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Code: T09E3MT 

The Influence of orally avallable drugs on the productivity of selected genes has been demonstrated manifold In the past. and an endogenoue substance termed AICAR has bean found to be of considerable Interest ln the treatment of the metabolic syndrome associated with type-II diabetes, obesity, etc. The stimulation of fat utillzatlon and the Increased production of mitochondria generated conslderabla concerns whether this substance might be misused in sports also since laboratory rodents demonstrated significantly enhanced endurance performance arter a 4-weaks treabnant with AICAR, and the Worid Anti-Doping Agency has banned Its use since January 2009. Dua to the natural occurrence of AICAR as a (by-)product in purine biosynthesis, a quantitative determination of natural urinary AICAR values In healthy Individuals Is necessary to provide a means to uncover the llllclt administration of this drug, which should increase the renal allmlnatlon and, thus, the urine concentration of AICAR, slgnlflcantly. 
Prellmlnary data have demonstrated that urinary AICAR levels vary consldarably depending on the studied population (athletes, healthy lndlvlduals, vitamin B12 or follc acid daflclent persons, etc.), and AICAR concentrations higher than 7,500 ng/mL ware detected In athletes' doping control samples. Since It can not be excluded that some athletes misuse AICAR already today, urinary AICAR levels of healthy Individuals shall be determined to obtain reliable reference values. Within the planned project. a cohort of approxlmately 500 sport students shall be analyzed for urinary AICAR levels including pre- and post-exercise samples, males and females, as well aa different sport dlsclpllnaa (endurance, strength, and game sporta) to allow a statlstlcal evaluation of the obtained results and enable the consideration of threshold value(s) that are Indicative for AICAR misuse ln sporta. Future projects might further elucldate the option to differentiate endogenous AICAR from the synthetically derived analog by Isotope-ratio mass spectrometry. 

Main Findings: 

The adenosine monophosphate activated protein kinase (AMPK) activator 5-amino-4-imidazolecarboxyamide ribonucleoside (AICAR) was found to significantly enhance the endurance of rodents even under sedentary conditions. Thus, usage of this substance was classified as gene doping and AICAR was added to the list of prohibited substances of the World Anti-Doping Agency (WADA). Due to the endogenous production of AICAR in healthy humans, considerable amounts are present in the circulation and, thus, are excreted into urine. Considering these facts, the present study was initiated to establish reference values of renally cleared AICAR in elite athletes. Therefore a quantitative analytical method by means of isotope-dilution liquid chromatography coupled to tandem mass spectrometry, following a sample preparation consisting of a gentle dilution of native urine, was developed. Doping control samples of 500 healthy volunteers were analysed and AICAR concentrations in urine were determined. The statistical evaluation showed a significantly better distribution (normality for log-transformed values) for creatinine corrected data compared to density correction. Data evaluation of the analysis of 500 urine samples yielded a mean concentration of 863 ng/mL with sd=462 ng/mL (corrected via density) or 552 ng/mg with sd=290 ng/mg (corrected via creatinine). Computing the 99.99% reference intervals values for the creatinine corrected amounts of 3361 ng/mg were obtained and these calculations for the present dataset suggest that amounts of urinary AICAR higher than 3500 ng/mg are not consistent with an endogenous production in healthy humans.

Code: 09A23CH

To facilitate the continued anti-doping efforts it is becoming important to make the best use of the limited analysis resources available for doping detection. The primary tools for doping analysis consist of equipment that requires highly skilled operators and the analyses are often expensive and/or time consuming. As such the number of samples that can be tested for any one event is limited; affording those doping a possibility of not being tested. To overcome this deficiency we propose a method to both increase the amount of testing and focus the high quality testing on those samples most likely to be actual doping cases. 
This will be accomplished through the use of highly rapid capillary electrophoretic separations; capable of identifying key indicators of blood transfusion and other methods of enhancing oxygen delivery in the blood. The benefits of capillary electrophoretic separations are numerous; the high speed of the separations, the capability of having portable instruments and the low cost of operation. Capillary electrophoresis based separations can act as frontline screening systems to identify those blood samples that present signs of doping; flagging them for further analysis, while eliminating the bulk of the clean samples from further required testing. The advantage in the size of the capillary electrophoretic systems is the ability to perform multiple analysis with as little as a single drop of blood from the athlete; a much less invasive and rapid sampling method. 

