Projets de recherche

Code: 14A15RH

Over the last decades, an increasing number of pharmaceutical peptides and proteins have entered the market. Unfortunately, protein drugs are also misused by athletes to illicitly enhance their performance. As these biopharmaceuticals often are highly similar to natural endogenous proteins, reliably tracing of protein doping products in urine or blood is very challenging. Current anti-doping methods can detect prohibited protein substances, but they do not provide unambiguous information on the molecular structure of the detected species, leaving room for errors. Therefore, advanced analytics such as liquid chromatography-mass spectrometry (LC-MS), have gained popularity in doping analysis. However, LC of intact protein molecules is troublesome, and so far few suitable LC-MS methodologies for distinguishing endogenous from manufactured proteins have been developed.

In this project, we will develop a highly selective and generic analytical platform for the unambiguous characterization of prohibited protein substances in biofluids, like urine and blood. To this end, capillary electrophoresis (CE) will be combined with high-resolution MS. In contrast to LC, CE can provide highly efficient separations of intact protein species. High-end MS detection will yield accurate molecular masses of the separated proteins and their isoforms. This way, CE-MS will allow consistent discrimination of banned protein species from endogenous proteins. In order to meet the requirement to measure very low protein concentrations, a novel ultrasensitive interface will be employed for coupling CE and MS. Moreover, for sample pretreatment, advanced affinity extraction and in-capillary preconcentration strategies will be exploited to achieve very low detection limits. Ultimate goal is to aid the fight against doping by delivering a new reliable method for high-throughput assessment of prohibited performance- enhancing proteins.      

Main findings: 

Results. Major efforts have been put in developing a robust and sensitive capillary electrophoresis method to allow hGH isoform separation and to live up to the requirements for doping analysis. Separation of the two main hGH isoforms can be achieved using a volatile alkaline background electrolyte (BGE). Evaporation of volatile BGE components, leading to severe method instability, could be circumvented by the use of a mineral oil overlay. The nature and order of conditioning showed to have a big impact on the migration time (MT) stability. After verifying several combinations, MT RSDs for the electroosmotic flow and analyte remained constant and were below 0.3% and 0.5%, respectively, for 48 consecutive injections. 
High resolution mass spectrometry (MS) in combination with CE separation enabled structural characterization of endogenous hGH. Next to the differentiation of the 20 kDa and 22 kDa isoform, two known deamidated forms of asparagine were observed. Furthermore, two truncated forms, resulting from the loss of the first two amino acids from the N-terminus, were distinguished. All these many other modifications are, most probably, formed during the long lifetime of this standard (over 30 years). Interestingly, the hGH isoform ratio observed in commercial human standards found with CE-MS (1:15-25) correlated well with reported values (~1:20). 
The CE method enabled for the first time to assess the affinity of commercial hGH antibodies on an individual isoform level. Careful optimization of the binding and elution steps revealed a good recovery for the 22 kDa isoform using one mAb. However, in all other cases low recovery was obtained. This raises the question whether current mAbs are able to accurately distinguish hGH isoform and should be used for sample clean-up and biochemical assays. Division of BioAnalytical Chemistry WADA final report 
Conclusions. A simple and robust CE method has been developed that can separate the two main isoforms of hGH under MS compatible conditions. The hyphenation with MS detection ensures detailed characterization of hGH isoforms. It is the first time that unambiguous assignment of the 20 and 22 kDa variant was made possible showing a clear improvement over current methodologies. Unfortunately, the current commercial interfaces do not provide the sensitivity or robustness without further optimization. The developed CE method did indicate that using the available commercial antibodies raised against the two main isoforms of hGH do not show the same affinity.

Code: 14D16JB

Glucocorticoids are widely used among athletes in treatment of acute inflammation and in chronic inflammatory diseases such as asthma. However, glucocorticoids may be misused to increase recovery and performance during competition, and several former Tour de France cyclists have admitted use of glucocorticoids as performance-enhancing agents during races. The current prohibited list allows glucocorticoids to be applied as a skin cream or inhaled from an inhalator, whereas injections and intake of pills are prohibited. In order to distinguish between prohibited misuse and
therapeutic use, and to establish urinary thresholds, it is necessary to investigate differences in urine concentrations between the different administration forms. The aim of the current study is thus to investigate differences in the urine concentration of the two most common glucocorticoids following different routes of administration during exercise.

