Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT.

Longitudinal monitoring of endogenous steroids in human serum by UHPLC-MS/MS as a tool to detect testosterone abuse in sports.

The detection of testosterone abuse in sports is routinely achieved through the 'steroidal module' of the Athlete Biological Passport by GC-MS(/MS) quantification of selected endogenous anabolic androgenic steroids (EAAS) from athletes' urines. To overcome some limitations of the "urinary steroid profile" such as the presence of confounding factors (ethnicity, enzyme polymorphism, bacterial contamination, and ethanol), ultrahigh performance liquid chromatography (UHPLC) measurements of blood concentrations of testosterone, its major metabolites, and precursors could represent an interesting and complementary strategy. In this work, two UHPLC-MS/MS methods were developed for the quantification of testosterone and related compounds in human serum, including major progestogens, corticoids, and estrogens. The validated methods were then used for the analyses of serum samples collected from 19 healthy male volunteers after oral and transdermal testosterone administration. Results from unsupervised multiway analysis allowed variations of target analytes to be assessed simultaneously over a 96-h time period. Except for alteration of concentration values due to the circadian rhythm, which concerns mainly corticosteroids, DHEA, and progesterone, significant variations linked to the oral and transdermal testosterone administration were observed for testosterone, DHT, and androstenedione. As a second step of analysis, the longitudinal monitoring of these biomarkers using intra-individual thresholds showed, in comparison to urine, significant improvements in the detection of testosterone administration, especially for volunteers with del/del genotype for phase II UGT2B17 enzyme, not sensitive to the main urinary marker, T/E ratio. A substantial extension of the detection window after transdermal testosterone administration was also observed in serum matrix. The longitudinal follow-up proposed in this study represents a first example of 'blood steroid profile' in doping control analysis, which can be proposed in the future as a complement to the 'urinary module' for improving steroid abuse detection capabilities.

Antidoping programme and biological monitoring before and during the 2014 FIFA World Cup Brazil.

BACKGROUND: The FIFA has implemented an important antidoping programme for the 2014 FIFA World Cup. AIM: To perform the analyses before and during the World Cup with biological monitoring of blood and urine samples. METHODS: All qualified players from the 32 teams participating in the World Cup were tested out-of-competition. During the World Cup, 2-8 players per match were tested. Over 1000 samples were collected in total and analysed in the WADA accredited Laboratory of Lausanne. RESULTS: The quality of the analyses was at the required level as described in the WADA technical documents. The urinary steroid profiles of the players were stable and consistent with previously published papers on football players. During the competition, amphetamine was detected in a sample collected on a player who had a therapeutic use exemption for attention deficit hyperactivity disorder. The blood passport data showed no significant difference in haemoglobin values between out-of-competition and postmatch samples. CONCLUSIONS: Logistical issues linked to biological samples collection, and the overseas shipment during the World Cup did not impair the quality of the analyses, especially when used as the biological passport of football players.

A novel ultra-high-performance supercritical fluid chromatography hyphenated to tandem mass spectrometry method for the analysis of urinary endogenous steroids in the anti-doping context.

The first step in the detection of testosterone (T) doping is to measure the urinary steroid profile for the athlete biological passport (ABP). To harmonise the analysis between anti-doping laboratories, urinary steroid profiling is parametrised in deep detail and shall be performed by gas chromatography hyphenated to mass spectrometry (GC-MS). However, due to its requirement for extensive sample preparation, alternatives to GC-MS are being actively pursued. The aim of this study was the evaluation of Ultra-High-Performance Supercritical Fluid Chromatography hyphenated to tandem Mass Spectrometry (UHPSFC-MS/MS) as an alternative for the quantification of endogenous urinary steroids. In this context, we developed a high throughput sample extraction method, followed by a novel UHPSFC-MS/MS method for the analysis of 10 endogenous urinary steroids which are relevant for doping control analysis. Depending on the steroid, the herein presented method is capable of quantification from 0.5 ng/mL up to 10 µg/mL. After validation, the applicability of the method was evaluated by analysing 132 authentic urine samples, which demonstrated results similar to classical GC-MS analysis. Steroid concentrations determined by UHPSFC-MS/MS were slightly overestimated in comparison with GC-MS, but the ratios had <10 % difference between the two methods. As the ABP considers the steroid ratios for passport evaluation, the herein presented method could be used for steroid profiling without reducing the sensitivity of the ABP. Thus, we would propose to consider UHPSFC-MS/MS as an alternative to GC-MS after more tests would have been performed to support our findings. Furthermore, we have also investigated the potential of this technology for sample purification prior to Isotope Ratio Mass Spectrometry (IRMS) for the differentiation between exogenous and endogenous origin of T and its metabolites. While the achieved separation was sufficient to purify urine samples for IRMS analysis in our proof-of-concept study, the instrumental parameters should be further refined for future use.

