Projets de recherche

Code: 09C1JB

The primary problems associated with routine detection of illegal doping with recombinant human growth hormone (rhGH) have been the identical amino acid sequences of natural and rhGH and the extremely low levels of hGH in urine (0.1% to 1% of serum levels). 
Operational Technologies Corp. (OpTech) proposes to develop DNA aptamer-based assays capable of detecting and discriminating:  1) natural hGH,
2) 20kD, 22kD, and heavier glycosylated hGH isoforms, and
3) E. coli-modified rhGH “markers” which exist as ~ 2% “contaminants” of the total hGH in recombinant hormone preparations (Hepner et al., 2005). The development of aptamer reagents for detection and discrimination of hGH and rhGH or its E. coli-modified forms is novel and carries certain advantages vs. traditional immunoassays including potentially improved sensitivity and specificity, reduced cost (due to obviating of animal hosts and expensive antibody production and purification procedures), and an assured supply of identical reagent from lot-to-lot once the aptamer DNA sequences are identified. 
In addition to the potential advantages of DNA aptamers over antibodies, OpTech intends to couple its aptamers to tosyl-magnetic beads (MBs) to concentrate the hGH-associated analytes in serum or urine and use either quantum dots (QDs) or electrochemiluminescence (ECL) to produce assays with lower than femtogram detection limits. The combination of aptamers, MBs, and QDs or ECL may produce assays capable of detecting the ~ 2% modified marker forms of rhGH in urine to eliminate invasive blood draws and facilitate frequent testing of urine. If successful, the assays may even be performed on-site at sporting events with a handheld fluorometer such as the PicofluorTM or a portable ECL sensor. The preferred embodiment will use OpTech’s one step (homogeneous) aptamer plastic-adherent cuvette assay and handheld reader as shown on OpTech’s spin out biotech company’s website (www.pronucleotein.com) and recently published (Bruno JG, et al. J. Fluorescence 2008). 

Main Findings: 

Operational Technologies Corporation has conducted a successful pilot study for WADA in which 8 aptamers were identified that appear to distinguish recombinant from natural hGH. 
The rhGH used in these studies is not a pharmaceutical grade, but served to provide proof-of-concept suggesting that pharmaceutical targets such as Genotropin® could be detected by an aptamer-based ELISA-like plate assay. The aptamer assay may also be embodied in various formats including presumptive lateral flow test strips or dipsticks, fluorescence, chemiluminescence or ECL or other types of assays.

Code: T09E2WS

According to the prohibited list 2010, glycerol is specifically named as masking agent. Oral and intravenous application of glycerol is prohibited. Therefore it is necessary to establish threshold values for urinary glycerol concentrations because glycerol may also be of endogenous origin or may be taken up from other exogenous sources.
In sports glycerol is typically used in high doses in combination with excess fluid in order to increase total body water. However, glycerol plasma concentrations can also be elevated during fasting and endurance exercise.
Furthermore, there are natural sources of glycerol such as foodstuff (e.g. wine) and over-the-counter drugs. The urinary excretion of glycerol following administration has not been studied systematically at rest and during exercise.  Also, little is known about urinary glycerol following prolonged fasting, endurance exercise or the intake of glycerol-rich foods or drugs.
A quantification method for urinary glycerol has been published recently but urinary threshold values for the detection of glycerol misuse are lacking.
For the development of urinary threshold levels we intend to perform a) application studies of glycerol at rest and during exercise, b) to assess urinary glycerol excretion due to fasting and exercise and c) to determine reference values for urinary glycerol concentrations in normal doping control samples. Additionally, an IRMS method for glycerol will be implemented.

Main Findings: 