Main Findings: 

While the pharmacokinetics of methylprednisolone is well established in untrained individuals at rest, no studies have, to our best knowledge, investigated the pharmacokinetics of methylprednisolone during simulated competitive exercise applicable to real life sport settings in highly trained individuals. In a randomized open-label crossover study, we investigated urine pharmacokinetics of methylprednisolone after oral, intramuscuar (vastus lateralis and erector spinae muscles), and intravenous administration (16 mg for each route) in conjuction with exercise in 16 endurance-trained men [aged 25±4 years with a VO2max of 63±4 (mean±SD)]. After administration of methylprednisolone, subjects performed 3 hours of cycling exercise at 55-60% VO2max. Urine samples were collected prior and 0-25 hours following administration and were analysed for concentrations of unchanged methyprednisolone using HPLC-MS/MS. Urine excretion rate of unchanged methylprednisolone followed an exponential decline for the intravenous and intramuscular injection routes, peaking withine the first 1½ hours and reaching values close to zero 12-24 hours following injection. For the oral route, excretion rate peaked 1½-3 hours after ingestion, reaching values close to zero 12-24 hours following ingestion. Urine concentrations of methylprednisolone displayed substantial inter-individual variability. Following intravenous or intramuscular injection, urine concentrations peaked 1½ and 3 hours following administration and declined rapidly, displaying low concentrations 8-12 hours following injection. Urine concentrations were lower following oral ingestion during the firsst 1½ hours after administration, but slightly higher than the other routes 8-24 hours afer administration. These observations indicate that urine spot sampling cannot discriminate different systemic routes of methylprednisolone administration based on the concentrations of unchanged methylprednisolone.

Code: 14A34XD

Formestane is an anti-estrogenic drug used on the treatment of breast cancer. In humans, estrogens are strong pituitary inhibitors of gonadotrophins releasing factors. The inhibition of the estrogens synthesis produces an increase of luteinizing hormone (LH) and then a net increase of testosterone production is expected. In addition the combined administration with testosterone will reduce the side effects linked to aromatization like gynecomastia. For these reasons, anti-estrogenic substances were included in 2004 in the World Anti Doping Agency (WADA) List of Prohibited substances. 
The analytical methodologies developed so far are based GC/MS or LC/MS, targeting formestane itself. Traces of formestane can be produced endogenously and detected in urine samples in low concentrations(0.5-20 ng/mL) and thus, since 2011, it is mandatory according to WADA rules to perform a confirmation based on isotope ratio mass spectrometry (IRMS) in order to assess the synthetic origin of formestane before releasing an adverse analytical finding. 
The IRMS developed methods require two consecutive liquid chromatographic purifications (HPLC) before obtaining extracts of adequate purity, and not all laboratories are currently prepared to perform such IRMS analyses.  The metabolism of formestane analysis has been extensively described in a single male volunteer. It appears that among the high number of metabolites described 4a-hydroxy-epiandrosterone has a longer detection window. A metabolic approach based on the detection of specific metabolites of formestane may avoid the use of IRMS for the confirmation of formestane intake, thus reducing the cost and complexity of the analyses. Simultaneously in the case IRMS should still needed, the development of a method for the detection of long term metabolites, excreted for a longer time compared to formestane and in larger amounts in the elimination phase, will certainly improve the detection capacity of formestane.

