Deciphering the origin of total estrogenic activity of complex mixtures.

Introduction: Identifying compounds with endocrine properties in food is getting increasingly important. Current chemical analysis methodology is mainly focused on the identification of known substances without bringing insight for biological activity. Recently, the application of bioassays has been promoted for their potential to detect unknown bioactive substances and to provide information on possible interactions between molecules. From the toxicological perspective, measuring endocrine activity cannot inform on endocrine disruption and/or health risks without sufficient knowledge on the nature of the responsible factors. Methods: The present study addresses a promising approach using High Performance Thin-Layer Chromatography (HPTLC) coupled to bioassays were analyzed using the Liquid Chromatography Mass-Spectrometry (LC-MS). The estrogen receptor activation was assessed using the transcription activation Estrogen Receptor Alpha Chemical Activated LUciferase gene eXpression assay (ERα- CALUX) and the HPTLC coupled to the Estrogen Screen Yeast assay (p-YES). Results: Seven isoflavones were identified in the soy isolates. Estrogen receptor activation was assessed for both, the identified isoflavones and the soy isolates with ERα-CALUX test. Correlation between the soy isolates extracts and the identified isoflavones was shown. Moreover, p-YES revealed the presence of an estrogenic bioactive zone. Analysis of the bioactive zone through LCHRMS highlighted signals corresponding to several isoflavones already detected in the isolates as well as two additional ones. For all detected isoflavones, an estrogenic activity dose-response was established in both bioassays. Conclusion: Finally, genistein, daidzein, and naringenin were found as the most active substances. A concordance analysis integrating the analytical and bioassay data indicated that genistein and daidzein were the drivers of the estrogenic activity of these soy protein isolates. Altogether, these data suggest that the integration of HPTLC-bioassay together with chemical analysis is a powerful approach to characterize the endocrine activity of complex mixtures.

From Structural Alerts to Signature Fragment Alerts: A Case Study on Pyrrolizidine Alkaloids.

Even though modeling is considered a valid alternative to mutagenicity testing for substances with known structures, it can be applied for mixtures only if all of the single chemical structures are identified. Within the present work, we investigate a new avenue to exploit computational toxicology for mixtures, such as plant-based food ingredients. Indeed, considering that in the absence of toxicological information, an important early consideration is whether any substance may be genotoxic through the mutagenic mechanism of action, we tried to establish a correspondence between genotoxic structural alerts (SAs) and so-called signature fragment alerts (SFAs). Once this correspondence is established, chromatograms could be screened for chemical features associated with genotoxic alerts. Pyrrolizidine alkaloids (PAs), a large group of natural toxins (several of them known as genotoxic) were used as a case study because their early identification would bring significant benefits. The method was built using 56 PA pure standards, resulting in the characterization of signature fragment alerts. Finally, the approach was verified in real plant-based samples such as herbal tea and alfalfa, where the screening of signature fragment alerts allowed highlighting quickly the presence of genotoxic PAs in plant-based mixtures. Therefore, the SFA analysis can be used for risk prioritization of newly identified PAs and for their identification in mixtures, contributing to the unnecessary use of animal experimentation for genotoxicity testing.

Degradation of glyphosate along coffee roasting: Do residue levels in green beans mirror exposure derived from coffee consumption?

Robusta and Arabica green beans were supplemented with carbon 14-glyphosate labelled on each carbon position alternatively prior to roasting, up to 220 °C for Robusta and 200 °C for Arabica (2, 5 and 10 min). The results of the study point a significant decomposition of glyphosate that happens during roasting, from at least 42 % to > 74 % depending on roasting conditions (time, temperature) and coffee variety. The data obtained with 14C-labelled glyphosate materials suggest that the carboxymethyl branch of the compound degrades more effectively than the phosphonomethyl moiety. The degradation of glyphosate does not lead to the formation of aminomethylphosphonic acid (AMPA). The results of the study indicate that carbon dioxide, methylamine and dimethylamine are major degradation products of glyphosate formed along coffee roasting, likely released with the roasting gas. Ultimately, the results of the study show that levels of glyphosate in green beans do not mirror those in coffee beverages.

Limitations of currently available in vitro oestrogenicity bioassays for effect-based testing of whole foods as the basis for decision making.

