Publications by authors named "Raul Nicoli"

28 Publications

  • Page 1 of 1

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

J Pharm Biomed Anal 2021 Sep 26;204:114280. Epub 2021 Jul 26.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Switzerland.

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.
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http://dx.doi.org/10.1016/j.jpba.2021.114280DOI Listing
September 2021

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

Anal Chim Acta 2021 Aug 7;1175:338739. Epub 2021 Jun 7.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

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 β2-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.
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http://dx.doi.org/10.1016/j.aca.2021.338739DOI Listing
August 2021

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

Drug Test Anal 2021 Apr 5. Epub 2021 Apr 5.

Center of Research and Expertise in Anti-Doping Sciences-REDs, Institute of Sport Sciences, University of Lausanne, Lausanne, Switzerland.

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,17β-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.
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http://dx.doi.org/10.1002/dta.3040DOI Listing
April 2021

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.

Anal Chim Acta 2021 Apr 31;1152:338257. Epub 2021 Jan 31.

School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland (ISPSO), University of Geneva, CMU-Rue Michel Servet 1, 1211, Geneva 4, Switzerland. Electronic address:

In this series of two papers, 192 doping agents belonging to the classes of stimulants, narcotics, cannabinoids, diuretics, β2-agonists, β-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, CCS 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 CCS 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.
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http://dx.doi.org/10.1016/j.aca.2021.338257DOI Listing
April 2021

Development and validation of an UHPLC-MS/MS method for extended serum steroid profiling in female populations.

Bioanalysis 2020 Jun 1;12(11):753-768. Epub 2020 Jun 1.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne & Geneva, Lausanne University Hospital & University of Lausanne, Switzerland.

Quantitative endogenous steroid profiling in blood appears as a complementary approach to the urinary module of the World Anti-Doping Agency's Athlete Biological Passport Steroidal Module for the detection of testosterone doping. To refine this approach further, a UHPLC-MS/MS method was developed for the simultaneous determination of 14 free and 14 conjugated steroids in serum. The method was validated for quantitative purposes with satisfactory results in terms of selectivity, linearity range, trueness, precision and combined uncertainty (<20%). The validated method was then applied to serum samples from both healthy women and women diagnosed with mild hyperandrogenism. The UHPLC-MS/MS method showed promising capability in quantifying free and conjugated steroids in serum and determining variations of their concentration/distribution within serum samples from different populations.
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http://dx.doi.org/10.4155/bio-2020-0046DOI Listing
June 2020

Supercritical fluid chromatography-mass spectrometry in routine anti-doping analyses: Estimation of retention time variability under reproducible conditions.

J Chromatogr A 2020 Apr 12;1616:460780. Epub 2019 Dec 12.

Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

The aim of this study was to estimate the retention time variability under reproducible conditions of an SFC-MS analytical method for routine anti-doping analyses. For this purpose, a set of 51 doping agents, as neat standards and spiked in diluted urine, was used to assess their retention times variability over a period of four months, as well as the column inter-batch reproducibility. Three UHPSFC stationary phases have been employed, the Acquity UPC Torus 2-Picolylamine (2-PIC), UPC Viridis BEH and Acquity UPLC HSS C18 SB. Four columns, per column chemistry, have been purchased to represent three different production lots, with a total of twelve columns employed in this study. The two columns from the same lot were applied to the first part of the study (repeatability), whereas the representative of three different lots were employed in the second part (robustness). In terms of organic modifier, a mixture of 98% MeOH and 2% water containing 20 mM ammonium formate was selected in order to limit the formation of methyl-silyl ethers on the surface of the silica particles, thus potentially improving the repeatability of retention times. A comparison with an UHPLC reference analytical method was made with the same set of analytes. The average relative standard deviations (RSD%), represented in split violin plots, illustrate how two of the UHPSFC columns assessed in this study were able to generate an excellent repeatability of retention times, with results that are in a similar range of those generated by UHPLC. Moreover, the Torus 2-PIC has proven to be the best of the three stationary phases, with an impressive RSD% of 0.5% in diluted urine relative to the inter-month variability. Finally, the inter-batch reproducibility assessment has highlighted a good reproducibility of the same stationary phase belonging to different production lots for all three column chemistries assessed, with the Viridis BEH silica generating an RSD% of 0.7% in diluted urine. Higher values of RSD (%) were found for Torus 2-PIC and HSS C18 SB, respectively of 1.0% and 1.6%.
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http://dx.doi.org/10.1016/j.chroma.2019.460780DOI Listing
April 2020

Steroidomics for highlighting novel serum biomarkers of testosterone doping.