Main Findings:

Formestane confirmation by IRMS is not an easy task and the method is not yet implemented in all WADA accredited laboratories. IRMS confirmations are time consuming and generate additional cost to the Testing Authorities.  The aim of this work was to improve the knowledge on formestane detection and on its metabolism in order to find specific biomarkers that may reduce to the minimum or even discard the need of IRMS. To do so, oral and transdermal excretion studies were performed and the metabolism of formestane was studied. The first observation is that if no specific method is applied (MS/MS) the estimation of formestane concentration in urine is overestimated, its detection may interfere with the detection of 2-hydroxyandrostenedione. The 4-hydroxylation of steroids is residual while the 2- hydroxylation that is much more important.  The main specific metabolites of formestane (4OH-AED), detected in different proportions depending on the administration route, are 4αOH-epiandrosterone (4OHEA) and 4αOH-androsterone (4OH-A). These metabolites have not been detected in urine samples so far. Their endogenous origin can be most probably discarded, masking their detection specific to trace back a formestane administration. The detection of these metabolites and some metabolic ratios (4OH-EA/4OH-AED, 4OHA/4OH-AED and 4OH-EA/4OH-A) may allow to detect the exogenous administration of formestane and to discriminate the route of administration without the need of using IRMS.
Formestane confirmation by IRMS is not an easy task and the method is not yet implemented in all WADA accredited laboratories. IRMS confirmations are time consuming and generate additional cost to the Testing Authorities.  The aim of this work was to improve the knowledge on formestane detection and on its metabolism in order to find specific biomarkers that may reduce to the minimum or even discard the need of IRMS. To do so, oral and transdermal excretion studies were performed and the metabolism of formestane was studied. The first observation is that if no specific method is applied (MS/MS) the estimation of formestane concentration in urine is overestimated, its detection may interfere with the detection of 2-hydroxyandrostenedione. The 4-hydroxylation of steroids is residual while the 2- hydroxylation that is much more important.
The main specific metabolites of formestane (4OH-AED), detected in different
proportions depending on the administration route, are 4αOH-epiandrosterone (4OHEA) and 4αOH-androsterone (4OH-A). These metabolites have not been detected in urine samples so far. Their endogenous origin can be most probably discarded, masking their detection specific to trace back a formestane administration. The detection of these metabolites and some metabolic ratios (4OH-EA/4OH-AED, 4OHA/4OH-AED and 4OH-EA/4OH-A) may allow to detect the exogenous administration of formestane and to discriminate the route of administration without the need of using IRMS.

Code: 14C27DE 

The ABP was proposed by WADA nearly a decade ago to longitudinally monitor and define an athlete’s individual blood variables in an attempt to indirectly detect doping. The ABP relies on the monitoring of blood variables sensitive to the administration of performance enhancing drugs (PEDs) to identify abnormalities in an athlete’s profile that cannot be explained by a normal physiological or pathological condition. When reviewing irregularities in an athlete’s blood profile, the experts must consider the effect confounding factors such as physical exercise have on the ABP. Indeed, studies have shown decreases in the absolute blood volume and increases in hemoglobin (HGB) concentration when subjects were acutely dehydrated. The ability to characterize an athlete’s hydration status at the time of blood collection would assist experts when reviewing irregularities in the ABP. The most widely used indicator of hydration status is osmolality, a measurement of the electrolyte-water balance in the body. Albumin is the most abundant protein in plasma and is largely responsible for attracting water into the circulatory system. Elevated albumin is typically a sign of dehydration. As lactate and other metabolites accumulate in working muscles, plasma water will be pulled to the working muscles thereby reducing plasma volume and elevating albumin concentration. Interestingly, increases in blood lactate correlate to decreases in plasma volume after maximal exercise. Therefore, it may be possible to indirectly assess relative plasma volume by measuring blood lactate. The purpose of the present study is to compare the changes in serum albumin, osmolality and lactate, potential markers of fluid balance, in the context of the ABP when athletes are subjected to cycling trials of varying levels of dehydration and exercise intensity. The inclusion of additional biomarkers in the ABP responsive to whole body hydration will strengthen the sensitivity of the ABP.