Comparison of analytical approaches for the detection of oral testosterone undecanoate administration in men.

For antidoping laboratories, the determination of an illicit testosterone (T) administration in urine samples remains a difficult process as it requires the determination of the exogenous origin by carbon isotope ratios (CIRs) of testosterone and its metabolites. As a complement to the urinary analysis, targeting testosterone esters (e.g. testosterone undecanoate [TU]) in serum samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) could represent a simpler approach compared with isotope ratio mass spectrometry (IRMS). These two approaches both lead to the direct detection of the administration of exogenous T but with a difference in effort and complexity of the analysis. To compare the detection window obtained with the two strategies, serum and the corresponding urine samples collected from an administration study with oral TU were analysed. Results showed that, at all timepoints where the intact TU was detected in serum, the CIRs of urinary steroids were also not in agreement with an endogenous origin. IRMS analysis required more effort but resulted in slightly longer detection windows than the ester analysis. Finally, this comparison study showed that, in the presence of a suspicious urinary steroid profile, the LC-MS/MS steroid esters analysis in the corresponding serum samples can be very helpful. If steroid esters are not detected, the IRMS analysis can then be conducted on the urine sample afterwards. Overall, the combination of matrices might facilitate the detection of prohibited T administration in sports, especially for athletes with naturally low T/E ratios.

Effects of empagliflozin on reproductive system in men without diabetes.

Sodium-glucose cotransporter (SGLT) 2 inhibition is a well-known target for the treatment of type 2 diabetes, renal disease and chronic heart failure. The protein SGLT2 is encoded by SLC5A2 (Solute Carrier Family 5 Member 2), which is highly expressed in renal cortex, but also in the testes where glucose uptake may be essential for spermatogenesis and androgen synthesis. We postulated that in healthy males, SGLT2 inhibitor therapy may affect gonadal function. We examined the impact on gonadal and steroid hormones in a post-hoc analysis of a double-blind, randomized, placebo-controlled research including 26 healthy males who were given either placebo or empagliflozin 10 mg once daily for four weeks. After one month of empagliflozin, there were no discernible changes in androgen, pituitary gonadotropin hormones, or inhibin B. Regardless of BMI category, the administration of empagliflozin, a highly selective SGLT2 inhibitor, did not alter serum androgen levels in men without diabetes. While SGLT2 is present in the testes, its inhibition does not seem to affect testosterone production in Leydig cells nor inhibin B secretion by the Sertoli cells.

LC-MS/MS measurement of endogenous steroid hormones and phase II metabolites in blood volumetric absorptive microsampling (VAMS) for doping control purposes.

BACKGROUND: Volumetric Absorptive Microsampling (VAMS) is emerging as a valuable technique in the collection of dried biological specimens, offering a potential alternative to traditional sampling methods. The objective of this study was to assess the suitability of 30 µL VAMS for the measurement of endogenous steroid hormones. METHODS: A novel LC-MS/MS method was developed for the quantification of 18 analytes in VAMS samples, including main endogenous free steroids and phase II metabolites of androgens. The method underwent validation in accordance with ISO/IEC 17025:2017 and World Anti-Doping Agency (WADA) requirements. Subsequently, it was applied to authentic VAMS samples obtained from 20 healthy volunteers to assess the stability of target analytes under varying storage conditions. RESULTS: The validation protocol assessed method's selectivity, matrix effect, extraction recovery, quantitative performance, carry-over and robustness. The analysis of authentic samples demonstrated the satisfactory stability of monitored steroids in VAMS stored at room temperature, 4 °C, -20 °C and -80 °C for up to 100 days and subjected to up to 3 freezing-thawing cycles. CONCLUSIONS: The validated LC-MS/MS method demonstrated its suitability for the measurement of steroids in dried blood VAMS. The observed stability of steroidal compounds suggests promising prospects for future applications of VAMS, both in anti-doping contexts and clinical research.