Bioanalysis 2019 Jun 10;11(12):1171-1187. Epub 2019 Jun 10.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 1211 Geneve, Switzerland.

Quantification of testosterone (T) and 5α-dihydrotestosterone serum concentrations proved to be an efficient alternative to urinary steroid profiling for the detection of T doping. In this context, additional serum markers could be discovered by exploratory untargeted steroidomics studies. Endogenous steroid metabolites were monitored by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry in serum samples collected during a T administration clinical trial. A three-step workflow for accurate review of annotation was used and multifactorial data analysis allowed highlighting promising serum biomarkers. Longitudinal monitoring of selected compounds was performed to assess T abuse detection capabilities. Application of serum steroidomics showed high potential for biomarker discovery of T doping, suggesting longitudinal monitoring of steroid hormones in serum as a significant improvement in detection of endogenous steroids abuse.
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http://dx.doi.org/10.4155/bio-2019-0079DOI Listing
June 2019

UHPLC-HRMS Analysis for Steroid Profiling in Serum (Steroidomics).

Methods Mol Biol 2018 ;1738:261-278

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva 4, Switzerland.

The extraction and untargeted UHPLC-HRMS analysis of endogenous steroids in serum samples is described in this protocol. The employed full-scan acquisition mode provides the adequate sensitivity to highlight the main endogenous steroids present in blood, including mineralocorticoids, progestogens , and androgens. Technical aspects for both chromatography and mass spectrometry are discussed in detail, together with a proposition of setup for sample sequence and data analysis. Furthermore, general comments are given to help the assessment of data quality and system performance.
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http://dx.doi.org/10.1007/978-1-4939-7643-0_18DOI Listing
January 2019

Systematic evaluation of matrix effects in supercritical fluid chromatography versus liquid chromatography coupled to mass spectrometry for biological samples.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Mar 7;1079:51-61. Epub 2018 Feb 7.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland. Electronic address:

Matrix effects (ME) is acknowledged as being one of the major drawbacks of quantitative bioanalytical methods, involving the use of liquid chromatography coupled to mass spectrometry (LC-MS). In the present study, the incidence of ME in SFC-MS/MS and LC-MS/MS in the positive mode electrospray ionization (ESI+) was systematically compared for the analysis of urine and plasma samples using two representative sets of 40 doping agents and 38 pharmaceutical compounds, respectively. Three different SFC stationary phase chemistries were employed, to highlight the importance of the column in terms of selectivity. Biological samples were prepared using two different sample treatments, including a non-selective sample clean-up procedure (dilute and shoot (DS) and protein precipitation (PP) for urine and plasma samples, respectively) and a selective sample preparation, namely solid phase extraction (SPE) for both matrices. The lower susceptibility to ME in SFC vs. reversed phase LC (RPLC) was verified in all the experiments performed on urine, and especially when a simple DS procedure was applied. Also, with the latter, the performance strongly varied according to the selected SFC stationary phase, whereas the results were quite similar with the three SFC columns, in the case of SPE clean-up. The same trend was observed with plasma samples. Indeed, with the PP procedure, the occurrence of ME was different on the three SFC columns, and only the 2-picolylamine stationary phase chemistry displayed lower incidence of ME compared to LC-MS/MS. On the contrary, when a SPE clean-up was carried out, the results were similar to the urine samples, with higher performance of SFC vs. LC and limited discrepancies between the three SFC columns. The type of ME observed in LC-MS/MS was generally a signal enhancement and an ion suppression for urine and plasma samples, respectively. In the case of SFC-MS/MS, the type of ME randomly varied according to the analyzed matrix, selected column and sample treatment.
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http://dx.doi.org/10.1016/j.jchromb.2018.01.037DOI Listing
March 2018

High-resolution mass spectrometry as an alternative detection method to tandem mass spectrometry for the analysis of endogenous steroids in serum.