Main Findings: 

This study aimed to identify markers of dehydration not currently accounted for in the hematological passport, specifically serum albumin and serum osmolality, in addition to understanding the effect of exercise-induced dehydration on the current markers of the hematological passport (hemoglobin, reticulocyte%, OFF-score, and ABPS).  Twelve subjects underwent multiple controlled exercise trials designed to induce varying levels of dehydration.  Pre-exercise blood samples were collected to establish baseline values for individual passports.  During exercise interventions, blood samples were collected before the start of exercise and immediately following exercise at 10-minute, 1-hour, 2-hour, and 24-hour time points.  Plasma volume (PV) decreases, as calculated using the Dill and Costill method, caused by dehydration-inducing exercise resulted in significant increases in hematological parameters (hemoglobin ([Hb]), hematocrit (Hct), serum albumin concentration (ALB), serum osmolality (Osm), and calculated OFF-Hr score) at varying time points following exercise.  These changes (increases) in ALB were found to be highly correlated with changes in [Hb] (r = 0.784) and PV shifts (r = 0.809), while no correlation was identified between Osm and [Hb] or PV shifts.  Additionally, the effect of exercise-induced dehydration was analyzed for each individual in the context of the Athlete Biological Passport.  One case existed where the dehydration-induced increase in [Hb] triggered an atypical finding at 95% specificity, however no instances occurred at 99% specificity where increases in hematological variables or the athlete biological passport score (ABPS) exceeded the individual’s upper threshold.  

This study resulted in two key findings: 1) a strong correlation was identified between percentage changes in albumin and hemoglobin (and, thus, plasma volume) following dehydrating and euhydrated exercise; and 2) dehydration-inducing exercise resulted in one atypical finding at 95% specificity and many other unusual profiles when utilizing the Athlete Biological Passport program

Code: 14A18XD 

The detection of the exogenous administration of synthetic androgens (the so called “pseudo-endogenous” steroids) having the same chemical structure of the compounds produced endogenously (i.e testosterone, 5α-dihydrotestosterone or androstenedione) is primarily based on the alterations of the urinary endogenous steroid profiles. 
A Bayesian approach and adaptive model has been adopted by WADA for the management of the steroid profiles and all the parameters obtained by the Accredited Laboratories will be collected starting 1st January 2014 in a global database integrated in the Athletes Biological Passport (ABP), permitting to establish the individual reference ranges of every athlete. 
The detection capacity of the steroid profile can be improved by the inclusion of the urinary hydroxylated androgen metabolites excreted in lower amounts but whose diagnostic values become more significant after the administration of endogenous steroids. The additional inclusion of the isotope ratio mass spectrometric (IRMS) data has demonstrated to improve the statistical discrimination between basal samples and samples obtained after controlled administration of testosterone. 
Finally, the use of IRMS is a very powerful tool for the detection of pseudoendogenous steroids if the starting material for their synthesis has a 13C composition different from the endogenously produced molecule. The detection of formulations able to circumvent their detection by IRMS already occurred since the criteria are based on population data and not on athlete’s previous data. 
The main goal of this project is to establish the long term variability of the IRMS delta values of both target (TC) and endogenous reference compounds (ERC), and of the steroid profile parameters including hydroxylated androgens metabolites in order to improve the evaluation of the longitudinal profiles. This should permit to enlarge the detection capacity and to potentially detect the abuse of preparation.

Main Findings: 

There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.
There is a definite need of finding more efficient ways to detect the exogenous administration of pseudoendogenous steroids. Although the implementation of the ABP has been a clear step forward, there is still a gap between our capacity to suspect and to confirm the abuse of these substances. The use or more specific markers of the urinary steroid profile like some hydroxylated steroids has been investigated, although their implementation is not easy and would require big efforts to harmonize their detection and quantification as was done for the current markers of the ABP.
The potential inclusion of the IRMS data in the ABP as a direct evidence of doping by pseudoendogenous steroids has been evaluated. As for the ABP markers, instead of comparing the obtained data with reference population ranges (present approach as a confirmation strategy), we suggest to incorporate these data to the ABP and to evaluate the values to the reference data produced by every single athlete.

To reach this goal, the first step was to investigate the stability of the IRMS data in both healthy volunteers and athletes. 

The variability of the IRMS data in a short, medium and long term period in 8 males and 6 females volunteers when compared to data of athletes submitted to investigations by the respective NADO due to atypical steroid profiles, showed that the variability of the individuals’ absolute delta values of the parameters studied are at least one half lower than the population ones and  much lower than the markers of the steroid module of the ABP. The data obtained from real samples of athletes showing atypical steroid profiles, show a variability comparable to the non-athletes volunteers, demonstrating that sports practice and the use dietary supplements do not influence the delta values of the endogenously produce steroids. These IRMS values depend mainly on the individuals’ diet and metabolism. 