Glycerol is prohibited by the World Anti-Doping Agency (WADA) as masking agent since 2010. Exogenously administered glycerol is excreted in the urine so that the detection of the misuse of glycerol is possible in theory. However, little is known about the urinary excretion pattern of glycerol. In addition, glycerol may also occur endogenously during increased lipolysis and it can also be ingested from foodstuff or drugs. In order to contribute to the development of urinary threshold levels for the misuse of glycerol, two placebo-controlled application studies (glycerol dose: 1 g/kg body weight) were performed at rest and in combination with exercise. There was a rapid increase in urinary glycerol and maximum concentrations (50000-60000 µg/mL) were observed 2.5 to 4 h after the administration. Urinary concentrations remained significantly elevated for 12-15 h (exercise) to 15-18 h (rest). Plasma volume expansion (+1.5 to 3%) and the reduction in hemoglobin (-0.2 to -0.3 g/dL) and haematocrit (-1%) after glycerol administration were rather small when compared to the administration of placebo and statistically significant only after 2.5 h. 
After 18 h of food deprivation followed by 90 minutes of endurance exercise, highly elevated plasma glycerol concentrations were observed but urinary glycerol concentrations were only slightly increased (maximum concentration: 50.5 µg/mL). In 516 randomly selected routine doping control samples, it was confirmed that urinary concentrations are slightly higher in in-competition than in out-of-competition samples, but in all samples urinary glycerol concentrations remained below 200 µg/mL.
Further, a suitable IRMS-method was developed to measure δ13C-values of glycerol in urine. Directly after glycerol administration, δ13C-values were similar to the isotope ratio of the administered glycerol.
In conclusion, the urinary threshold of 200 µg/mL as suggested by Thevis et al. (2008) can be used to identify athletes, who have misused glycerol in relevant amounts. As shown in the study, this threshold is sufficiently high to minimize the risk of false-positive results due to exercise- or fasting induced lipolysis and unintentional intake from other sources.

Code: 09A19WS

As per list of the World Anti-Doping Agency (WADA) 2009 Boldenone and Boldione are explicitly listed in group S1 “anabolic androgenic steroids”, and are therefore prohibited in sports, while Androsta-1,4,6-triene-3,17-dione is classified as aromatase inhibitor (class S4. Hormone Antagonists and Modulators, particularised class S4.1. Aromatase Inhibitors). All these three substances are reported to be excreted as Boldenone and/or Boldenone metabolite in the urine. As class S1 substances are considered as “Non-Specified Substances” while class S4.1. substances are judged as “Specified Substances” the assignment to the administered substances is particularly important for the valuation of the adverse analytical finding. Thus, the project aims to investigate the urinary metabolism and pharmacokinetics using mass spectrometric analyses. Isotope ratio mass spectrometry (GC-C-IRMS) will be applied to identify origin of Testosterone and other endogenously occuring steroids. Based on the analytical data criteria to trace the administered substance have to be established. 

Main Findings: 

As per list of the World anti-soping agency (WADA) 2009 Boldenone and Boldione are explicitly listed in group S1 "anabolic androgenic steroids", and are therefore prohibited in sports, while Androsta-1,4,6-treine-3,17-dione is classified as aromatase inhibitor (class S4. Hormone antagonits and modulators, particularised class S4.1 aromatase inhibitors). All these three substance are reported to be excreted as Boldenone and/or Boldenone metabolite in the urine. As class S1 substances are considered as  " non-specified substances" while class S4.1. substances are judged as " specified substances" the assignment to the administered substances is particulary important for the valuation of the adverse analytical finding. Thus, the project aims to investigate the urinary metabolism and pharmacokinetics using mass spectrometric analyses. Isotope ratio mass spectrometry (GTC-C-IRMS) will be applied to identify origin of Testosterone and other endogenously occuring steroids. Based on the analytical data criteria to trace the administered substance have to be established. 

Code: 09E7FP 

Platelets are an intriguing autologous source of growth factors (GFs) which have been demonstrated to be able to modulate the recruitment, duplication, activation and differentiation of cells involved in bone- and soft-tissue healing. Doping related issues are still matter of debate when considering this therapeutic approach for the treatment of sport-related injuries in particular because of the IGF-1 content in the platelets alpha granules. Certainly, the use of PRP for the treatment of bone, tendon, cartilage and ligament injuries cannot be considered as a technique able of enhancing the physical performances but its muscle injection is still a matter of debate. Moreover, some authors suggested the hypothesis that the use of PRP for ameliorating muscle healing may lead to late muscle fibrosis. 
The aim of the present study is to analyse, in a murine model, the effect of the different techniques used for the preparation of platelets derived GFs [i.e. Platelet Rich Plasma (PRP) and Platelet Rich Fibrin (PRF)] on muscle repair processes. Muscle injuries, induced on animals, will be treated with the 2 different techniques. Morphological and morphometrical analysis as well as the evaluation of myonucleous with electron mycroscopy will be carried out on repaired muscles. The evaluation, both quantitative and qualitative, of the satellite cells will be performed with the immunohistochemistry technique combined with confocal microscopy. Functional evaluation will be performed with the grafting test. All of the analysis will be carried out with both internal and external controls represented by untreated muscles and muscles treated with recombinant growth factors as well as treated with adeno-associated-virus(AAV) mediated –VEGF gene transfer which has been demonstrated to be able to stimulate angiogenesis and repair processes when trasnfected into skeletal muscles. Long term follow up will be able to highlight eventual muscle fibrosis. 