The idea that previously unknown hazards can be readily revealed in complex mixtures such as foods is a seductive one, giving rise to the hope that data from effect-based assays of food products collected in market surveys is of suitable quality to be the basis for data-driven decision-making. To study this, we undertook a comparative study of the oestrogenicity of blinded cereal samples, both in a number of external testing laboratories and in our own facility. The results clearly showed little variance in the activities of 9 samples when using a single method, but great differences between the activities from each method. Further exploration of these findings suggest that the oestrogenic activity is likely an inherent part of the natural food matrix which the varying sample preparation methods are able to release and extract to differing degrees. These issues indicate the current poor suitability of these types of datasets to be used as the basis for consumer advice or food decision-making. Data quality must be improved before such testing is used in practice.

High resolution mass spectrometry workflow for the analysis of food contaminants: Application to plant toxins, mycotoxins and phytoestrogens in plant-based ingredients.

An analytical workflow including mass spectral library, generic sample preparation, chromatographic separation, and analysis by high-resolution mass spectrometry (HRMS) was developed to gain insight into the occurrence of plant toxins, mycotoxins and phytoestrogens in plant-based food. This workflow was applied to 156 compounds including 90 plant toxins (pyrrolizidine alkaloids, tropane alkaloids, glycoalkaloids, isoquinoline alkaloids and aristolochic acids), 54 mycotoxins (including ergot alkaloids and Alternaria toxins) and 12 phytoestrogens (including isoflavones, lignans and coumestan) in plant-based protein ingredients, cereal and pseudo-cereal products. A mass spectral library was built based on fragmentation spectra collected at 10 different collision energies in both positive and negative ionisation modes for each toxin. Emphasis was put on a generic QuEChERS-like sample preparation followed by ultra-high-pressure liquid chromatography using alkaline mobile phase allowing the separation of more than 50 toxic pyrrolizidine alkaloids. HRMS acquisition comprised a full-scan event for toxins detection followed by data-dependent MS2 for toxin identification against mass spectrum. Method performance was evaluated using fortified samples in terms of sensitivity, repeatability, reproducibility and recovery. All toxins were positively identified at levels ranging from 1 µg kg-1 to 100 µg kg-1. Quantitative results obtained by a standard addition approach met SANTE/12682/2019 criteria for 132 out of 156 toxins. Such a workflow using generic, sensitive and selective multi-residue method allows a better insight into the occurrence of regulated and non-regulated toxins in plant-based foods and to conduct safety evaluation and risk assessments when needed.

Fate of acrylamide during coffee roasting and in vitro digestion assessed with carbon 14- and carbon 13-labeled materials.

Acrylamide (AA) formation during coffee roasting happens rapidly, reaching a peak value within the first minutes of roasting followed by a fast decrease to reach an asymptote at approximately 200 µg/kg. Today, the mechanisms by which AA is reduced during roasting remain unclear. In this research, the fate of AA during roasting followed by drip brewed-like extraction was studied using 14C-radiolabeled (14C-AA) and 13C-labeled (13C3-AA) materials. Results showed that 28% of the spiked 14C-AA was lost during the roasting process, presumably by degradation to volatile compounds and 25% was non-extractable; therefore, appeared bound to the matrix. About 50% of initial AA went into the water extract, either unchanged or transformed by conjugation/binding. The release of bound acrylamide was further evidenced by increasing levels of 13C3-AA over prolonged roasting times. In addition, the absence of 14C activity in the hexane extracts suggested acrylamide not to bind to any lipophilic material.

Quantification of folpet and phthalimide in tea and herbal infusions by LC- high-resolution MS and GC-MS/MS.

Two methods based on a modified QuEChERS sample preparation and either LC coupled to atmospheric pressure ionisation and high-resolution MS or GC coupled to electron ionisation and tripled quadrupole MS have been assessed for the quantification of folpet and phthalimide in tea and other dry herbal infusions. Both methods have been fully validated in green tea and further checked in black tea, verbena and rooibos, and they performed according to the SANTE/11813/2017 criteria at the target LOQ concentration level (50 µg/kg). These methods allow the accurate quantification of folpet in the selected matrices according to the new EU residue definition, which includes phthalimide. Phthalimide is the main metabolite and degradation product of folpet, although according to recent studies, it could be generated from different sources than folpet breakdown, such as food processing or analysis by GC.

Quantification of folpet and phthalimide in food by gas chromatography and mass spectrometry: Overcoming potential analytical artefacts.