This would allow defining individual reference ranges much narrow than the currently applied ones. This would permit (1) to extend the detection window of the pseudoendogenous administration and (2) to potentially detect the use of steroids from pharmaceutical preparations showing delta values close to the endogenous values.

The results of the present project, suggest that the IRMS data obtained with the procedures already in place in the WADA accredited Laboratories, can be used in a more performing way. If the data instead of being only used during the confirmation evaluation of an atypical steroid profile would be evaluated longitudinally, the information obtained would permit to enlarge the detection window of pseudoendogenous steroids abuse and the potential detection of pharmaceutical preparations showing delta values in the endogenous region.
We are not proposing the implementation of a new method but the better exploitation of the data already obtained. By doing so, the gap between the ability of detecting suspicious steroid profiles and the capacity of confirming them by IRMS will be drastically reduced.

Code: 14B14ZW

Detection of insulin or insulin analogue doping is still a challenge for doping analysis laboratories. Until now no screening method is available providing the fast and reliable detection of doping with insulin analogue. The insulin analogues are modified insulin preparations to show faster acting or long acting effects compared to regular insulin, which are therefore broadly used in clinical therapy for diabetes and might also be intensively misused in sport and body building. The insulin analogues have amino acid sequences different to regular insulin. Such difference could be recognized by specially selected monoclonal antibodies, which have their binding site or epitopes involved in the positions where amino acid sequences are altered in insulin analogues.  
In this project, we will at first generate and select two monoclonal antibodies which bind all insulin analogues significantly differently. Thereafter a differential immunoassay based on immune-PCR Imperacer Technologie from Chimera Biotech will be constructed with these 2 antibodies. The immune-PCR is the ultra sensitive immunoassay using antibody-DNA conjugate. The DNA fragment can specifically amplify millions fold and the signal will be directly read out in a real-time PCR machine.  An insulin analogue is detected if the insulin concentrations determined by the two assays are significantly differently. This ultrasensitive real-time immune-PCR duplex assay could directly use small amount of urine samples for doping control. Furthermore, efforts will be made to produce specific monoclonal antibodies to each insulin analogue. Each monoclonal antibody will only bind one insulin analogue but not regular insulin or other analogues. 
Differential immunoassays, especially multiplex immune-PCR constructed with these antibodies will be able to identify quickly and unequivocally which insulin analogues are misused. 

Main Findings: 

In this research project, high affinity anti-insulin monoclonal antibodies (mAbs) including several regular insulin specific mAbs and glargine specific mAbs were produced and they were carefully examined for their binding to insulin and insulin analogues. More than 30 best hybridomas were chosen to be cloned with limited dilution. The antibodies were produced in protein free medium, purified with affinity column and biotinylated. The mAb pairs for the sandwich immunoassays were identified through examining more than 900 antibody combinations. A permissive insulin sandwich assay using mAb 5E10 and a regular insulin preferential assay using mAb 7F3 were constructed. A glargine specific sandwich assay with mAb 6E8 was also established. These three assays are ultra-sensitive (limit of detection <1 pg/ml) and together they can identify any insulin analogue in urine and serum directly with only a few hundred microliters of samples. In a pilot study, these assays showed to be able to identify low amount of insulin analogues in the urine samples of the diabetic patients treated insulin analogues alone or combined with regular insulin preparations. 