Main Findings: 

Background Ample evidences suggest that growth factors (GFs) may play a key role in the healing process, especially in the early stages of the inflammatory phase. Despite the reported clinical successes, there is still a lack of knowledge when considering the biological mechanism at the basis of platelet-rich plasma (PRP) activity during the muscle healing process. The aim of the present study was to analyze the early effects of PRP in an easily reproducible animal model.  Materials and methods 102 Wistar male adult rats were used in the present study. The muscle lesion was performed by scalpel on the flexor sublimis muscles. PRP was administered immediately after surgery. Treated, untreated and contralateral muscles were tested by morphological analysis, immunohistochemistry, RT-PCR analysis and Western Blot assay.  
Results In the PRP treated muscles, the leukocyte infiltration was significantly higher when compared to both untreated and contralateral samples. The latter showed a higher leukocyte infiltration when compared to the untreated muscles. PRP treatment also modified the cellular composition of the leukocyte infiltration leading to an increased expression of the CD3, CD8, CD19 and CD68 and to a decreased CD4 antigen expression in both PRP treated and contralateral muscles. The analysis of the blood vessel density and the blood vessel diameters showed no statistically significant differences when comparing the three groups analyzed. At day 2 and 5 after PRP administration there was a significant expression of Pax7 and MyoD1. The analysis of pro- and anti-inflammatory cytokines expression showed that PRP induced a more pronounced and/or early inflammatory response by expression of IL-1β and TGF-1β.  
Discussion The results of the present study showed that PRP treatment increased the physiological early inflammatory response further to a muscle injury with a parallel modification of the pattern of cellular recruitment. Moreover, the local PRP treatment may exert, directly or, more plausibly, indirectly, a systemic effect when healing processes were concerned, at least limited to the very first inflammatory phase. The results of the present study might suggest the hypothesis that PRP promoted an early inflammatory response together with a more efficient production of pre-myogenic progenitor population, satellite cell and myoblasts activation/proliferation during muscle regeneration. Conversely, the terminal differentiation markers (i.e., myogenin, Mrf4) as well as others pro-inflammatory cytokines (i.e., IL-6, IL-10, TNFα) and VEGF-A were not additionally modulated by PRP treatment. Further experimental studies are needed to fully understand the local and systemic mechanism of action before apply PRP in routine clinical practice as well as in order to deeply understand possible systemic effects on muscle performance.

Code: T09E1MB

Research in recent years demonstrated that the analysis of changes in the hGH isoform pattern occurring after administration of recombinant hGH can be used to detect hGH doping. Two pairs of immunoassays based on monoclonal antibodies have been developed, which can be used as two independent tests to measure the relative abundance of the monomeric 22 kD isoform over all other isoforms in a serum sample. Practically, a ratio is calculated between the results from the “rec” assay (employing antibodies preferentially recognizing the 22kDa isoform) and the “pit” assay (employing antibodies recognizing a wide spectrum of pituitary isoforms of GH). Details of the method have been published (Wu et al., 1999; Bidlingmaier et al. 2000, 2001, 2003, 2007, 2009). The method has been implemented in most WADA accredited laboratories and could theoretically be used for detecting cheating athletes.

An important aspect to make test results court proof is the reliability and quality of the normative data underlying the cut-off criteria developed to define an adverse analytical finding. Several studies have been performed to investigate the isoform composition and the respective rec/pit ratios in samples taken at rest and after exercise in healthy subjects, recreational and elite athletes from several sports disciplines and from different ethnic background. The differences found in the rec/pit ratios between the groups were comparably small, and it could be clearly shown that injection of rhGH leads to a strong increase in the ratio exceeding the normal variability.