J Chromatogr B Analyt Technol Biomed Life Sci 2017 May 18;1052:34-42. Epub 2017 Mar 18.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland. Electronic address:

Recently, steroid hormones quantification in blood showed a promising ability to detect testosterone doping and interesting complementarities with the urinary module of the Athlete Biological Passport (ABP). In this work, an ultra-high pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) method was developed for the quantification of eleven endogenous steroids in serum. The performance of the full scan and targeted SIM acquisition modes was evaluated and compared to the performance of tandem mass spectrometry (MS/MS). Passing-Bablok regressions and Bland-Altman plots were assessed for each analyte of interest, and concentration values measured by HRMS showed high correlation with the ones obtained by MS/MS for all target hormones, with low absolute differences in the majority of cases. A slight decrease in terms of sensitivity was observed with HRMS in both acquisition modes, but performing an analysis of variance multiblock orthogonal partial least squares (AMOPLS) on the dataset obtained with all three methods revealed that only 0.8% of the total variance was related to instrumentation and acquisition methods. Moreover, the evaluation of the testosterone administration effect over time highlighted testosterone itself and dihydrotestosterone as the most promising biomarkers of exogenous testosterone administration. This conclusion suggests that HRMS could provide suitable performance for blood steroid analysis in the anti-doping field.
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http://dx.doi.org/10.1016/j.jchromb.2017.03.016DOI Listing
May 2017

Hepcidin as a potential biomarker for blood doping.

Drug Test Anal 2017 Jul 18;9(7):1093-1097. Epub 2016 Nov 18.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland.

The concentration of hepcidin, a key regulator of iron metabolism, is suppressed during periods of increased erythropoietic activity. The present study obtained blood samples from 109 elite athletes and examined the correlations between hepcidin and markers of erythropoiesis and iron metabolism (i.e., haemoglobin, erythropoietin (EPO), ferritin, erythroferrone (ERFE), and iron concentration). Furthermore, an administration study was undertaken to examine the effect of recombinant human EPO (rhEPO) delta (Dynepo™) on hepcidin concentrations in healthy male volunteers. The effects on hepcidin were then compared with those on reticulocyte percentage (Ret%) and ferritin concentration. There was a significant positive correlation between hepcidin and ferritin, iron, and haemoglobin levels in athletes, whereas hepcidin showed an inverse correlation with ERFE. Administration of rhEPO delta reduced hepcidin levels, suggesting that monitoring hepcidin may increase the sensitivity of the Athlete Biological Passport (ABP) for detecting rhEPO abuse. Copyright © 2016 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/dta.2122DOI Listing
July 2017

Methods for Doping Detection.

Front Horm Res 2016 27;47:153-67. Epub 2016 Jun 27.

Over the past few years, the World Anti-Doping Agency (WADA) has focused its efforts on detecting not only small prohibited molecules, but also larger endogenous molecules such as hormones, in the view of implementing an endocrinological module in the Athlete Biological Passport (ABP). In this chapter, the detection of two major types of hormones used for doping, growth hormone (GH) and endogenous anabolic androgenic steroids (EAASs), will be discussed: a brief historical background followed by a description of state-of-the-art methods applied by accredited anti-doping laboratories will be provided and then current research trends outlined. In addition, microRNAs (miRNAs) will also be presented as a new class of biomarkers for doping detection.
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http://dx.doi.org/10.1159/000445177DOI Listing
September 2017

Liquid chromatography and supercritical fluid chromatography as alternative techniques to gas chromatography for the rapid screening of anabolic agents in urine.