Furthermore, a regular insulin specific sandwich assay was also constructed with mAb 1B7. This assay together with the permissive insulin assay can identify insulin analogues, bovine and porcine insulin. Due to its high specificity but moderate sensitivity, this assay would be more suitable for the confirmation assay. Additionally, a universal ultrasensitive Immuno-PCR assay format using real-time PCR cycler was constructed, which can easily be converted to the ultra-sensitive duplex assays

Code: 14A21RN 

Ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC-MS) could represent in the near future an orthogonal technique to LC-MS(/MS) and GC-MS(/MS) for routine doping analysis. 
UHPSFC is a normal phase separation technique that uses carbon dioxide as mobile phase, with the addition of small amounts of methanol as co-solvent. This technology is considered to be a green approach. Using appropriate stationary phase chemistries and dimensions, different pharmaceutical compounds (non-polar and polar) have already been analyzed. However, up to date, only few applications of this technique for the analysis of compounds in biological fluids (urine and blood) exist in scientific literature. Furthermore, no studies are available for the analysis of forbidden substances in anti-doping area. 
In this study, the performance of UHPSFC-MS(/MS) will be evaluated for the analysis of different classes of forbidden substances present in the prohibited list, such as exogenous and endogenous steroids, stimulants, diuretics, narcotics, corticoids, small peptides, etc.  Particular attention will be focused to endogenous and exogenous steroids in urine and blood samples as well as to the optimization of analytical conditions (separation and detection) for both screening and confirmation purposes. Results will be evaluated in terms of selectivity, matrix effect, sensitivity and limits of detection in both biological matrices. Benefits and drawbacks of this technique will be discussed and compared to conventional LC-MS(/MS) and GC-MS(/MS) analyses. An improvement of detection capabilities for different forbidden substances hardly detectable by conventional techniques could be expected.

Main Findings: 

Today, LC-MS(/MS) and GC-MS(/MS) remain the most widely used analytical strategies for doping control analysis. The goal of this project was to evaluate the potential of an alternative chromatographic strategy, namely SFC-MS/MS, for the determination of a wide range of doping agents present in the WADA prohibited list. For this purpose, a UHPSFC-MS/MS screening method was developed for 110 stimulants, diuretics and narcotics in urine samples. Then, the same analytical platform was used for the screening of about 100 analytes belonging to the difficult classes of anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids in urine samples. 
From these two large screening methods, we were able to demonstrate that SFC-MS/MS was extremely reliable and could be considered as an ideal alternative to the widely used LC-MS(/MS) and GC-MS(/MS) platforms for the determination of diverse substances in biological matrices. In this project, the following benefits were noticed in SFC-MS/MS: 1. The retention of SFC is mostly based on polar interactions with the stationary phases (i.e. dipole-dipole and hydrogen bonding), which is the opposite of RPLC, where the retention is driven by hydrophobic interactions. Then, some polar doping agents present in the WADA prohibited list (e.g. methylecgonine, octopamine, oxilofrine, synephrine, phenylephrine…) were sufficiently retained in SFC, while it was not the case in RPLC under generic screening conditions.
2. Because the retention mechanisms in SFC and RPLC are orthogonal, some problematic coelutions in RPLC can be “easily” resolved in SFC and vice-versa. This feature was particularly useful for the determination of anabolic agents. In this particular class of compounds, there were indeed a significant number of isobaric substances that deserve special chromatographic attention, since they cannot be resolved by MS/MS detection.
3. In SFC-MS/MS, and contrary to what happen for GC-MS(/MS), the urine samples can be directly analyzed, without the need for a chemical derivatization strategy. Therefore, the sample treatment was simplified and the analytical throughput was strongly enhanced in SFC vs. GC. In view of the workload undergone by a doping control analysis laboratory, such a rapid procedure could be of great value.
4. The instrumental sensitivities achieved in SFC-MS/MS were adequate for screening purposes. Indeed, the LOD achieved were systematically below the MRPLs fixed by the WADA. For the 100 anabolic agents, hormones and metabolic modulators, synthetic cannabinoids and glucocorticoids, there were only one metabolite of each of these substances (norethisterone, turinabol and norbolethone) that did not meet the MRPL criteria among the 100 tested compounds. Through this project, we demonstrated that the sensitivities achieved in SFCMS/MS were comparable and often improved with regard to RPLC-MS/MS and GC-MS/MS, due to the very limited amount of water in the SFC mobile phase.
5. The susceptibility of the developed SFC-MS/MS methods towards matrix effects was evaluated at different concentration levels and the obtained results were compared to that of LC-MS/MS and GC-MS/MS (in some cases). The matrix effects were very low in UHPSFCMS/ MS, and slightly higher in UHPLC-MS/MS, and unacceptable in GC-MS/MS, due to “matrix induced chromatographic response enhancement”, that could jeopardize the reliability of the method. hanks to this project, UHPSFC was for the first time proven to be applicable for the screening of doping agents in urine samples. The application range that can be managed in SFC-MS/MS was found to be similar and even larger than RPLC-MS/MS. Compared to GC-MS/MS, a much higher throughput can be achieved, as there was no need for chemical derivatization. The benefits reported in UHPSFC–MS/MS make this approach very attractive and promising for doping control analysis, as a screening strategy or possibly as a confirmatory approach.