To further strengthen the scientific evidence for the cut-off, the project proposed here aims to investigate the rec/pit ratio under clinical conditions which might be used as an argument by an athlete to explain an adverse analytical finding (=increased ratio) in front of a court. Investigations of molecular isoforms of hGH under different pathological conditions are scarce. Some papers exist about the 20 kD isoform in acromegaly, when endogenous GH secretion is increased (Murakami 2000, 2004; Tsushima 1999; Boguszewski 1997). The findings were not consistent, pointing to a similar variability in the isoforms in acromegalic patients as in normal subjects. However, the assays used in these papers are completely independent from the isoform assays used in WADA accredited laboratories. So, it remains to be clarified if specific pituitary diseases, but also other conditions where hGH secretion is affected, have an influence on the rec/pit ratios.

In the proposed project, samples from different clinical studies will be investigated. The conditions of interest are:

- early pregnancy (40 samples).

- acromegaly (30 samples)

- other pituitary diseases (non-functioning adenomas, Cushing adenomas etc., 20 samples total)

- diabetes (both, type I and type II, 20 samples total)

The samples will be analyzed by both rec/pit assay combinations (termed “kit1” and “kit2”). The two kits involve different antibodies and are used for screening and confirmation test procedures in WADA accredited laboratories.

Main Findings

From the data obtained during the project it is reasonable to conclude that diabetes and cortisol producing pituitary adenomas (Cushings disease) have no detectable impact upon the GH isoform pattern in serum samples as measured by the rec- and pit-assays used for the differential immunoassay approach.

During pregnancy, where pituitary GH secretion is gradually reduced and placental GH secretion increased with increasing gestational age, the situation is different for kit 1 and 2: Whereas the antibodies used in the rec2- and pit2-assay as well as the pit1-assay apparently are not influenced by the changes in the molecular forms of circulating GH, the presence of GHV seems to reduce the signal obtained from the rec1-assay. Overall, this leads to a remarkable reduction in the rec1/pit1-ratio especially after gestational week 10/11, probably because GHV concentrations reach higher levels. However, it is important that there is no risk of a false positive doping test result arising from this situation: Although for ethical reasons it is impossible to formally test recombinant GH applications in pregnant females, the only possible impact of the phenomenon on a doping test would be false negative test results because of falsely low ratios.

In active acromegaly, there was an intriguing difference between the findings in females and males: Whereas the ratios in female patients were completely normal, the mean ratios in males where somewhat higher than expected. However the samples were collected over several years, and not in a controlled doping test setting. Therefore, results have to be interpreted with caution. Nevertheless - although the vast majority of the ratio results were in the expected range, and even the highest ratios were far below the cut offs used to define an AAF - the observation in males deserves further investigation. It is noteworthy that the highest ratio was observed in a patient with a particular aggressive and big tumor, which was continuously growing and required repeated surgery plus cranial irradiation therapy in addition to medical treatment. Because of the paucity of scientific studies investigating the GH isoform composition in acromegaly it cannot be ruled out a priori that some subtypes of pituitary tumors preferentially synthesize the 22 kD GH isoform. Alternatively, the continuous presence of high circulating GH levels might affect the formation of dimers and multimers of GH in selected patients, thereby potentially leading to altered rec/pit ratios.

However, it is clear that the results presented here come from severely ill patients suffering from an extremely rare, but easily detectable disease because of the dramatic consequences of the pituitary tumors. Even if this is a population of extreme cases, no rec/pit ratio came into the range seen after recombinant GH administration. Therefore, the observation seems to be of some scientific interest but very likely does not represent a real practical problem in doping controls.

Code: 09B7CR

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin – Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF) in carrier ampholytes. 
Due to the risk of suffering a stroke or heart attack some athletes already admitted (e.g. the triathlet Lisa Huetthaler; published interview) having used anticoagulants (e.g. aspirin) in combination with Epo-doping. Heparin is one of the oldest and cheapest anticoagulants. 
Our preliminary results showed that heparin has an at least threefold destructive effect on the Epo IEF-method, i.e. either (1) the Epo-profile gets completely destroyed or smeared, (2) the NESP-profile gets shifted to the endogenous area, or (3) a negative profile gets shifted to the basic area and thus leads to a false positive result.
The project investigates the effect of different heparin pharmaceuticals (unfractionated heparins,  fractionated “low molecular mass” heparins) on both the Epo IEF-PAGE and SDS/Sarcosyl-PAGE methods and after administration of heparin to humans. Both biological matrices will be studied, urine and blood serum/plasma. Additionally, the capability of Epo immunoaffinity purification on the removal of heparin will be studied. What we already know is that ultrafiltration is not able to remove higher molecular mass heparins from urinary Epo. 
Hence, the effect of a targeted enzymatic degradation of heparins on various Epo IEF and SDS-PAGE profiles (endogenous, recombinant) will be studied. This latter strategy might be a solution to the destructive effect of heparins on Epo analysis. 