Main Findings:

The objective of this project is to investigate how effectively capillary electrophoresis (CE) can be applied to the analysis of athlete blood samples for the identification of blood dopants. This work has specifically targeted three blood dopants: autologous blood transfusions, hemoglobin based oxygen carriers (HBOCs), and perfluorocarbon emulsions (PFC). The benefits of working with CE include the ability to perform very rapid separations, and the versatility to handle both molecular samples (i.e. HBOCs) and cellular samples (i.e. red blood cells). Furthermore, the benefits of CE separations can be translated from bench-top instruments to lab-on-a-chip devices, for what is often termed “point of care” analyses. This could be highly advantageous in anti-doping analyses, where the samples could be rapidly tested with instrumentation brought to the site of the athletic competition, likely reducing the delay in detecting those using performance enhancing agents. 
For our work, we have focused on the use of traditional, bench-top, CE instruments, as they provide the greatest flexibility in method development. In investigating the three above mentioned blood dopants we have been able to successfully apply CE to two of the three dopant methods. The one method that has been found to be incompatible with CE analysis were the PFC; we found that their inability to remain suspended in a blood sample lead to difficulties in obtaining reproducible injections into the CE. Furthermore, their significantly greater density than red blood cells (RBCs) allows for their rapid, initial identification simply through centrifugation, rendering CE analysis superfluous. The remaining dopants, HBOCs and autologous blood transfusions, have shown much better success in detection through CE. In this past year our work on the detection of HBOCs, mixed into fresh blood samples in vitro, was published in Electrophoresis (DOI:10.1002/elps.201100506). In that work we were able to detect the presence of HBOCs at concentrations down to 5.5 g/L of whole blood, an amount below a 5% increase in total hemoglobin concentration. The CE method that we have developed for the detection of autologous blood transfusions continues to show great promise. This analysis is based on the different electrophoretic mobilities experienced by RBCs of different sizes. As circulating RBCs in athletes tend to be newer, larger cells, whereas RBCs taken from storage tend to be substantially smaller, we are able to exploit the differences in size and mobility of the RBCs to identify the presence of RBCs from storage.  

Code: 09A10GG 

Determining the origin of testosterone and other steroids in human urine is a major issue in doping control. Looking at the latest published laboratory statistics of 2007 from the World-Anti-Doping Agency (WADA), testosterone was by far the most frequent reported adverse analytical finding among the anabolic steroids. Since it is well known that natural outliers of a normal steroid profile exist, a further investigation is recommended after a finding of an elevated testosterone/epitestosterone ratio or high testosterone levels. Commonly, an isotope ratio mass spectrometric (IRMS) analysis is conducted. Of the samples submitted to IRMS analysis, however, surprisingly few are reported as positive findings. 
In the proposed project confiscated testosterone preparations will be investigated. Through a close collaboration with the Special Task Force “Doping” of the Austrian Ministry of Internal Affairs, the Doping Control Laboratory in Seibersdorf is in the unique position to have access to a significant amount of seized testosterone and other anabolic preparations. 
The aim is to determine whether these products have been manipulated, with respect to carbone isotope ratios, in order to prevent a positive IRMS finding. Additionally, it will be investigated to what extent the preparations contain the masking agent epitestosterone. Epitstosterone may be used as a measure to manipulate a doping control result, by lowering the testosterone/epitestosterone ratio in urine steroid profiles.  Thereby, a further investigation of the sample may not come into consideration. 