Straightforward quantification of endogenous steroids with liquid chromatography-tandem mass spectrometry: Comparing calibration approaches.

Different calibration strategies are used in liquid chromatography hyphenated to mass spectrometry (LC-MS) bioanalysis. Currently, the surrogate matrix and surrogate analyte represent the most widely used approaches to compensate for the lack of analyte-free matrices in endogenous compounds quantification. In this context, there is a growing interest in rationalizing and simplifying quantitative analysis using a one-point concentration level of stable isotope-labeled (SIL) standards as surrogate calibrants. Accordingly, an internal calibration (IC) can be applied when the instrument response is translated into analyte concentration via the analyte-to-SIL ratio performed directly in the study sample. Since SILs are generally used as internal standards to normalize variability between authentic study sample matrix and surrogate matrix used for the calibration, IC can be calculated even if the calibration protocol was achieved for an external calibration (EC). In this study, a complete dataset of a published and fully validated method to quantify an extended steroid profile in serum was recomputed by adapting the role of SIL internal standards as surrogate calibrants. Using the validation samples, the quantitative performances for IC were comparable with the original method, showing acceptable trueness (79%-115%) and precision (0.8%-11.8%) for the 21 detected steroids. The IC methodology was then applied to human serum samples (n = 51) from healthy women and women diagnosed with mild hyperandrogenism, showing high agreement (R2 > 0.98) with the concentrations obtained using the conventional quantification based on EC. For IC, Passing-Bablok regression showed proportional biases between -15.0% and 11.3% for all quantified steroids, with an average difference of -5.8% compared to EC. These results highlight the reliability and the advantages of implementing IC in clinical laboratories routine to simplify quantification in LC-MS bioanalysis, especially when a large panel of analytes is monitored.

Variability of the urinary and blood steroid profiles in healthy and physically active women with and without oral contraception.

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17ß-diol/5ß-androstane-3α,17ß-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module.

Longitudinal Profiling of Endogenous Steroids in Blood Using the Athlete Biological Passport Approach.

CONTEXT: Detection of endogenous anabolic androgenic steroids (EAAS), like testosterone (T), as doping agents has been improved with the launch of the Steroidal Module of the Athlete Biological Passport (ABP) in urine samples. OBJECTIVE: To target doping practices with EAAS, particularly in individuals with low level of biomarkers excreted in urine, by including new target compounds measured in blood. DESIGN: T and T/androstenedione (T/A4) distributions were obtained from 4 years of anti-doping data and applied as priors to analyze individual profiles from 2 T administration studies in female and male subjects. SETTING: Anti-doping laboratory. Elite athletes (n = 823) and male and female clinical trials subjects (n = 19 and 14, respectively). INTERVENTION(S): Two open-label administration studies were carried out. One involved a control phase period followed by patch and then oral T administration in male volunteers and the other followed female volunteers during 3 menstrual cycles with 28 days of daily transdermal T application during the second month. MAIN OUTCOME MEASURE(S): Serum samples were analyzed for T and A4 and the performance of a longitudinal ABP-based approach was evaluated for T and T/A4. RESULTS: An ABP-based approach set at a 99% specificity flagged all female subjects during the transdermal T application period and 44% of subjects 3 days after the treatment. T showed the best sensitivity (74%) in response to transdermal T application in males. CONCLUSIONS: Inclusion of T and T/A4 as markers in the Steroidal Module can improve the performance of the ABP to identify T transdermal application, particularly in females.