Code: 14A03KP 

Since years high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS) gained importance for the detection of various classes of doping relevant substances. In contrast to the classical GC-MS technique it allows for separation of analytes with different functional properties without derivatization. 
However some analytes are still challenging as HPLC-MS/MS shows limited resolution capabilities and highly polar analytes interact only insufficiently on the conventional analytical columns. Thus, especially the HPLC analysis of several steroidal doping substances and their metabolites but also the polar stimulants are dissatisfactory. Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC may help to overcome these issues. 
During the project it will be tested for the analysis of stimulants. In this class some of the very polar compounds already proved to be nicely analyzed by SFC-MS/MS. A multi-analyte method will be developed and compared to the currently used method. Special focus is given to robustness, identification power and turn around times. 

Main Findings: 

High performance liquid chromatography (HPLC) is considered as method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact only insufficiently on conventional reversed phase (RP) columns. Especially in combination with mass spectrometric detection limitations apply for alterations of mobile phase composition and thereby also stationary phases. Some highly polar sympathetic drugs and their metabolites showed almost no retention on different reversed phase columns. Even on phenylhexyl phases, that show different selectivity due to π-π-interaction their retention remains poor. 
Supercritical fluid chromatography (SFC) as orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. In method development software assistance was used to create a robust separation. Thereby the response surface was generated as full central composite in quadratic design model focusing on the critical peak pairs, that show the same ion transitions in tandem mass spectrometry (MS/MS) and therefore require chromatographic separation for proper assignment. In the final SFC-MS/MS method, all compounds showed sharp peaks and good retention also for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations due to the orthogonality. Furthermore, short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, a precise temperature and backpressure regulation is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC solvent consumption and solvent waste are considerably reduced.

Code: 14A08OM

For sports drug testing, the errorless discrimination of rEPOs from endogenous human EPO is important. The current gel electrophoretic methods are also labour-intensive for anti-doping laboratories. 
Mass spectrometry-based procedures provide strong identification power, robustness and reproducibility in sports drug testing. Recently, we have developed a high-sensitivity and high-throughput qualitative detection method for human urinary NESP using LC-MS/MS. This was the first report of successful practical application of the mass spectrometric identification of NESP in human urine. 
Following this outcome, the aims of this study are to optimize the NESP detecting method for a robust implementation in WADA accredited doping laboratories, and further to establish a novel detection method for other rEPOs, such as CERA in human matrices by LC-MS/MS for doping control purposes. 
CERA is a long-acting erythropoietin derivative with methoxy polyethylene glycol butanoic acid linking to the N-terminal amino group or the ε-amino group of any lysine, predominantly Lys45 and Lys52, in the protein molecule. The structure of CERA is different from that of human EPO and epoetins. After enzymatic digestion, the target Pegylated peptides will be searched by the LTQ Orbitrap high resolution hybrid mass spectrometer for discriminating between CERA and endogenous EPO. Furthermore mass spectrometric glycoform profiling of rEPOs (e.g. epoetins) will be performed by LC-ESI-MS/MS. After enzymatic digestion of core protein and glycan of rEPOs, the target digested glycosylated peptides will be identified by the LTQ Orbitrap high resolution hybrid mass spectrometer (Thermo) and SynaptG2 HDMS TOFMS (Waters) for discriminating between epoetins and endogenous EPO. We focus the differences of syalylation, N-glycosilation, O- acetylation of sialic acids and O-glycosilation between epoetins and human EPO, and we have an optimized CID technique for discriminating N-glycopeptides by LC-MS. 