Main Findings: 

The detection of doping with recombinant peptide and protein hormones (e.g. erythropoietin - Epo, human growth hormone - hGH) is one of the most challenging analytical problems in doping control. The WADA-accredited method for the detection of doping with recombinant human erythropoietins (rhEpo) is based on isoelectric focusing (IEF) in carrier ampholytes.
Due to the risk of suffering a stroke or heart attack some athletes already admitted (e.g. the triathlete Lisa Hütthaler; published interview) having used anticoagulants (e.g. aspirin) in combination with Epo-doping. Heparin is one of the oldest and cheapest anticoagulants. Our results showed that heparin has an at least threefold destructive effect on the Epo IEF-method, i.e. either (1) the Epo profile gets completely destroyed or smeared, (2) the NESP profile gets shifted towards the endogenous area, or (3) a negative profile gets shifted to the basic area and thus leads to a false positive result. The project investigated the effect of different heparin pharmaceuticals (unfractionated heparins, fractionated “low molecular mass” heparins) on the analysis of endogenous and recombinant epoetins by IEF-PAGE and SDS/SAR-PAGE with spiked samples and after administration of heparin to humans. Both, urine and blood serum were used as sample matrices. It  was demonstrated that Epo immunoaffinity purification is able to remove heparin and prevents its harmful effect on the Epo IEF profile. Alternatively, targeted enzymatic degradation of heparins prior to IEF- or SDS/SAR-PAGE can be used to reverse the destructive effect of heparin. No destructive effects of heparin were observed for SDS- and SAR-PAGE of EPO standards and urine/serum samples (independently of that result, urine and serum samples have to be immunoaffinity purified in order to avoid gel overloading with high abundant proteins)

Code: 09A11GR 

Many new compounds—chemically different drugs—have recently been included in the list of prohibited substances published by the World Anti-Doping Agency. Current applications of HPLC–MS in today’s anti-doping laboratories are based predominantly on the utilization of high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) systems. With these systems, known drugs or metabolic products are determined by measuring precursor/product ion pairs, providing the utmost sensitivity but also restricting the technique to a limited number of compounds.
To overcome this problem, Georgakopoulos and colleagues have recently proposed the use of orthogonal-axis high-resolution time-of-flight mass spectrometry (OTOF), which allows a broader range of doping agents. Significantly improving this methodology, we have evaluated HPLC/in-source CID atmospheric pressure chemical ionization (APCI) orbitrap mass spectrometry with accurate mass measurements for its screening potential for agents with antiestrogenic activity, beta2-agonists, exogenous anabolic steroids, and other anabolic agents to take advantage of its high resolution, sensitivity, and full scan acquisition. Furthermore, we plan to refine significantly this methodology in our proposed study.
The aims of the proposed research study are the following:
Development and optimization of HPLC/ESI–, APCI–, and APPI HRMS (MS/MS) methods with respect to eluent composition, ion source parameters, and fragmentation. The detection limits and specificity of the methods will be compared. A full description and validation as a general screening method based on SPE and HPLC/orbitrap mass spectrometry with different ionization techniques for doping agents, including anabolic steroids, beta2-adrenergic agonists, SARMs, agents with antiestrogenic activity, diuretics, stimulants, beta-blockers, and cannabinoids. Validation of the method will consist of investigation of specificity, analytical
recovery, limit of detection, and repeatability.
Finally, a comparison of the HPLC/orbitrap mass spectrometry and HPLC/MS/MS methods for the qualitative screening of prohibited substances in urine will be performed.