Main Findings:

In the present study, the content of a number of black marked testosterone products collected in Austria has been analyzed. Additionally, δ 13C‰ values were measured for testosterone in the products after cleavage of the testosterone ester. The aim was to determine whether these products had been manipulated, with respect to carbon isotope ratios, in order to prevent a positive isotopic ratio mass spectrometric (IRMS) finding in doping control. Moreover, it was investigated to what extent the preparations were containing the masking agent epitestosterone, in order to lower the testosterone/epitestosterone ratio in urine steroid profiles. Out of 30 analyzed products, the declared ingredients differed from the actual content in 10 cases. Epitestosterone, however, could not be found in any of the products. The products displayed δ 13C‰ values between –23.6 and —29.4. For more than half of these products, the δ 13C‰ values were above – 26 and within a range reported for endogenous urinary steroids. Hence, the the current study clearly shows the extent of availability of testosterone products with endogenous-like carbon isotope profiles on the black market. Consequently, the applicability of the IRMS – technique to detect the use of these products is currently reduced. It is therefore considered important to continue monitoring this development and intensify research on alternatives for the detection of testosterone misuse. 

Code: 09A12JM

Testosterone is an anabolic steroid that is naturally present in everyone at various concentrations. Therefore it is not a simple task to determine whether it has been used by athletes for doping. A level of testosterone greater than four times that of its close analogue epitestosterone can point to steroid abuse in many subjects, but others have naturally high or low T/E ratios. Samples with high T/E ratios need further investigation by carbon isotope ratio mass spectrometry (IRMS) to confirm whether the steroid is from a natural or synthetic origin. This however is a relatively complex and time consuming technique.

Long-term monitoring of the T/E ratio and concentrations of related steroids over time can reveal unusual changes in metabolite levels and/or an atypical T/E ratio for a particular athlete that may be indicative of doping. For such longitudinal studies to be effective it is imperative that the results being produced by different laboratories around the world are accurate and comparable over extended periods of time. Reference materials of urine with concentration values traceable to the international system of measurement units (SI) are an excellent tool to verify the comparability of laboratory results.

The NMI Australia, with a research grant from WADA, has already produced a certified reference material (CRM) of human urine (NMIA MX005) with accurately known values for the concentration of testosterone and epitestosterone and a T/E ratio close to 4. The certification of this material is currently being extended to a range of other natural steroids related to testosterone. In this project, a pre-existing freeze-dried urine CRM (NMIA MX002) will become the basis of a second urine CRM with a different T/E ratio and concentration of these steroids. This will assist labs to maintain accuracy and comparability of results over a range of concentrations and T/E ratios.

Main Findings

An existing freeze-dried urine certified reference material (NMIA MX002) with property values for the mass fraction and mass concentration of 19-norandrosterone glucuronide has been provided with further certification of the concentrations of testosterone and epitestosterone glucuronides, the testosterone/epitestosterone ratio and the concentrations of four important testosterone metabolites used in longitudinal profiling studies; 5α-androstane-3α,17β-diol glucuronide, 5β-androstane-3α,17β-diol glucuronide, androsterone glucuronide and etiocholanolone glucuronide. The provision of these additional property values traceable to the international measurement system will provide an unequivocal benchmark for key measurement parameters in the detection of testosterone abuse. The NMIA MX002 certified reference material is the second reference material certified at NMIA for testosterone, epitestosterone and the key metabolites of testosterone at a range of concentrations to assist laboratories in longitudinal profiling measurements and in the detection of testosterone abuse.

Two independent reference methods were employed to certify the mass fractions of 5α-androstane-3α,17β-diol, 5β-androstane-3α,17β-diol, androsterone, etiocholanolone, testosterone and epitestosterone glucuronide conjugates in the urine CRM. The two independent reference methods employ different high-efficiency, two-dimensional HPLC clean-up techniques with determination by the complementary techniques of GC with high resolution mass spectrometry (GC/HRMS) and liquid chromatography with tandem mass spectrometry (LC/MS/MS). In both cases the primary ratio technique of isotope dilution mass spectrometry (IDMS) was employed with an exact-matching calibration protocol to minimize bias in quantification.

Studies have been conducted on the homogeneity and stability of the urine material during long-term storage, transport and use with respect to the mass fractions of testosterone, epitestosterone and the testosterone metabolites. Sample analysis was performed using both the GC/HRMS and LC/MS/MS reference methods to investigate possible measurement biases.