Accurate quantification of folpet is problematic because it degrades into phthalimide during sample preparation and analysis by gas chromatography (GC). Thus, EU regulation was recently modified to include phthalimide in the folpet residue definition. However, recent studies have shown that phthalimide could also be generated from different sources, which could lead to an overestimation of the phthalimide content and therefore to false positives. GC coupled with either negative chemical ionisation and single quadrupole mass spectrometry, or electron ionisation with triple quadrupole mass spectrometry (GC-EI-MS/MS), were evaluated for the determination of folpet and phthalimide in food. Both methods were validated in 4 different matrices namely apple puree, rice flour, raspberry puree and infant formula. Better selectivity and precision were obtained with GC-EI-MS/MS. Negligible amounts of phthalimide was found in blank matrices, and validation results met the SANTE/11813/2017 criteria in all matrices at the LOQ concentration levels by using GC-EI-MS/MS.

PEMT, Δ6 desaturase, and palmitoyldocosahexaenoyl phosphatidylcholine are increased in rats during pregnancy.

DHA is important for fetal neurodevelopment. During pregnancy, maternal plasma DHA increases, but the mechanism is not fully understood. Using rats fed a fixed-formula diet (DHA as 0.07% total energy), plasma and liver were collected for fatty acid profiling before pregnancy, at 15 and 20 days of pregnancy, and 7 days postpartum. Phosphatidylethanolamine methyltransferase (PEMT) and enzymes involved in PUFA synthesis were examined in liver. Ad hoc transcriptomic and lipidomic analyses were also performed. With pregnancy, DHA increased in liver and plasma lipids, with a large increase in plasma DHA between day 15 and day 20 that was mainly attributed to an increase in 16:0/DHA phosphatidylcholine (PC) in liver (2.6-fold) and plasma (3.9-fold). Increased protein levels of Δ6 desaturase (FADS2) and PEMT at day 20 and increased Pemt expression and PEMT activity at day 15 suggest that during pregnancy, both DHA synthesis and 16:0/DHA PC synthesis are upregulated. Transcriptomic analysis revealed minor changes in the expression of genes related to phospholipid synthesis, but little insight on DHA metabolism. Hepatic PEMT appears to be the mechanism for increased plasma 16:0/DHA PC, which is supported by increased DHA biosynthesis based on increased FADS2 protein levels.

Multi-omics Integrative Investigation of Fatty Acid Metabolism in Obese and Lean Subcutaneous Tissue.

White adipose tissue (WAT) plays a central role in whole-body energy homeostasis through storage and release of fatty acids. A deeper understanding of the complex and highly integrated pathways regulating WAT fatty acid metabolism, and how they are altered with obesity, is necessary for diagnostic and therapeutic innovations in nutritional disorders. In this multi-omics study, we investigated the influence of obesity on fatty acid metabolism in human subcutaneous adipose tissue (SAT) using an approach that integrated transcriptomic, peptidomic, and fatty acid analyses. Notably, all analyses were conducted in the same adipose tissue sample from each participant, thus minimizing the chance of spurious results. In a sample of SAT from the periumbilical abdominal region of obese (n = 11, mean body mass index [BMI] = 35.0 ± 1.2 kg/m2) and lean subjects (n = 9, mean BMI = 22.1 ± 0.5 kg/m2), we found that obese SAT tended to have higher relative amounts of specific monounsaturated fatty acids and n-6 polyunsaturated fatty acids, and lower amounts of saturated fatty acids (p < 0.05). These changes were associated with differential regulation of lipogenic and lipolytic pathways in obese SAT. Fatty acid analysis showed changes in estimated fatty acid desaturase and elongase activities between lean and obese SAT (p < 0.05). Biomarkers of lipogenesis (e.g., fatty acid synthase protein) were differentially regulated between lean and obese SAT. These changes were noted in conjunction with increases in extracellular matrix remodeling proteins. Transcriptomic data revealed that the key regulators of lipolysis were reduced in obese SAT. This integrative multi-omics analysis collectively shows that obese SAT has a distinct fatty acid signature compared to lean SAT and the pathways underlying fatty acid metabolism are broadly regulated at the level of gene expression and protein abundance.

Metabolomics Reveals Metabolically Healthy and Unhealthy Obese Individuals Differ in their Response to a Caloric Challenge.