J Chromatogr A 2016 Jun 3;1451:145-155. Epub 2016 May 3.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

This work describes the development of two methods involving supported liquid extraction (SLE) sample treatment followed by ultra-high performance liquid chromatography or ultra-high performance supercritical fluid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS and UHPSFC-MS/MS) for the screening of 43 anabolic agents in human urine. After evaluating different stationary phases, a polar-embedded C18 and a diol columns were selected for UHPLC-MS/MS and UHPSFC-MS/MS, respectively. Sample preparation, mobile phases and MS conditions were also finely tuned to achieve highest selectivity, chromatographic resolution and sensitivity. Then, the performance of these two methods was compared to the reference routine procedure for steroid analyses in anti-doping laboratories, which combines liquid-liquid extraction (LLE) followed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS). For this purpose, urine samples spiked with the compounds of interest at five different concentrations were analyzed using the three analytical platforms. The retention and selectivity of the three techniques were very different, ensuring a good complementarity. However, the two new methods displayed numerous advantages. The overall procedure was much faster thanks to high throughput SLE sample treatment using 48-well plates and faster chromatographic analysis. Moreover, the highest sensitivity was attained using UHPLC-MS/MS with 98% of the doping agents detected at the lowest concentration level (0.1ng/mL), against 76% for UHPSFC-MS/MS and only 14% for GC-MS/MS. Finally, the weakest matrix effects were obtained with UHPSFC-MS/MS with 76% of the analytes displaying relative matrix effect between -20 and 20%, while the GC-MS/MS reference method displayed very strong matrix effects (over 100%) for all of the anabolic agents.
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http://dx.doi.org/10.1016/j.chroma.2016.05.004DOI Listing
June 2016

Fast and sensitive supercritical fluid chromatography - tandem mass spectrometry multi-class screening method for the determination of doping agents in urine.

Anal Chim Acta 2016 Apr 12;915:102-10. Epub 2016 Feb 12.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

This study shows the possibility offered by modern ultra-high performance supercritical fluid chromatography combined with tandem mass spectrometry in doping control analysis. A high throughput screening method was developed for 100 substances belonging to the challenging classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, which should be detected at low concentrations in urine. To selectively extract these doping agents from urine, a supported liquid extraction procedure was implemented in a 48-well plate format. At the tested concentration levels ranging from 0.5 to 5 ng/mL, the recoveries were better than 70% for 48-68% of the compounds and higher than 50% for 83-87% of the tested substances. Due to the numerous interferences related to isomers of steroids and ions produced by the loss of water in the electrospray source, the choice of SFC separation conditions was very challenging. After careful optimization, a Diol stationary phase was employed. The total analysis time for the screening assay was only 8 min, and interferences as well as susceptibility to matrix effect (ME) were minimized. With the developed method, about 70% of the compounds had relative ME within the range ±20%, at a concentration of 1 and 5 ng/mL. Finally, limits of detection achieved with the above-described strategy including 5-fold preconcentration were below 0.1 ng/mL for the majority of the tested compounds. Therefore, LODs were systematically better than the minimum required performance levels established by the World anti-doping agency, except for very few metabolites.
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http://dx.doi.org/10.1016/j.aca.2016.02.010DOI Listing
April 2016

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Am J Hematol 2016 May 6;91(5):467-72. Epub 2016 Apr 6.

Centre Hospitalier Universitaire Vaudois (CHUV), Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland.

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.
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http://dx.doi.org/10.1002/ajh.24313DOI Listing
May 2016

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

Anal Bioanal Chem 2016 Jan 16;408(3):705-19. Epub 2015 Dec 16.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine Geneva and Lausanne, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Chemin des Croisettes 22, 1066, Epalinges, Switzerland.

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.
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http://dx.doi.org/10.1007/s00216-015-9185-1DOI Listing
January 2016

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

Transfusion 2016 Mar 18;56(3):571-8. Epub 2015 Nov 18.

Swiss Laboratory for Doping Analyses, University Centre of Legal Medicine, Lausanne and Geneva, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland.

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.
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http://dx.doi.org/10.1111/trf.13408DOI Listing
March 2016

Analytical Strategies for Doping Control Purposes: Needs, Challenges, and Perspectives.

Anal Chem 2016 Jan 24;88(1):508-23. Epub 2015 Nov 24.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne , Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

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http://dx.doi.org/10.1021/acs.analchem.5b03994DOI Listing
January 2016

Systematic evaluation of matrix effects in hydrophilic interaction chromatography versus reversed phase liquid chromatography coupled to mass spectrometry.