Code: 09E22RG

The aim of all kinds of blood manipulation is to increase the total haemoglobin mass (tHb-mass), which is directly correlated to maximum aerobic power and hence performance. When using the current doping tests it is not yet possible to detect autologous blood transfusions or the application of all kinds of erythropoiesis boosting stimulants.

To minimize these illegal practices we recommend monitoring tHb-mass of endurances athletes over time. If the individual profile deviates substantially from the expected, the athlete has to undergo further follow-ups testing. Serial measurements of tHb-mass can also be used to demonstrate objectively that an athlete has or has not used blood doping practices.

Practical experience demonstrates that the recently developed method (optimized CO-rebreathing method) is valid, very reproducible and suitable to measure routinely an athlete’s tHb-mass. The practicability and significance of the method was evaluated in two scientific projects by Prof. Schmidt (WADA 2006-2007 and 2008-2010).

Nevertheless, the acceptance of this method is low in federations and athletes due to the relatively high amount of CO applied during the CO-rebreathing test exceeding the internationally existing threshold limits. The use of another tracer instead of CO, i.e. the isotopically labelled nitric oxide, has several advantages compared to the established CO-rebreathing method. The amount of inhaled tracer gas can be 4000-fold reduced, avoiding a toxic load for the athlete. This reduction is a combination of the isotopic ratio of 15NO/14NO (300) and the high sensitivity of the used detection method (Faraday-Rotation-Spectroscopy) for the measurement of 15NO.

Furthermore, NO has a 200-fold higher affinity to hemoglobin reducing the influence of possible confounding factors. We expect the NO-rebreathing technique using 15NO as innovative tracer gas as an optimal method to determine tHb-mass. As a consequence tHb-mass can be introduced as a key parameter into the athlete’s biological blood passport. 

Main Findings

The direct transfer of the determination of tHb-mass changing only the applied tracer gas from carbon monoxide to nitric oxide is not possible, even if the experimentally derived NO number for the saturation value is used instead of Hüfner’s number. The formula assumes that the applied NO amount binds completely to HbNO. Reactions with other haemoglobin forms and different binding positions have to be quantified in order not to underestimate tHb-mass.

The estimated reaction of oxyHb with nitric oxide prevents high toxic concentrations of NO in the blood leading to metHb, which is converted back to haemoglobin with a certain time constant by the enzyme cytochrome b5-reductase. Therefore for the routine tHb-mass measurement increasing the inhaled NO amount is not useful, since it only increases the metHb and the nitrate concentration in the blood.

As a consequence the use of nitric oxide instead of carbon monoxide has an additional physiological advantage. The endogenous concentration of the target haemoglobin form (HbNO) for the routine method of tHb-mass detection is extremely small. The interaction of NO with other haemoglobin forms prevents an increase of the HbNO concentration leading to a physiological range where the HbNO is relevant. Unfortunately with the laser breakdown after the first project year we were not able to measure neither the endogenous nor the increased or even saturated HbNO concentration since it was below our sensitivity limit of the chemiluminescence sensor and the Faraday-Rotation-Spectrometer (FRS). The only way is the improvement of the 15NO sensitivity of the FRS.

Considering our measurements with NEM/EDTA, the HbNO concentrations should be measured directly after drawing the blood samples. Storing the samples at -80°C lead to a decrease in HbNO concentration compared to the native samples, even when stabilizing them with NEM measurements.

Code: 09E6DH

This study evaluates the threat for sports doping of a class of hormones called gonadotrophin releasing hormone (GnRH) analogs.  These are synthetic, small peptide superactive analogs of the natural hypothalamic hormone GnRH, a decapeptide which is a major regulator of the reproductive system. GnRH stimulates pituitary LH and testicular testosterone (T) secretion but exogenous use of both LH and T are banned in sports. Consequently, GnRH and its analogs are also prohibited in sports for their potential to act as doping and/or masking agents. 
Used as intended for prolonged periods, GnRH analogs suppress gonadal function in hormone dependent diseases like breast or prostate cancers. However, when used for short periods they stimulate gonadal function, creating a so-called “flare” reaction, before suppression sets in. This “flare” reaction could be used by athletes for indirect androgen doping by stimulating the body’s own LH and testosterone secretion to unnaturally high levels. So far, although GnRH analogs are banned and their use by athletes is suspected, there has been no detailed evaluation of how GnRH analogs affect sports doping tests, when to suspect their use and how to detect them in urine specimens. 
This study will examine in detail the hormonal effects of a superactive GnRH analog (leuprolide) on serum and urine LH and T when used for short periods, and when repeated with a drug-free interval and well as with an androgen-suppressed gonadal axis. Our preliminary evidence proves major hormonal effects are produced so a detection test for this GnRH analog is required. We will develop suitable LC/MS/MS methodology, apply it to the clinical study and to a set of urine samples from athletes with high-normal urine testosterone but normal T/E ratio where the suspicion of GnRH analog use might be highest. 