Main Findings: 

1) The Multipole RF Amplitude, Multipole 00, Lens 0, Multipole 1 and Front lens voltages have dramatic effect on the Orbitrap sensitivity.
2) The research results shown that at least 113 compounds(including metabolites) out of 131 can be detected in urine at concentrations corresponding the MRPL by HPLC-APCI/Orbitrap mass spectrometry.
3) It was observed that most diuretics containing double bonded sulfur atoms could not be detected by HPLC–APCI/Orbitrap mass spectrometry because they did not ionize under APCI conditions used.
4) The proposed HPLC–ESI/Orbitrap mass spectrometry with high-pH mobile phase was demonstrated to be effective for comprehensive screening of banned substances and their metabolites.
5) Ammonium hydroxide (pH=10.3) was found to be necessary for the sensitive detection of anabolic agents and other doping substances in terms of the S/N ratio.
6) Contrary to common expectations, high-pH mobile phase do not affect the responses of β-blockers and corticosteroids in positive ESI.
7) The results demonstrated that at least 121 compounds (including metabolites) out of 131 can be detected in urine at concentrations corresponding the MRPL by HPLC–ESI/Orbitrap mass spectrometry with high-pH mobile phase.
8) It was observed that steroids without any protonable function (e.g 17α-methyl5α-androstane-3α,17β-diol) gave good photoionization yields in solvent mixtures of 2-propanol/acetonitrile/water and propanol/ethanol/water.
9) 2-propanol/acetonitrile/water (37.5:12.5:50) was found to be necessary for the sensitive detection of doping substances in terms of the S/N ratio.
10) For maximum sensitivity, the APCI probe nebulizer (shared by APPI) is titled toward the cone face as close as possible but far enough to minimize the nebulized mobile phase spraying directly on the cone face.
11) The research results shown that doping substances (including metabolites) with diverse structures and polarities can be analyzed in urine with 100% detection rate at concentrations corresponding the MRPL by HPLC-APPI/Orbitrap mass spectrometry.
12) The results demonstrated that 225 compounds (including metabolites) out of 225 can be detected in urine at concentrations corresponding the MRPL by HPLC– APPI/Orbitrap mass spectrometry with Hypercarb column.
13) Our results confirm the previous findings about the lower susceptibility to matrix effect of APPI.
14) The specificity of the method HPLC–APPI/Orbitrap mass spectrometry with hypercarb column was extremely good and did not represent a limiting factor for selectivity.
15)Our results shows that APPI is a valuable tool for day-to-day usage in doping control because it is able to successfully to ionize more compounds, with greater structural diversity, than the other two ionization techniques

Code: 09D5CG

Luteinizing hormone (LH) is a peptide hormone which is abused by athletes to either induce the production of endogenous hormones such as testosterone to hide the use of anabolic agent use which would normally suppress the production of LH. In the case of the low molecular weight LH receptor agonists they would induce the production of endogenous hormones such as testosterone in a similar way as LH. These compounds are banned in the WADA list (S2. Hormones and related substances) as they have a similar biological effect as the use of LH. 
A method of detection for these compounds needs to be direct as their effect, the excess production of endogenous hormones, cannot be proven by analysis techniques which test for the endogenous hormones. In this proposal several compounds which have been identified as having high LH receptor agonist activity will be synthesized. An instrumental analysis technique based on liquid chromatography with tandem mass spectrometry will be developed for the analysis of urine samples. 

Main Findings: 

Luteinizing hormone (LH) is a peptide hormone which is abused by athletes to either induce the production of endogenous hormones such as testosterone or it can also be used to hide the use of anabolic agents which would normally suppress the production of LH. LH is a naturally occurring hormone which is secreted by the anterior pituitary gland. In males LH acts on the Leydig cells of the testis, stimulating testosterone production, while in females, LH plays an integral part in the ovulation cycle. In the case of the low molecular weight (LMW) LH receptor agonists they would induce production of endogenous hormones such as testosterone in a similar way as LH. These compounds are banned in the WADA list (S2. Hormones and related substances) as they have a similar biological effect as the use of LH.  
The method of detection for these compounds needed to be direct as their effect, the excess production of endogenous hormones, cannot be proven easily by analysis techniques which test for the endogenous hormones. In this project several compounds which had been identified as having high LH receptor agonist activity were synthesised. These compounds fall into two groups, pyrazoles and the Org series. For this project Pyrazole 10, Pyrazole 25, Org 41841 and Org 43553 were synthesised. An instrumental analysis technique was developed using High Resolution Mass Spectrometry coupled with Liquid Chromatography (LC/HRMS) for the analysis of urine samples spiked with the LMW LH receptor agonists. In vitro enzyme analysis was undertaken to identify possible metabolites for the LMW LH receptor agonists. The proposed analysis methodology was validated using the parameters of specificity, linearity, limit of detection (LOD), precision and recovery. The LOD for each compound was determined at 2 ng/mL for Pyrazole 10, Pyrazole 25 and Org 43553 and 5 ng/mL for Org 41841. The analysis technique will be able to be immediately implemented into laboratory screening tests for small molecules using liquid chromatography with mass spectrometry detection. A sample of Org 43553 has been distributed to all WADA laboratories.