J Chromatogr A 2016 Mar 14;1439:42-53. Epub 2015 Sep 14.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d⿿Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

Reversed phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) is the gold standard technique in bioanalysis. However, hydrophilic interaction chromatography (HILIC) could represent a viable alternative to RPLC for the analysis of polar and/or ionizable compounds, as it often provides higher MS sensitivity and alternative selectivity. Nevertheless, this technique can be also prone to matrix effects (ME). ME are one of the major issues in quantitative LC-MS bioanalysis. To ensure acceptable method performance (i.e., trueness and precision), a careful evaluation and minimization of ME is required. In the present study, the incidence of ME in HILIC-MS/MS and RPLC-MS/MS was compared for plasma and urine samples using two representative sets of 38 pharmaceutical compounds and 40 doping agents, respectively. The optimal generic chromatographic conditions in terms of selectivity with respect to interfering compounds were established in both chromatographic modes by testing three different stationary phases in each mode with different mobile phase pH. A second step involved the assessment of ME in RPLC and HILIC under the best generic conditions, using the post-extraction addition method. Biological samples were prepared using two different sample pre-treatments, i.e., a non-selective sample clean-up procedure (protein precipitation and simple dilution for plasma and urine samples, respectively) and a selective sample preparation, i.e., solid phase extraction for both matrices. The non-selective pretreatments led to significantly less ME in RPLC vs. HILIC conditions regardless of the matrix. On the contrary, HILIC appeared as a valuable alternative to RPLC for plasma and urine samples treated by a selective sample preparation. Indeed, in the case of selective sample preparation, the compounds influenced by ME were different in HILIC and RPLC, and lower and similar ME occurrence was generally observed in RPLC vs. HILIC for urine and plasma samples, respectively. The complementary of both chromatographic modes was also demonstrated, as ME was observed only scarcely for urine and plasma samples when selecting the most appropriate chromatographic mode.
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http://dx.doi.org/10.1016/j.chroma.2015.09.035DOI Listing
March 2016

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

Br J Sports Med 2015 May;49(9):614-22

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Geneva & Lausanne, Epalinges, Switzerland.

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.
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http://dx.doi.org/10.1136/bjsports-2015-094762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413745PMC
May 2015

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. II: Analysis of biological samples.

Anal Chim Acta 2015 Jan 13;853:647-659. Epub 2014 Oct 13.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard D'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

The potential and applicability of UHPSFC-MS/MS for anti-doping screening in urine samples were tested for the first time. For this purpose, a group of 110 doping agents with diverse physicochemical properties was analyzed using two separation techniques, namely UHPLC-MS/MS and UHPSFC-MS/MS in both ESI+ and ESI- modes. The two approaches were compared in terms of selectivity, sensitivity, linearity and matrix effects. As expected, very diverse retentions and selectivities were obtained in UHPLC and UHPSFC, proving a good complementarity of these analytical strategies. In both conditions, acceptable peak shapes and MS detection capabilities were obtained within 7 min analysis time, enabling the application of these two methods for screening purposes. Method sensitivity was found comparable for 46% of tested compounds, while higher sensitivity was observed for 21% of tested compounds in UHPLC-MS/MS and for 32% in UHPSFC-MS/MS. The latter demonstrated a lower susceptibility to matrix effects, which were mostly observed as signal suppression. In the case of UHPLC-MS/MS, more serious matrix effects were observed, leading typically to signal enhancement and the matrix effect was also concentration dependent, i.e., more significant matrix effects occurred at the lowest concentrations.
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http://dx.doi.org/10.1016/j.aca.2014.10.007DOI Listing
January 2015

Ultra high performance supercritical fluid chromatography coupled with tandem mass spectrometry for screening of doping agents. I: Investigation of mobile phase and MS conditions.

Anal Chim Acta 2015 Jan 14;853:637-646. Epub 2014 Oct 14.