OBJECTIVE: To determine if metabolically healthy obese (MHO) individuals have a different metabolic response to a standardized diet compared to lean healthy (LH) and metabolically unhealthy obese (MUO) individuals. METHODS: Thirty adults (35-70 yrs) were classified as LH, MHO, and MUO according to anthropometric and clinical measurements. Participants consumed a standardized high calorie meal (~1330 kcal). Blood glucose and insulin were measured at fasting, and 15, 30, 60, 90 and 120 min postprandially. Additional blood samples were collected for the targeted analysis of amino acids (AAs) and derivatives, and fatty acids (FAs). RESULTS: The postprandial response (i.e., area under the curve, AUC) for serum glucose and insulin were similar between MHO and LH individuals, and significantly lower than MUO individuals (p < 0.05). Minor differences were found in postprandial responses for AAs between MHO and MUO individuals, while three polyunsaturated FAs (linoleic acid, γ-linolenic acid, arachidonic acid) showed smaller changes in serum after the meal in MHO individuals compared to MUO. Fasting levels for various AAs (notably branched-chain AA) and FAs (e.g., saturated myristic and palmitic acids) were found to correlate with glucose and insulin AUC. CONCLUSION: MHO individuals show preserved insulin sensitivity and a greater ability to adapt to a caloric challenge compared to MUO individuals.

Molecular insights into the role of white adipose tissue in metabolically unhealthy normal weight and metabolically healthy obese individuals.

Obesity is a risk factor for the development of type 2 diabetes and cardiovascular disease. However, it is now recognized that a subset of individuals have reduced cardiometabolic risk despite being obese. Paradoxically, a subset of lean individuals is reported to have high risk for cardiometabolic complications. These distinct subgroups of individuals are referred to as metabolically unhealthy normal weight (MUNW) and metabolically healthy obese (MHO). Although the clinical relevance of these subgroups remains debated, evidence shows a critical role for white adipose tissue (WAT) function in the development of these phenotypes. The goal of this review is to provide an overview of our current state of knowledge regarding the molecular and metabolic characteristics of WAT associated with MUNW and MHO. In particular, we discuss the link between different WAT depots, immune cell infiltration, and adipokine production with MUNW and MHO. Furthermore, we also highlight recent molecular insights made with genomic technologies showing that processes such as oxidative phosphorylation, branched-chain amino acid catabolism, and fatty acid ß-oxidation differ between these phenotypes. This review provides evidence that WAT function is closely linked with cardiometabolic risk independent of obesity and thus contributes to the development of MUNW and MHO.

Untargeted profiling of urinary steroid metabolites after testosterone ingestion: opening new perspectives for antidoping testing.

AIM: Antidoping procedures are expected to greatly benefit from untargeted metabolomic approaches through the discovery of new biomarkers of prohibited substances abuse. RESULTS: Endogenous steroid metabolites were monitored in urine samples from a controlled elimination study of testosterone undecanoate after ingestion. A platform coupling ultra-high pressure LC with high-resolution quadrupole TOF MS was used and high between-subject metabolic variability was successfully handled using a multiblock data analysis strategy. Links between specific subsets of metabolites and influential genetic polymorphisms of the UGT2B17 enzyme were highlighted. CONCLUSION: This exploratory metabolomic strategy constitutes a first step toward a better understanding of the underlying patterns driving the high interindividual variability of steroid metabolism. Promising biomarkers were selected for further targeted study.

Serum and adipose tissue amino acid homeostasis in the metabolically healthy obese.

A subgroup of obese individuals, referred to as metabolically healthy obese (MHO), have preserved insulin sensitivity and a normal lipid profile despite being obese. The molecular basis for this improved cardiometabolic profile remains unclear. Our objective was to integrate metabolite and gene expression profiling to elucidate the molecular distinctions between MHO and metabolically unhealthy obese (MUO) phenotypes. A subset of individuals were selected from the Diabetes Risk Assessment study and classified into three groups using anthropometric and clinical measurements: lean healthy (LH), MHO, and MUO. Serum metabolites were profiled using gas chromatography coupled to mass spectrometry. Multivariate data analysis uncovered metabolites that differed between groups, and these were subsequently validated by capillary electrophoresis coupled to mass spectrometry. Subcutaneous adipose tissue (SAT) gene expression profiling using microarrays was performed in parallel. Amino acids were the most relevant class of metabolites distinguishing MHO from MUO individuals. Serum levels of glutamic acid, valine, and isoleucine were positively associated (i.e., LH < MHO < MUO) with homeostasis model assessment-insulin resistance (HOMA-IR) and glycated hemoglobin (HbA1c) values, while leucine was only correlated with HOMA-IR. The glutamine-to-glutamic acid ratio and glycine were inversely correlated (i.e., LH > MHO > MUO) with HbA1c values. Concomitantly, SAT gene expression profiling revealed that genes related to branched-chain amino acid catabolism and the tricarboxylic acid cycle were less down-regulated in MHO individuals compared to MUO individuals. Together, this integrated analysis revealed that MHO individuals have an intermediate amino acid homeostasis compared to LH and MUO individuals.

