Steroid profiling in urine of intact glucuronidated and sulfated steroids using liquid chromatography-mass spectrometry

Wilde, Laurie De; Roels, K.; Renterghem, Pieter Van; Eenoo, Peter Van; Deventer, Koen

doi:10.1016/j.chroma.2020.461231

Cited by 20 publications

(16 citation statements)

References 34 publications

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“…Three hundred microliters of urine was analyzed using our LC method for the steroid profiling of intact steroid conjugates, which includes 5ααβdiol3G, 5ααβdiol17G, 5βαβdiol3G and 5βαβdiol17G, androsterone glucuronide (AG), androsterone sulfate (AS), dehydroepiandrosterone sulfate (DHEAS), epiandrosterone sulfate (EpiAS), epitestosterone glucuronide (EG), epitestosterone sulfate (ES), etiocholanolone glucuronide (EtioG), etiocholanolone sulfate (EtioS), testosterone glucuronide (TG), and testosterone sulfate (TS) 43 . The origin of all used reference standards can be found in the publication on the steroid profile 43 . Measured concentrations below the limit of quantification (LOQ) were excluded from the statistics.…”

Section: Methodsmentioning

confidence: 99%

“…Three hundred microliters of urine was analyzed using our LC method for the steroid profiling of intact steroid conjugates, which testosterone glucuronide (TG), and testosterone sulfate (TS). 43 The origin of all used reference standards can be found in the publication on the steroid profile. 43 Measured concentrations below the limit of quantification (LOQ) were excluded from the statistics.…”

Section: Sample Preparationmentioning

confidence: 99%

“…43 The origin of all used reference standards can be found in the publication on the steroid profile. 43 Measured concentrations below the limit of quantification (LOQ) were excluded from the statistics. Urine samples with steroid concentrations exceeding the highest calibrator were appropriately diluted with water.…”

Section: Sample Preparationmentioning

confidence: 99%

“…Recently, our laboratory has developed a steroid profiling method using LC–MS/MS, 43 which is compliant with the current quality criteria established by WADA for the measurement of the steroid profile 16 . In this method, the intact steroid conjugates are analyzed by direct injection, omitting hydrolysis, and derivatization.…”

Section: Introductionmentioning

confidence: 99%

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Long‐term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

Wilde

Renterghem

Eenoo

2021

Drug Testing and Analysis

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The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called "steroid profiling" method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses.The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.

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Section: Methodsmentioning

confidence: 99%

Section: Sample Preparationmentioning

confidence: 99%

Section: Sample Preparationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Long‐term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

Wilde

Renterghem

Eenoo

2021

Drug Testing and Analysis

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“…Especially the latter, which was constructed from (EpiT sulfate /EpiT glucuronide) divided by (T sulfate/T glucuronide), proved competitive and, occasionally, superior compared with conventionally recorded T/EpiT ratios in indicating the administration of testosterone esters, resulting in the proposal to include the (EpiT sulfate/EpiT glucuronide)/(T sulfate/T glucuronide) into the list of ABP biomarkers. In a complementary manner, De Wilde et al presented a dilute‐and‐inject approach towards the quantification of steroid sulfates and glucuronides, targeting 17 analytes, where further to the aforementioned protocols, the inclusion and chromatographic separation of 5αAdiol and 5βAdiol 3‐ and 17‐ O ‐glucuronides was accomplished 76 . Here, a C‐18 analytical column (100 × 2.1 mm, 1.8‐μm particle size) was operated with gradient elution using water (solvent A) and methanol (solvent B), both containing 0.01% formic acid and 5‐mM ammonium formate.…”

Section: Anabolic Agentsmentioning

confidence: 99%

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

Thevis

Kuuranne

Geyer

2020

Drug Testing and Analysis

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Analytical chemistry-based research in sports drug testing has been a dynamic endeavor for several decades, with technology-driven innovations continuously contributing to significant improvements in various regards including analytical sensitivity, comprehensiveness of target analytes, differentiation of natural/ endogenous substances from structurally identical but synthetically derived compounds, assessment of alternative matrices for doping control purposes, and so forth. The resulting breadth of tools being investigated and developed by anti-doping researchers has allowed to substantially improve anti-doping programs and data interpretation in general. Additionally, these outcomes have been an extremely valuable pledge for routine doping controls during the unprecedented global health crisis that severely affected established sports drug testing strategies. In this edition of the annual banned-substance review, literature on recent developments in antidoping published between October 2019 and September 2020 is summarized and discussed, particularly focusing on human doping controls and potential applications of new testing strategies to substances and methods of doping specified the World Anti-Doping Agency's 2020 Prohibited List.

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Rapid UHPLC–MS/MS measurement of pregnanediol 3‐glucuronide in spot urine samples for detecting ovulation

Leoni,

Rosmini,

Ledda

et al. 2024

Biomedical Chromatography

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Biochemical confirmation of ovulation typically involves measuring serum progesterone levels during the mid‐luteal phase. Alternatively, this information could be obtained by monitoring urinary excretion of conjugated metabolites of ovarian steroids such as pregnanediol 3‐glucuronide (PDG) using immunoassay techniques that have methodological limitations. The aim of the present study was to develop a mass spectrometry (MS)‐based method for the rapid and accurate measurement of urinary PDG levels in spot urine samples. A “dilute and shoot” ultra‐high‐performance liquid cromatography tandem mass spectrometry (UHPLC‐MS/MS) method was developed for measuring PDG urinary concentration with a 6‐min analysis time. The method underwent validation in accordance with ISO 17025 documentation for quantitative methods, proving an efficient separation of PDG from other structurally similar glucuro‐conjugated steroid metabolites and ensuring a sufficient sensitivity for detecting the target analyte at concentrations as low as 0.01 μg/mL. The validation protocol yielded satisfactory results in terms of accuracy, repeatability, intermediate precision, and combined uncertainty. Additionally, the stability of both the samples and calibration curves was also conducted. The application to real urine samples confirmed the method's capability to measure PDG levels throughout an entire menstrual cycle and detecting ovulation. The rapidity of the analytical platform would therefore enable high throughput analysis, which is advantageous for large cohort clinical studies.

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Steroid profiling in urine of intact glucuronidated and sulfated steroids using liquid chromatography-mass spectrometry

Cited by 20 publications

References 34 publications

Long‐term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

Long‐term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

Annual banned‐substance review: Analytical approaches in human sports drug testing 2019/2020

Rapid UHPLC–MS/MS measurement of pregnanediol 3‐glucuronide in spot urine samples for detecting ovulation

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