Single-run UHPLC-MS/MS method for simultaneous quantification of endogenous steroids and their phase II metabolites in serum for anti-doping purposes.

Anti-doping rule violations related to the abuse of endogenous anabolic androgenic steroids can be currently discovered by the urinary steroidal module of Athlete Biological Passport. Since this powerful tool is still subjected to some limitations due to various confounding factors altering the steroid profile, alternative strategies have been constantly proposed. Among these, the measurement of blood concentrations of endogenous steroid hormones by LC-MS is currently of increasing interest in anti-doping, bringing significant advantages for the detection of testosterone abuse in females and in individuals with deletion of UGT2B17 enzyme. Although various research groups have made significant efforts in method development, there is currently no accepted or harmonized anti-doping method for quantitative analysis of the various testosterone doping markers in blood. In this study we present a UHPLC-MS/MS method for the quantification of major circulating steroid hormones together with an extended panel of glucuro- and sulpho-conjugated phase II metabolites of androgens. Chromatographic setup was optimized by comparing the performance of three different C18 stationary phases and by the careful selection of mobile phases with the aim of separating all the target steroids, including numerous isomeric/isobaric compounds. MS parameters were fine-tuned to obtain the sensitivity needed for measuring the target analytes, that show specific serum concentrations ranging from low pg/mL for less abundant compounds to µg/mL for sulpho-conjugated steroids. Finally, sample preparation protocol was developed for the extraction of steroid hormones from 200 µL of serum and the performance was evaluated in terms of extraction recovery and matrix effect. The final method was then applied to authentic serum samples collected from healthy volunteers (40 males and 40 females) at the Blood Bank of the City of Health and Science University Hospital of Turin. The analysis of these samples allowed to obtain results on serum concentrations of the targeted steroids, with particular emphasis on previously undiscovered phase II metabolites, such as the isomers of 5-androstane-3,17-diol glucuronide. This preliminary application also enabled measuring dihydrotestosterone sulphate in male samples, efficiently separating this analyte from its isomer, epiandrosterone sulphate, which circulates in blood at high concentrations. The promising results of this study are encouraging for the measurement of blood steroid profile markers in serum and plasma samples for Athlete Biological Passport purposes.

A new multimodal paradigm for biomarkers longitudinal monitoring: a clinical application to women steroid profiles in urine and blood.

BACKGROUND: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application. RESULTS: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring. SIGNIFICANCE AND NOVELTY: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping.

A comprehensive UHPLC-MS/MS method for the analysis of endogenous and exogenous steroids in serum for anti-doping purposes.

In the context of steroid analyses, the use of blood could represent a valuable complement to urine. While the blood steroid profile is currently being established to aid unveiling testosterone (T) doping, this matrix is also well suited for detection of exogenous anabolic steroids and steroid esters. In this study, a method to determine a simplified blood steroid profile in combination with the direct detection of exogenous anabolic steroids and steroid esters using just one serum aliquot was developed to obtain a comprehensive analytical workflow. Following the first chromatographic analysis of endogenous and exogenous steroids, samples were derivatised with Girard's reagent T (GT) to improve the ionisation of steroid esters and re-injected. The quantitative performance for T, androstenedione (A4) and 5α-dihydrotestosterone (DHT) was evaluated and the method was validated for qualitative analysis of exogenous analogues with estimated limits of detection (LOD) between 50 and 500 pg/ml. To demonstrate the applicability of the method, samples collected from a clinical study with an oral administration of testosterone undecanoate (TU) to 19 male volunteers were then analysed. The individual serum steroid profiles with the endogenous markers T, A4 and DHT were established as well as the concentrations of TU. TU was detected in all 19 volunteers up to 24 h, while DHT represented the most promising biomarker in endogenous steroid profile for the detection of oral TU administration. These results showed that the selected approach to combine exogenous and endogenous steroid analysis has the potential to strengthen T doping detection in the future.

Comparison of three different blood matrices for the quantification of endogenous steroids using UHPLC-MS/MS.