Main Findings:

Non-steroidal drugs that increase endogenous testosterone may be used to exploit ergogenic effects of androgens in power sports. While superactive GnRH analog use is suspected, neither screening nor detection tests are developed.  
Objective: To determine if (a) stimulation for 5 days by leuprolide of serum and urine steroids and urine LH is reproducible at a 2 week interval, (b) nandrolone decanoate (ND) co-administration masks responses to leuprolide administration, (c) performance of urine measurement of leuprolide and M1, its major metabolite, as a detection test.  
Healthy men randomized into a 4 week parallel group, open label clinical study. Leuprolide (1mg) was injected sc daily for 4 days in 1st & 3rd week with hormone-free 2nd & 4th weeks. In the 3rd week, men were randomized to either N decanoate injections or no extra treatment. Serum and urine steroids and urine leuprolide and M1 and LH.  
Results: Leuprolide stimulated striking, reproducible increases in serum and urine LH and steroids (serum T, DHT, 3α diol; urine T, E & A). ND suppressed basal serum T, E2, 3α diol, and urinary E but did not mask or change the magnitude of responses to leuprolide. Urine leuprolide and M1 measurement had 100% sensitivity and specificity in detecting leuprolide administration up to one day after cessation of injections with the detection window between 1 to 3 days after last dose. Screening using urine steroid and LH measurements, optimally by urinary log10(LH x T), correctly classified 82% of urine samples.  
Conclusions: Leuprolide stimulation of endogenous testosterone is reproducible after a 10 day interval, is not masked by ND and is reliably detected by urine leuprolide or M1 measurement for up to 1 to 3 days after administration. 
Publications 
Detection and effects on serum and urine steroid and LH of repeated GnRH analog (leuprolide) stimulation. Handelsman DJ, Idan A, Grainger J, Goebel C, Turner L, Conway AJ. J Steroid Biochem Mol Biol. 2014 May; 141:113-20. doi: 10.1016/j.jsbmb.2014.01.011. Epub 2014 Feb 2.

Code: 09A15PV

Gas chromatography mass spectrometry (GC-MS) is a technique routinely used for screening of doping control samples on the misuse of anabolic steroids. The current project would use a novel type of instrument, namely a triple quadrupole tandem mass spectrometer(GCQqQ-MSn) to develop a method which is faster and is capable of detecting lower concentrations of prohibited anabolic steroids. This would allow for longer detection times of steroid misuse and higher sample throughput (i.e. faster reporting times). 

Main Findings: 

The use of performance enhancing drugs in sports is prohibited. For the detection of misuse of such substances gas chromatography or liquid chromatography coupled to mass spectrometry are the most frequently used detection techniques. In this work the development and validation of a fast gas chromatography tandem mass spectrometric method for the detection of a wide range of doping agents was developed and validated.  
The method is capable to detect quantitatively 13 endogenous steroids (the steroid profile), 19-norandrosterone, salbutamol and 11-nor-9-tetrahydrocannabinol.9carboxylic acid in the applicable ranges and to detect qualitatively over 140 substances in accordance with the minimum required performance levels of the World Anti-Doping Agency in 1 ml of urine. The classes of substances included in the method are anabolic steroids, β2-agonists, stimulants, narcotics, hormone antagonists and modulators and beta-blockers. At these levels the identification according to WADA’s criteria for identification using chromatography and mass spectrometry was achieved. 
 Moreover, by using a short capillary column and hydrogen as a carrier gas the run time of the method is less than 8 min. This means the method is –in general- three times faster than most methods used routinely for the analysis of doping control samples. Hence, the method allows for a high throughput allowing faster reporting times and reduced instrument costs. 
Hence, this method is capable of detecting and confirming a wide range of doping substances at very low concentration in a short time. Such improvements increase the efficiency in anti-doping laboratories and allow for faster reporting. Additionally, the use of multiple internal standards allows for the evaluation of the quality of every single step in the analytical methodology. 

