The untargeted detection of phase II metabolites is a key issue for the study of drug metabolism in biological systems. Sensitive and selective mass spectrometric (MS) techniques coupled to ultrahigh performance liquid chromatographic (UHPLC) systems are the most effective for this purpose. In this study, we evaluate different MS approaches with a triple quadrupole instrument for the untargeted detection of bis-sulfate metabolites. Bis-sulfates of 23 steroid metabolites were synthesized and their MS behavior was comprehensively studied. Bis-sulfates ionized preferentially as the dianion ([M - 2H]) with a small contribution of the monoanion ([M - H]). Product ion spectra generated from the [M - 2H] precursor ions were dominated by the loss of HSO to generate two product ions, that is, the ion at m/z 97 (HSO) and the ion corresponding to the remaining monosulfate fragment. Other product ions were found to be specific for some structures. As an example, the loss of [CH + SO] was found to be important for several compounds with unsaturation adjacent to the sulfate. On the basis of the common behavior of the bis-sulfate metabolites two alternatives were evaluated for the untargeted detection of bis-sulfate metabolites (i) a precursor ion scan method using the ion at m/z 97 and (ii) a constant ion loss (CIL) method using the loss of HSO. Both methods allowed for the untargeted detection of the model compounds. Eight steroid bis-sulfates were synthesized in high purity in order to quantitatively evaluate the developed strategies. Lower limits of detection (2-20 ng/mL) were obtained using the CIL method. Additionally, the CIL method was found to be more specific in the detection of urinary bis-sulfates. The applicability of the CIL approach was demonstrated by determining progestogens altered during pregnancy and by detecting the bis-sulfate metabolites of tibolone.

In order to improve the detection capabilities of anabolic androgenic steroids (AAS) in sports, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method for the simultaneous detection of AAS phase I and phase II intact urinary metabolites (glucuronides and sulfates) was developed. A total of 36 metabolites (7 unconjugated; 19 glucuronides and 10 sulfates) corresponding to 15 of the most reported AAS were included. Analytes were extracted from urine using C18 cartridges. LC and MS conditions were studied in-depth to determine the most sensitive and selective conditions for each analyte. A selected reaction monitoring method was set up. The optimization of the experimental parameters for 13 metabolites not available as standards was performed using excretion study urines. Extraction recoveries were above 77% for all 23 validated analytes. Intra-day precision was lower than 21%, and LODs were in the range 0.25-4ng/mL for 18 of the 23 analytes. Matrix effect was evaluated using post column infusion and ranged from 92 to 147%. The method was successfully applied to excretion study urines of different exogenous AAS. The suitability of the strategy was demonstrated with methyltestosterone and stanozolol excretion study urines by achieving detection times of 22 and 21 days, respectively. The method is compliant with the World Antidoping Agency requirements for most of the studied compounds. It represents a cost-effective approach that improves the detection capabilities of AAS by increasing the sensitivity for some metabolites and by including recently described phase II long-term metabolites not detectable using the current screening strategy.

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.

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.

name: HEPACARE; AECC post-doctoral fellowship to MA (POSTD18014AREC); Ministerio de Educación FPU fellowship to MGR (FPU18/01461); Ministerio de Educación FPI fellowship to MR (BES-2017-079883); Ramón y Cajal Program contract to MGFB (RYC2018-024475-1); Fundación Eugenio Rodríguez Pascual; Fundación Mario Losantos; Fundación M Torres and the generous donation of Mr. Eduardo Avila.

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.

This procedure for routine quantification of albumin in urine is based on the dye-binding properties of albumin with bromphenol blue. The absorbance of 100 microL of urine mixed with 3 mL of color reagent is measured against blank reagent at 610 nm after 30 s. Results vary linearly with albumin concentration up to 6 g/L. The reaction is pH independent in the physiological range. It is not subject to substantial interference by uric acid, creatinine, calcium, sodium chloride, or bilirubin. The presence of globulins produces a small positive error. Within-run precision (CV) was 4.8, 1.5, and 0.9%, and day-to-day precision was 11.2, 2.0, and 1.9%, for samples containing albumin at about 0.1, 1.0, and 6.0 g/L, respectively. Results by a radial-immunodiffusion method (x) correlated well with those by the proposed method (y): r = 0.986; y = 0.98x + 0.096; n = 64. The method can also be used to detect globulins, such as Bence Jones protein, by measuring the ratio of the absorbance at 30 min to that at 30 s.