A distinct fatty acid profile underlies the reduced inflammatory state of metabolically healthy obese individuals.

BACKGROUND: Obesity is associated with numerous health complications; however, a subgroup of obese individuals (termed the metabolically healthy obese or MHO) appear to have lower risk for complications such as type 2 diabetes and cardiovascular disease. Emerging evidence suggests that MHO individuals have reduced inflammation compared to their metabolically unhealthy obese (MUO) counterparts. As it is recognized that fatty acids (FAs) have a strong relationship with inflammation, the current study aimed to uncover if the reduced inflammation observed in MHO individuals is mirrored by a more favourable FA profile. METHODS: Fasted serum samples were collected from lean healthy (LH), MHO, and MUO participants (n = 10/group) recruited from the Diabetes Risk Assessment study. A panel of pro- and anti-inflammatory markers were measured by immunoassay. Total serum FA profiling, as well as the FA composition of circulating phospholipids (PL) and triglycerides (TG), was measured by gas chromatography. ANOVA and Mann-Whitney-Wilcoxon tests were used to assess statistical significance between the groups (P<0.05). RESULTS: MHO and MUO individuals had similar BMI and body fat %; however, lipid parameters in MHO individuals more closely resembled that of LH individuals. MHO individuals had circulating levels of high sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) similar to LH individuals, while levels of platelet derived growth factor-ßß (PDGF-ßß) were intermediate to that of LH and MUO individuals. FA profiling analysis combined with discriminant analysis modelling highlighted a panel of nine FAs (consisting of three saturated, three monounsaturated, and three polyunsaturated FAs) in PL and TG fractions that distinguished the three groups. Specifically, saturated FA (myristic and stearic acids) levels in MHO individuals resembled that of LH individuals. CONCLUSION: Our results suggest that the reduced inflammatory state of MHO individuals compared to MUO individuals may stem, in part, from a more favourable underlying FA profile.

Human urinary biomarkers of dioxin exposure: analysis by metabolomics and biologically driven data dimensionality reduction.

Untargeted metabolomic approaches offer new opportunities for a deeper understanding of the molecular events related to toxic exposure. This study proposes a metabolomic investigation of biochemical alterations occurring in urine as a result of dioxin toxicity. Urine samples were collected from Czech chemical workers submitted to severe dioxin occupational exposure in a herbicide production plant in the late 1960s. Experiments were carried out with ultra-high pressure liquid chromatography (UHPLC) coupled to high-resolution quadrupole time-of-flight (QTOF) mass spectrometry. A chemistry-driven feature selection was applied to focus on steroid-related metabolites. Supervised multivariate data analysis allowed biomarkers, mainly related to bile acids, to be highlighted. These results supported the hypothesis of liver damage and oxidative stress for long-term dioxin toxicity. As a second step of data analysis, the information gained from the urine analysis of Victor Yushchenko after his poisoning was examined. A subset of relevant urinary markers of acute dioxin toxicity from this extreme phenotype, including glucuro- and sulfo-conjugated endogenous steroid metabolites and bile acids, was assessed for its ability to detect long-term effects of exposure. The metabolomic strategy presented in this work allowed the determination of metabolic patterns related to dioxin effects in human and the discovery of highly predictive subsets of biologically meaningful and clinically relevant compounds. These results are expected to provide valuable information for a deeper understanding of the molecular events related to dioxin toxicity. Furthermore, it presents an original methodology of data dimensionality reduction by using extreme phenotype as a guide to select relevant features prior to data modeling (biologically driven data reduction).

Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

Clenbuterol is a ß2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10µg) of clenbuterol-enriched yogurts.