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland. Electronic address:

The conditions for the analysis of selected doping substances by UHPSFC-MS/MS were optimized to ensure suitable peak shapes and maximized MS responses. A representative mixture of 31 acidic and basic doping agents was analyzed, in both ESI+ and ESI- modes. The best compromise for all compounds in terms of MS sensitivity and chromatographic performance was obtained when adding 2% water and 10mM ammonium formate in the CO2/MeOH mobile phase. Beside mobile phase, the nature of the make-up solvent added for interfacing UHPSFC with MS was also evaluated. Ethanol was found to be the best candidate as it was able to compensate for the negative effect of 2% water addition in ESI- mode and provided a suitable MS response for all doping agents. Sensitivity of the optimized UHPSFC-MS/MS method was finally assessed and compared to the results obtained in conventional UHPLC-MS/MS. Sensitivity was improved by 5-100-fold in UHPSFC-MS/MS vs. UHPLC-MS/MS for 56% of compounds, while only one compound (bumetanide) offered a significantly higher MS response (4-fold) under UHPLC-MS/MS conditions. In the second paper of this series, the optimal conditions for UHPSFC-MS/MS analysis will be employed to screen >100 doping agents in urine matrix and results will be compared to those obtained by conventional UHPLC-MS/MS.
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http://dx.doi.org/10.1016/j.aca.2014.10.004DOI Listing
January 2015

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

Bioanalysis 2014 ;6(19):2523-36

School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva, Switzerland.

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.
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http://dx.doi.org/10.4155/bio.14.200DOI Listing
July 2015

Serum androgen levels in elite female athletes.

J Clin Endocrinol Metab 2014 Nov 19;99(11):4328-35. Epub 2014 Aug 19.

International Association of Athletics Federations Medical and Anti-Doping Department and Commission (S.B., P.Y.G., M.S., G.D.), 98000 Monaco; Laboratoire Motricité Humaine Education Sport Santé (S.B.), Nice Sophia Antipolis University, 06107 Nice, France; and Monaco Institute of Sports Medicine and Surgery (S.B.), 98000 Monaco; Department of Women's and Children's Health (A.L.H., M.R.), Karolinska Institutet and University Hospital, SE-141 86 Stockholm, Sweden; Swiss Laboratory for Doping Analyses (N.R., S.G., R.N., N.B., M.S.), University Center of Legal Medicine, Geneva and Lausanne, and Centre Hospitalier Universitaire Vaudois and University of Lausanne, 1005 Lausanne, Switzerland; Department of Reproductive Endocrinology, and INSERM Unité 1065 (P.F.), Hôpital l'Archet, University Hospital of Nice, 06-003 Nice, France; Department of Clinical Chemistry (S.J.B., H.H.), Centre Hospitalier Universitaire, University Hospital of Lausanne, Vaudois, 1011 Lausanne, Switzerland.

Objective: Prior to the implementation of the blood steroidal module of the Athlete Biological Passport, we measured the serum androgen levels among a large population of high-level female athletes as well as the prevalence of biochemical hyperandrogenism and some disorders of sex development (DSD).

Methods And Results: In 849 elite female athletes, serum T, dehydroepiandrosterone sulphate, androstenedione, SHBG, and gonadotrophins were measured by liquid chromatography-mass spectrometry high resolution or immunoassay. Free T was calculated. The sampling hour, age, and type of athletic event only had a small influence on T concentration, whereas ethnicity had not. Among the 85.5% that did not use oral contraceptives, 168 of 717 athletes were oligo- or amenorrhoic. The oral contraceptive users showed the lowest serum androgen and gonadotrophin and the highest SHBG concentrations. After having removed five doped athletes and five DSD women from our population, median T and free T values were close to those reported in sedentary young women. The 99th percentile for T concentration was calculated at 3.08 nmol/L, which is below the 10 nmol/L threshold used for competition eligibility of hyperandrogenic women with normal androgen sensitivity. Prevalence of hyperandrogenic 46 XY DSD in our athletic population is approximately 7 per 1000, which is 140 times higher than expected in the general population.

