatography coupled to tandem mass spectrometry, achieving instrumental detection limits down to 0.1 pg.

A method based on ultra-high performance liquid chromatography (UHPLC) is proposed for the determination of chloramphenicol (CAP), its related compounds, thiamphenicol (TAP) and florfenicol (FF), and the polar metabolite florfenicol-amine (FFA), in animal-derived foods (chicken and pork meat, fish, prawns and honey). For the retention of FFA and its simultaneous analysis with the parent compounds a phenyl-hexyl column is proposed. A fast separation is achieved in less than 2 minutes using a methanol : acetic acid-ammonium acetate buffer (5 mM, pH 5) and gradient elution. Under these conditions, the FFA is retained at more than twice the dead volume, as recommended by the legislation. For the coupling with mass spectrometry, heated-electrospray (H-ESI) is used as ionisation source improving vaporization efficiency. To prevent interferences using selected-reaction monitoring (SRM) both quantitation and confirmation transitions were carefully selected. Two different sample treatments based on solid-phase extraction with mixed-mode cartridges for fish and meat products and hydrophilic-lipophilic-balanced cartridges for honey are proposed, providing limits of quantitation (LOQs) below μg kg(-1) level.