Urine is currently the matrix of choice for the detection of exogenous substances but also for the application of the steroidal module of the Athlete Biological Passport (ABP) consisting in a longitudinal monitoring of steroid biomarkers. To fill the limitations related to urine, the longitudinal monitoring of serum steroids concentration in the so-called 'blood steroid profile' has recently been proposed. Although serum samples are collected much less than urine samples, plasma derived from ABP whole blood samples used for the full blood count could be exploited for the quantification of endogenous steroids. Alternatively, dried blood spots (DBS) that are much easier to collect could also serve as matrix for the steroid profile. In this study, we compared the concentration levels of several endogenous steroids measured in three different blood matrices (serum, plasma and DBS) collected from 100 elite athletes participating in the 2019 Doha World Athletics Championships using UHPLC-MS/MS. Plasma and serum samples were collected by venipuncture, whereas DBS were generated from whole blood samples. Although steroids demonstrated a good agreement between the three matrices, a slight but acceptable underestimation (10%-20%) was observed in plasma compared with serum. The difference between DBS and the two other matrices was dependent of the bias between serum and plasma. We also showed that a generic HCT correction for DBS could be a valuable approach for quantitative measurements. This study demonstrates the possibility to use three different matrices for the quantification of endogenous steroids although the slight discrepancies should be considered for longitudinal evaluation.

Longitudinal evaluation of multiple biomarkers for the detection of testosterone gel administration in women with normal menstrual cycle.

In women, hormonal fluctuations related to the menstrual cycle may impose a great source of variability for some biomarkers of testosterone (T) administration, which can ultimately disrupt the sensitivity of their longitudinal monitoring. In this study, the sensitivity of the current urinary and haematological markers of the Athlete Biological Passport (ABP), as well as serum steroid biomarkers, was investigated for the monitoring of a 28-day T gel treatment combined with endogenous fluctuation of the menstrual cycle in 14 healthy female subjects. Additionally, the analysis of urinary target compounds was performed on a subset of samples for endogenous/exogenous origin via isotope ratio mass spectrometry (IRMS). In serum, concentrations of T and dihydrotestosterone (DHT) increased significantly during the treatment, whereas in urine matrix the most affected biomarkers were found to be the ratios of testosterone/epitestosterone (T/E) and 5α-androstane-3α,17ß-diol/epitestosterone (5αAdiol/E). The detection capability of both urinary biomarkers was heavily influenced by [E], which fluctuated depending on the menstrual cycle, and resulted in low sensitivity of the urinary steroidal ABP module. On the contrary, an alternative approach by the longitudinal monitoring of serum T and DHT concentrations with the newly proposed T/androstenedione ratio showed higher sensitivity. The confirmatory IRMS results demonstrated that less than one third of the tested urine samples fulfilled the criteria for positivity. Results from this study demonstrated that the 'blood steroid profile' represents a powerful complementary approach to the 'urinary module' and underlines the importance of gathering bundle of evidence to support the scenario of an endogenous prohibited substance administration.

Ion mobility-high resolution mass spectrometry in doping control analysis. Part II: Comparison of acquisition modes with and without ion mobility.

In the second part of this study, a systematic comparison was made between two ion fragmentation acquisition modes, namely data-independent acquisition (DIA) and DIA with ion mobility spectrometry (IMS) technology. These two approaches were applied to the analysis of 192 doping agents in urine. Group I included 102 compounds such as stimulants, diuretics, narcotics, and ß2-agonists, while Group II contained 90 compounds included steroids, glucocorticoids, and hormone and metabolic modulators. Important method parameters were examined and compared, including the fragmentation, sensitivity, and assignment capability with the minimum occurrence of false positive hits. The results differed between Group I and II in number of detected fragments when exploring the MS/MS spectra. In Group I only 13%, while in the Group II 64% of the substances had a higher number of fragments in DIA-IMS mode vs. DIA. In terms of sensitivity, the performance of the two modes with and without activated IMS dimension was identical for about 50% of the doping agents. The sensitivity was higher without IMS, i.e. in simple DIA mode, for 20-40% of remaining doping agents. Despite this sensitivity reduction with IMS, 82% of compounds from both Groups met the minimum required performance level (MRPL) criteria of the World Anti-Doping Agency (WADA) when the DIA-IMS mode was applied. Automated data processing is important in routine doping analysis. Therefore, processing methods were optimized and evaluated for the prevalence of false peak assignments by analysing the target substances at different concentrations in urine samples. Overall, a significantly higher number of misidentified compounds was observed in Group II, with an almost 2-fold higher number of misidentifications in DIA compared to DIA-IMS. This result highlights the benefit of the IMS dimension to reduce the rate of false positive in screening analysis. The optimized UHPLC-IM-HRMS method was finally applied to the analysis of urine samples from administration studies including nine doping agents from both Groups. However, to limit the number of interferences from the biological matrix, an emphasis is needed on the adequate settings of the data processing method.