Analytical aspects in doping control: challenges and perspectives.

Since the first anti-doping tests in the 1960s, the analytical aspects of the testing remain challenging. The evolution of the analytical process in doping control is discussed in this paper with a particular emphasis on separation techniques, such as gas chromatography and liquid chromatography. These approaches are improving in parallel with the requirements of increasing sensitivity and selectivity for detecting prohibited substances in biological samples from athletes. Moreover, fast analyses are mandatory to deal with the growing number of doping control samples and the short response time required during particular sport events. Recent developments in mass spectrometry and the expansion of accurate mass determination has improved anti-doping strategies with the possibility of using elemental composition and isotope patterns for structural identification. These techniques must be able to distinguish equivocally between negative and suspicious samples with no false-negative or false-positive results. Therefore, high degree of reliability must be reached for the identification of major metabolites corresponding to suspected analytes. Along with current trends in pharmaceutical industry the analysis of proteins and peptides remains an important issue in doping control. Sophisticated analytical tools are still mandatory to improve their distinction from endogenous analogs. Finally, indirect approaches will be discussed in the context of anti-doping, in which recent advances are aimed to examine the biological response of a doping agent in a holistic way.

A steroidomic approach for biomarkers discovery in doping control.

Anti-doping authorities have high expectations of the athlete steroidal passport (ASP) for anabolic-androgenic steroids misuse detection. However, it is still limited to the monitoring of known well-established compounds and might greatly benefit from the discovery of new relevant biomarkers candidates. In this context, steroidomics opens the way to the untargeted simultaneous evaluation of a high number of compounds. Analytical platforms associating the performance of ultra-high pressure liquid chromatography (UHPLC) and the high mass-resolving power of quadrupole time-of-flight (QTOF) mass spectrometers are particularly adapted for such purpose. An untargeted steroidomic approach was proposed to analyse urine samples from a clinical trial for the discovery of relevant biomarkers of testosterone undecanoate oral intake. Automatic peak detection was performed and a filter of reference steroid metabolites mass-to-charge ratio (m/z) values was applied to the raw data to ensure the selection of a subset of steroid-related features. Chemometric tools were applied for the filtering and the analysis of UHPLC-QTOF-MS(E) data. Time kinetics could be assessed with N-way projections to latent structures discriminant analysis (N-PLS-DA) and a detection window was confirmed. Orthogonal projections to latent structures discriminant analysis (O-PLS-DA) classification models were evaluated in a second step to assess the predictive power of both known metabolites and unknown compounds. A shared and unique structure plot (SUS-plot) analysis was performed to select the most promising unknown candidates and receiver operating characteristic (ROC) curves were computed to assess specificity criteria applied in routine doping control. This approach underlined the pertinence to monitor both glucuronide and sulphate steroid conjugates and include them in the athletes passport, while promising biomarkers were also highlighted.

Quantification of glucuronidated and sulfated steroids in human urine by ultra-high pressure liquid chromatography quadrupole time-of-flight mass spectrometry.

The urinary steroid profile is constituted by anabolic androgenic steroids, including testosterone and its relatives, that are extensively metabolized into phase II sulfated or glucuronidated steroids. The use of liquid chromatography coupled to mass spectrometry (LC-MS) is an issue for the direct analysis of conjugated steroids, which can be used as urinary markers of exogenous steroid administration in doping analysis, without hydrolysis of the conjugated moiety. In this study, a sensitive and selective ultra high-pressure liquid chromatography coupled to quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) method was developed to quantify major urinary metabolites simultaneously after testosterone intake. The sample preparation of the urine (1 mL) was performed by solid-phase extraction on Oasis HLB sorbent using a 96-well plate format. The conjugated steroids were analyzed by UHPLC-QTOF-MS(E) with a single-gradient elution of 36 min (including re-equilibration time) in the negative electrospray ionization mode. MS(E) analysis involved parallel alternating acquisitions of both low- and high-collision energy functions. The method was validated and applied to samples collected from a clinical study performed with a group of healthy human volunteers who had taken testosterone, which were compared with samples from a placebo group. Quantitative results were also compared to GC-MS and LC-MS/MS measurements, and the correlations between data were found appropriate. The acquisition of full mass spectra over the entire mass range with QTOF mass analyzers gives promise of the opportunity to extend the steroid profile to a higher number of conjugated steroids.