Main Findings: 

The current analytical methods for recombinant human erythropoietin (rEPO) are mainly gell electrophoretic methods. Mass spectrometry is necessary for  the reliable identification of rEPOs in doping control. The aim of this research project is to develop the mass spectrometric detection method for human sports drug testing.  
The research comprises three topics, i.e., 'Mass spectrometric characterization of rEPOs and their biosimilars', 'Improvement of the LC-MS/MS method for detecting darbepoetin alfa in human urine', and 'Mass spectrometric approach for detecting CERA'. 
It has been recognized that EPO biosimilars typically have the same amino acid sequence as the innovator product, and that they have slight differences in overall chemical structure.  As expected, the biosimilars of darbepoietin alfa have different glycan distributions compared with the originator. Interestingly, the biosimilars of darbepoietin alfa and a biosimilar of epoetin alfa contain not only des-arginine product comprising 165 amino acids, but also a C-terminal arginine product comprising 166 amino acids, in contrast to the results in which the originator recombinant EPO products. darbepoetin alfa and CERA contain only des-arginine EPO comprising 165 amino acids. The presence of C-terminal arginine EPO in human matrices might be evidence of administration of EPO biosimilars.  
We established a high-quality and more robust UPLC-MS/MS method for detecting darbepoetin alfa using a deutrated internal standard. The lower detection limit was 1 pg/ml. The detection window in urine from patients treated with NESP was estimated to be up to 16 days after administration. The present method could be a useful tool for detecting the biosimilars of darbepoetin alfa for doping control testing because the method does not depend on the glycan profiles.  CERA and epoetin beta representing human EPO could be successfully discriminated using  LC-MS/MS with in-source CID. However, this mass spectrometry-based technology needs to be improved in terms of sensitivity for detecting CERA in human biological fluids. 

Code: T14M01GP 

Human chorionic gonadotropin (hCG) may be abused by male athletes in sports and is included in the banned substance list of the World Anti-Doping Agency. An ti - soping laboratories mainly use immunoassays to quantify hCG but cross-reactivity with the different forms of hCG can constitute a poblem for quantitative hCG determination especially in urine samples. Choriogonadotropin protein is a heterodimer composed of an alpha chain, whjich is also common to thyrotropin, lutropin, follitropin, and a beta chain, which confers its specific biological activity. The alpha and beta subunits are non-covalently linked and carry numerous disulfide bonds. the 2 chains also harbour carbohydrate moieties: 2 N-glycosylations ans 4O- glycosylations on the beta subunit and 2 N-glycosylations on the alpha subunit. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers analytical specificity superior to that of immunoassays and can be alternative method for wuantification of hCG in athletes biological samples. for optimal assay accuracy and reliability, a stable isotope labeled internal standard should be used. In a precious project, we developed a protpcol to produce an isotopically-labeled version of hCG (PSAQ standard). The choriogonadotrophin heretodimer was successfully expressed in mammalian cells and purified. Stable isotope incorporation was determined to be greater than 98%. The goal of this project is to produce 200 to 500μg of hCG PSAQ standard according to the protocol previously developed to deliver different anti-doping laboratories. 

Main Findings: 

Recombinant hCG protein was produced and labelled in HEK293 cells. The choriogonadotrophin heterodimer was successfully expressed and purified using Ni-IMAC resin. To obtain higher purity level, the sample was submitted to a second purification step using Ion Exchange Chromatography (IEX). The purity was estimated to be greater than 95%.

In this program, to increase the sequence coverage, a sample of hCG was treated with a  combination of five enzymes to remove N-linked glycans and many common O-linked glycans.  Following deglycosylation, hCG alpha and beta subunits sequences were verified by LC-MS/MS analysis after reduction/alkylation and in-gel trypsin digestion of the purified protein.  The sequence coverage of CGB is greatly improved after treatment with the Deglycosylation Mix compared to the previous feasibility study where the protein was only digested with PNGase F.  Stable isotope incorporation was estimated using MS data generated by LC-MS/MS analysis.  
hCG PSAQ™ standard will be delivered in aliquots of 20 μg.  We recommend to store at -80°C upon receipt.