Steroid profiling by UHPLC-MS/MS in dried blood spots collected from healthy women with and without testosterone gel administration.

The quantification of a large panel of endogenous steroids in serum by LC-MS/MS represents a powerful clinical tool for the screening or diagnosis of diverse endocrine disorders. This approach has also demonstrated excellent sensitivity for the detection of testosterone misuse in the anti-doping field, especially in female athlete population. In both situations, the use of dried blood spots (DBS) could provide a viable alternative to invasive venous blood collection. Here, the evaluation of DBS sampling for the quantification of a panel of endogenous steroids using UHPLC-MS/MS is described. The UHPLC-MS/MS method was validated for quantitative analysis of eleven free and eight conjugated steroids and was then used for the analysis of DBS samples collected in 14 healthy women during a normal menstrual cycle (control phase) followed by a 28-days testosterone gel treatment (treatment phase). Results were compared with those obtained from serum matrix. Satisfactory performance was obtained for all compounds in terms of selectivity, linearity, accuracy, precision, combined uncertainty, stability as well as extraction recovery and matrix effects. In control phase, high correlation was observed between DBS and serum concentrations for most compounds. In treatment phase, higher testosterone concentrations were observed in capillary than in venous DBS, suggesting a possible interference resulting from testosterone contamination on finger(s) used for gel application. Steroid profiling in capillary DBS represents a simple and efficient strategy for monitoring endogenous steroid concentrations and their fluctuation in clinical context of steroid-related disorders, or for the detection of testosterone abuse in anti-doping.

Ion mobility-high resolution mass spectrometry in anti-doping analysis. Part I: Implementation of a screening method with the assessment of a library of substances prohibited in sports.

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, ß2-agonists, ß-blockers, anabolic agents, and hormone and metabolic modulators were investigated, with the aim to assess the benefits and limitations of ion mobility spectrometry (IMS) in combination with ultra-high performance liquid chromatography (UHPLC) and high resolution mass spectrometry (HRMS) in anti-doping analysis. In this first part, a generic UHPLC-IM-HRMS method was successfully developed to analyze these 192 doping agents in standard solutions and urine samples, and an exhaustive database including retention times, TWCCSN2 values, and m/z ratios was constructed. Urine samples were analyzed using either a simple "dilute and shoot" procedure or a supported liquid-liquid extraction (SLE) procedure, depending on the physicochemical properties of the compounds and sensitivity criteria established by the World Anti-Doping Agency (WADA) as the minimum required performance levels (MRPL). Then, the precision of the generic UHPLC-IM-HRMS method was assessed as intraday, interday as well as interweek variation of UHPLC retention times and TWCCSN2 values, for which RSD the values were always lower than 2% in urine samples. The possibility to filter MS data using IMS dimension was also investigated, and in average, the application of IMS filtration provided low energy MS spectra with 86% less interfering peaks in both standard and urine samples. Therefore, the filtered MS spectra allowed for an easier interpretation and a lower risk of false positive result interpretations. Finally, IMS also offers additional selectivity to the UHPLC-HRMS enabling to separate isobaric and isomeric substances. Among the selected set of 192 doping agents, there were 30 pairs of isobaric or isomeric compounds, and only two pairs could not be resolved under the developed conditions. This illustrates the potential of adding ion mobility to UHPLC-HRMS in anti-doping analyses.