Code: 09A20RV 

There is a need for doping control laboratories to improve the detection capabilities of the administration of anabolic androgenic steroids (AAS). Most AAS are extensively metabolized. Studies on steroid metabolism have been traditionally performed using GC-MS methods. Recently, LC-MS systems have demonstrated wide possibilities for the elucidation of new phase I metabolites and the use of this technology has resulted in the detection of previously unreported metabolites. Phase II metabolic reactions of AAS have been normally studied by using specific hydrolysis of the conjugated metabolites present in urine extracts. In most cases, enzymes with –glucuronidase activity were used, thus mainly conjugates with glucuronic acid have been studied up to now using both GC-MS and LC-MS/MS approaches.
Sulfate metabolites are known to be important for some endogenous steroids and they have also been described for exogenous AAS. However, the study of sulfates has been limited by the difficulties of their efficient hydrolysis to the phase I metabolites detected by GC-MS or LC-MS/MS. In spite of this, long-term sulfated metabolites have been reported for some AAS.
The objective of the project will be the evaluation of the phase II metabolism of AAS to look for sulfate conjugates of steroid metabolites that could be used as long-term markers of AAS misuse. A methodology based on the direct analysis of sulfate conjugates by LC-MS/MS will be developed. The different scan methods will be developed to identify unknown sulfate metabolites in administration study samples of different AAS. The excretion profiles of the identified sulfate metabolites will be compared with those of other steroid metabolites targeted in conventional screening procedures, in order to evaluate their interest as long-term markers of steroid misuse. Finally, a methodology addressed to the reliable detection of identified sulfate metabolites in routine antidoping analysis will be developed.

Main Findings: 

The objective of the project was to study the phase II metabolism of anabolic androgenic steroids (AAS) to look for sulphate conjugated metabolites that could be used to improve detection capabilities of AAS misuse. A methodology based on the direct analysis of sulphate conjugates by LC-MS/MS was developed. A liquid-liquid extraction with ethyl acetate was used to extract steroid sulphates from urine samples. Based on the common mass spectrometric behavior of steroid sulphates, precursor ion and neutral loss scan methods, and selected reaction monitoring methods including theoretical transitions of potential metabolites, were used to detect new sulphate metabolites in post-administration samples. AAS studied were boldenone, boldione, methyltestosterone and metandienone. Boldenone sulphate and epiboldenone sulphate were identified as minor metabolites of boldenone in humans. These metabolites were detected in urine during the same time as the main metabolites (boldenone and 5β-androst-1-en-17β-ol-3-one, BM1, detected in the glucuronide fraction). The analysis for the absence of these sulphates could be used as additional criterion of the endogenous origin of boldenone and BM1 in samples with low concentrations of these metabolites before going to GC/C/IRMS analysis. For boldione, seven metabolites conjugated with sulphate were detected. Three novel sulphate metabolites of methyltestosterone were identified and one of them was detected in urine for long time after administration, increasing the retrospectivity of the detection between two and three times with respect to other metabolites described. For metandienone, seven sulphate metabolites were detected in post-administration samples and one of them, was detected in urine for long time after administration, doubling the detection time compared to the last long-term metabolite described (the same phase I metabolite excreted in the glucuronide fraction). 
The results of the project demonstrate the importance of sulphatation as a phase II metabolic pathway for AAS and the interest to study this metabolic fraction to look for new metabolites of AAS to improve the capabilities (e.g. long-term metabolites) of the detection of these compounds in doping controls.