Conclusion: This is the first study to establish normative serum androgens values in elite female athletes, while taking into account the possible influence of menstrual status, oral contraceptive use, type of athletic event, and ethnicity. These findings should help to develop the blood steroidal module of the Athlete Biological Passport and to refine more evidence-based fair policies and recommendations concerning hyperandrogenism in female athletes.
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http://dx.doi.org/10.1210/jc.2014-1391DOI Listing
November 2014

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

J Chromatogr A 2013 May 19;1292:142-50. Epub 2012 Dec 19.

Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, West Switzerland, Chemin des Croisettes 22, 1066 Epalinges, Switzerland.

Clenbuterol is a β2 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.
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http://dx.doi.org/10.1016/j.chroma.2012.12.008DOI Listing
May 2013

Advances in LC platforms for drug discovery.

Expert Opin Drug Discov 2010 May 5;5(5):475-89. Epub 2010 Apr 5.

University of Geneva, University of Lausanne, School of Pharmaceutical Sciences, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland +41 22 379 33 59 ; +41 22 379 33 60 ;

Importance Of The Field: The major application areas of liquid chromatography (LC) in modern drug discovery are the identification and structural characterization of new potential lead compounds from natural and/or synthetic sources and the determination of their physico-chemical and pharmacokinetic properties.

Areas Covered In This Review: Significant advances in terms of LC platforms achieved in the last 5 years are highlighted in this review. Special attention is paid to novel LC strategies used in the discovery of new bioactive molecules and the determination of lipophilicity, pK(a) values, passive drug permeability and in vitro metabolism of new chemical entities.

What The Reader Will Gain: Many improvements were achieved in terms of LC instrumentation, columns technology and analytes detection to attain ultra-fast and/or high-resolution chromatographic separations. These advances are particularly beneficial to face the complexity and high number of samples studied in the early phases of the discovery process. Advantages and drawbacks of each strategy are discussed.

Take Home Message: LC and ultra high pressure liquid chromatography coupled with mass spectrometry detection constitute the most promising strategies to achieve high-throughput and/or high-resolution analyses in a drug discovery environment.
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http://dx.doi.org/10.1517/17460441003733874DOI Listing
May 2010

Development of an in-capillary approach to nanoscale automated in vitro cytochromes p450 assays.

J Med Chem 2009 Apr;52(8):2192-5

School of Pharmaceutical Sciences, University of Geneva and University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

A method to perform nanoscale automated CYP450-based drug metabolism studies using a capillary as a reaction vessel is described. In-capillary assays consumed only approximately 30 nL of recombinant human CYP450 solution. Ultrafast analysis of substrates and metabolites was achieved off-line by ultrahigh-pressure liquid chromatography coupled to mass spectrometry. This approach was successfully applied to qualitative metabolism studies of six major CYP450 isozymes and CYP2D6 inhibition experiments.
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http://dx.doi.org/10.1021/jm900201bDOI Listing
April 2009

Coupling ultra high-pressure liquid chromatography with single quadrupole mass spectrometry for the analysis of a complex drug mixture.

Talanta 2009 Apr 27;78(2):377-87. Epub 2008 Nov 27.

Laboratory of Pharmaceutical Analytical Chemistry, School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Boulevard d'Yvoy 20, 1211 Geneva 4, Switzerland.

The coupling of ultra high-pressure liquid chromatography with a single quadrupole mass spectrometer was investigated for the analysis of several cytochromes P450 (CYP450) substrates and respective metabolites. The effect of numerous operating parameters (e.g. mobile phase pH, flow rate, gradient length, MS acquisition mode, dwell time, polarity switching, etc.) on selectivity, sensitivity and acquisition rate was studied. It was demonstrated that basic pH conditions provided the best compromise in terms of sensitivity and chromatographic selectivity with both acidic and basic compounds. The optimal mobile phase flow rate for UHPLC-MS experiments should be comprised between 300 and 600 microL/min for 2.1mm ID columns, while a higher flow rate generated up to 3-fold loss in sensitivity. It was also demonstrated that the fast polarity switching mode represented a valuable tool to improve throughput, maintaining acceptable performance. Finally, limits of detection were included in the range [1-50 ng/mL] in positive ionization mode and [50-250 ng/mL] in negative ionization mode, for investigated compounds.
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http://dx.doi.org/10.1016/j.talanta.2008.11.029DOI Listing
April 2009
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