High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis

Abushareeda, Wadha; Vonaparti, Ariadni; Saad, Khadija; Almansoori, Moneera; Meloug, Mbarka; Saleh, Amal; Aguilera, Rodrigo; Angelis, Yiannis S.; Horvatovich, Péter; Lommen, Arjen; Alsayrafi, Mohammed; Georgakopoulos, Costas

doi:10.1016/j.jpba.2017.12.025

Cited by 50 publications

(67 citation statements)

References 28 publications

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“…13,14,[20][21][22]24,35 In this work, an Acquity UPLC BEH C18 column (1.7 μm) using MeOH:water with 0.01% formic acid as mobile phase and gradient elution was initially tested. 13,14,[20][21][22]24,35 In this work, an Acquity UPLC BEH C18 column (1.7 μm) using MeOH:water with 0.01% formic acid as mobile phase and gradient elution was initially tested.…”

Section: Liquid Chromatographymentioning

confidence: 99%

“…18,19,[25][26][27] Therefore, unlike glucuronides, there is no universal method for the cleavage of all steroid sulfates and this is an important limitation of the indirect analysis of these conjugates by gas chromatography coupled to mass spectrometry (GC-MS). 13,14,[20][21][22][23][24][28][29][30][31][32][33][34][35] However, the LC-MS reported methods for endogenous sulfates are only focused on some of the possible sulfate metabolites (eg, sulfates of T, E, A, and Etio), and do not cover other metabolites that could be of interest. 27 As a result, in recent years, some methods have been described to directly analyze sulfate metabolites in urine using LC-MS systems.…”

Section: Introductionmentioning

confidence: 99%

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Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Esquivel

Alechaga²,

Monfort³

et al. 2018

Drug Testing and Analysis

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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.

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Section: Liquid Chromatographymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Esquivel

Alechaga²,

Monfort³

et al. 2018

Drug Testing and Analysis

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“…32 The method could be optimized to incorporate new steroid sulfates in the future. Therefore, steroid sulfate metabolites should be included in the steroid profile and methods for their quantitation should be incorporated in the initial testing procedures used in doping control.…”

Section: Individual Thresholdsmentioning

confidence: 99%

Sulfate metabolites improve retrospectivity after oral testosterone administration

Esquivel

Alechaga²,

Monfort³

et al. 2018

Drug Testing and Analysis

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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.

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“…WADA Accredited Laboratories perform the initial testing procedure (ITP) or screening of small molecules using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS), in parallel for all urine samples, to comply with the analytical needs created by the List. For the LC/MS part of ITP, the analysis performed using Orbitrap high‐resolution full‐scan LC/MS (LC/HR‐FS‐MS) has particular advantages, because (1) of the sensitive qualitative detection of the analytes, (2) the full‐scan MS acquisition can be conducted in both positive and negative ionization polarity within a single analysis for all analytes, and (3) it can be combined in future with quantitative intact Phase II metabolite analysis . Because of its untargeted nature, this technology may not only help in ITP of a large list of known doping substances, but also acquire MS signals from doping substances that are not known yet, but belong to the prohibited pharmacological classes of the List.…”

Section: Introductionmentioning

confidence: 99%

“…The use of HR‐FS‐MS with LC and GC in anti‐doping has been reported and applied recently . However reprocessing of thousands of data files is also a logistically difficult task, because the size of high‐resolution MS data files per sample is in the range 100–500 MB.…”

Section: Introductionmentioning

confidence: 99%

Ultra‐fast retroactive processing of liquid chromatography high‐resolution full‐scan Orbitrap mass spectrometry data in anti‐doping screening of human urine

Lommen

Elaradi

Vonaparti

et al. 2019

Rapid Comm Mass Spectrometry

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Rationale Retroactive analysis of previously tested urine samples has become an important sports anti‐doping tool. Retroactive reprocessing of old data files acquired from a generic screening procedure can reveal detection of initially unknown substances, like illegal drugs and newly identified metabolites. Methods To be able to efficiently search through hundreds to thousands of liquid chromatography high‐resolution full‐scan Orbitrap mass spectrometry data files of anti‐doping samples, a combination of MetAlign and HR_MS_Search software has been developed. MetAlign reduced the data size ca 100‐fold making possible local storage of a massive volume of data. Results The newly developed HR_MS_Search module can search through the reduced data files for new compounds (mass or isotope pattern) defined by mass windows and retention time windows. A search for 33 analytes in 940 reduced data files lasted 10 s. The output of the automatic search was compared to the standard manual routine evaluation. The results of searching were evaluated in terms of false negatives and false positives. The newly banned b2‐agonist higenamine and its metabolite coclaurine were successfully searched in reduced data files originating from a testing period for which these substances were not banned, as an example of retroactive analysis. Conclusions The freeware MetAlign software and its automatic searching module HR_MS_Search facilitated the retroactive reprocessing of reduced full‐scan high‐resolution liquid chromatography/mass spectrometry screening data files and created a new tool in anti‐doping laboratories' network.

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High resolution full scan liquid chromatography mass spectrometry comprehensive screening in sports antidoping urine analysis

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Cited by 50 publications

References 28 publications

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Direct quantitation of endogenous steroid sulfates in human urine by liquid chromatography‐electrospray tandem mass spectrometry

Sulfate metabolites improve retrospectivity after oral testosterone administration

Ultra‐fast retroactive processing of liquid chromatography high‐resolution full‐scan Orbitrap mass spectrometry data in anti‐doping screening of human urine

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