Code: 09B8FG 

Erythropoietin (EPO) is an hormone regulating the synthesis of red blood cells, thus increasing the ability of blood to carry oxygen. Its analogues could be used by athletes in endurance sport and therefore appear in the WADA list of banned substances. Nowadays there are direct and indirect methods for the detection of erythropoietin. 
The direct method developed by Lasne, is divided into four steps: urine concentration, isoelectric focusing separation, double blotting and detection by chemiluminescence. The development of other forms of recombinant EPO, and the need to increase the window of detectability of EPO administration, led to the need of updating the Lasne method to ensure the highest efficacy and efficiency of the anti-doping analysis. For instance, due to the differences in the apparent molecular mass of recombinant and endogenous erythropoietins, the administration of some recombinant EPOs can also be detected by SDS PAGE, another tool to clarify the profile of the suspicious samples. 
The overall aim of the project is to study the effect of some specific modifications of the Lasne method, in order to (i) reduce the overall time of the analysis, (ii) simplify the sample treatment process, (iii) improve the limit of detection, and (iv) increase the robustness of the results.  Particularly, we plan to evaluate the effect of some modification at the level of (i) the preparation of the gel, (ii) the incubation steps respective to the blotting process, and (iii) the antibody incubations following the second blotting step. The effectiveness of the proposed solutions will be tested on a significant number of reference samples, of spiked urines and of negative and positive samples coming from our routine activity.

Main Findings: 

In the first part of this project we focused on the possibility of reducing the overall analytical times of the method for the detection of erythropoietins (EPOs), possibly completing the procedure in less than one working day from the start of the analytical process. The method is basically the same originally proposed by Lasne et al. and presently implemented by the WADA accredited laboratories, with few significant modifications involving the use of a novel blotting system based on vacuum technologies (namely, the SNAP i.d.® system). The system allows in principle a significant shortening of the time required for blocking, washing and antibody incubations, and we have considered its potential effectiveness if used after the first blotting step of the isoelectrofocusing (IEF) procedure. By applying a vacuum to actively drive reagents through the blotting membrane, the time of incubation for the blocking step and the washing step are indeed drastically shortened. 
Furthermore, since the differences between the apparent molecular masses of endogenous and recombinant erythropoietins and analogues make possible to discriminate the endogenous and some exogenous EPOs by SDS-PAGE, the utility of the SNAP i.d.® system was verified also for this procedure. Finally, due to the recent developments in the field of electrophoretic EPOs detection, the utility for the SDS/Sarcosyl-PAGE procedure was also investigated.  The alternative procedures involving the SNAP i.d.® system in the IEF and SDS/Sarcosyl-PAGE methods were validated in terms of sensitivity, specificity and robustness using blank urine samples spiked with biological reference preparation of exogenous erythropoietin (BRP, rEpo) or darbepoetin α (NESP) or epoetin δ (Dynepo) or CERA and urine samples from antidoping controls found to be negative during the screening analysis. The effectiveness of the newly developed approach has been also evaluated analyzing urine samples from excretion studies. 
The results showed that even if the time of each step is drastically reduced (the use of SNAP i.d.® system reduces the time for the complete analysis five to six hours) the limits of detection, the specificity and the robustness of the newly developed procedure are very similar to those obtained by the method currently used by the WADA accredited laboratory of Rome.  We have validated both the IEF method and the SDS/Sarcosyl-PAGE method with the use of the SNAP i.d.® system.  The shortening of the time required to complete the procedure may reveal particularly valuable especially on the occasion of major international events, where the analytical workload drastically increase and, in parallel, the reporting times are very compressed. 
In the second part of the study we have evaluated the effect of some specific modifications of the procedure, in particular different reagents: a secondary antibody goat anti-mouse IgG (H+L) biotin conjugated different from the one currently in use in our laboratory purchased from Pierce/Thermo, two different streptavidin-peroxidase adducts (one complexed and one conjugated) and a specific monoclonal antibody anti-hEPO from mouse (Clone 23H2) on a gelatinous support inside a column to purify the urine samples were tested.  The results obtained showed no significant advantage with respect to the reference procedure, although the results obtained the monoclonal antibody anti-hEPO used for immunopurification step is not good to purify urine samples but we have preliminarily obtained good results using it as primary antibody.