Code: 09D8TM

Nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance, however, only one study has to our knowledge attempted to replicate these findings to exercise capacity. We previously observed an improved exercise duration by 17% with transdermal nicotine, and in the absence of any effect on peripheral markers concluded that nicotine prolongs endurance by a central mechanism. This finding, coupled with increased anecdotal evidence of nicotine ‘experimentation’ amongst competitive cyclists and the fact that as yet it is neither a banned substance nor is its access restricted, raises an ethical dilemma over athletes gaining an unfair advantage and/or serious concerns over the safety of its use during competitive exercise/sport. We are currently finishing a follow-up study to determine whether nicotine administration (patch, gum vs. placebo) can improve ~1h time-trial performance in trained cyclists. The present project proposes to analyze frozen human plasma samples this study. Samples at rest, prior to and following exercise will be analysed for nicotine and cotinine, its’ major metabolite, using reverse-phase HPLC. Results will shed light onto concentrations of nicotine and its major metabolite when taken in the quantities that are commonly available ‘over-the-counter’ in endurance-trained cyclists competing in a simulated event 

Main Findings: 

Nicotine is widely reported to increase alertness, improve coordination and enhance cognitive performance; however, only one study has attempted to replicate these findings to exercise capacity, observing an improved exercise duration by 17% with transdermal nicotine administration. This finding, coupled with increased anecdotal evidence of ‘experimentation’ amongst athletes and the fact that as yet it is neither a banned substance nor is its access restricted, raises an ethical dilemma over athletes gaining an unfair advantage and serious concerns over the safety of its use during competitive sport and exercise.  
Therefore, we conducted a follow-up study to determine whether nicotine administration can improve a 1h time-trial performance in trained cyclists, a more face-valid protocol. We recruited 10 competitive male cyclists who attended the laboratory on three occasions one week apart in a randomized order. A 48-hr period of dietary and exercise control preceded each visit, which comprised a work-dependent time-trial on a cycle ergometer to simulate 40km or about 1 hr. On one occasion they received a nicotine patch (PAT, 7 mg·24hr-1) the evening before, on another nicotine gum (GUM, 2 mg) 30 mins prior to exercise, and finally placebo gum and patch (PLA). Venous blood samples were taken at rest, prior to and following exercise and analysed for nicotine and its major metabolite, cotinine, in plasma using high-performance liquid chromatography (HPLC).  
GUM (-0.6 ± 4.4%) and PAT (-1.0 ± 4.8%) resulted in no significant improvement in performance time compared to PLA (62.9 ± 4.1, 62.6 ± 4.5 and 63.3 ± 4.1 min, respectively), with mean power outputs of 264 ± 31 (GUM), 265 ± 32 (PAT) and 263 ± 33 W (PLA), respectively. None of the variables measured (core body temperature, heart rate, ratings of perceived exertion) were different between trials. Despite concerted efforts we were unable to recover sufficient nicotine from plasma samples, most likely due to its tendency to fluctuate, relatively short half-life of about 2h, or perhaps sensitivity of the HPLC (cf. mass spectrometry). Recovery of cotinine - nicotine’s major metabolite (70%) and with a longer retention time of 18-20h - was 100% and lends itself better to accurately and sensitively assessing blood concentrations following nicotine administration. For the baseline samples, traces of cotinine were found in 2 subjects during PLA, 2 subjects during GUM, and unsurprisingly all 10 when they had been wearing PAT overnight. During PLA, negligible concentrations were found at baseline (0.3 ± 0.3 ng/ml), pre-exercise (0.5 ± 0.2 ng/ml) and post-exercise (1.0 ± 0.6 ng/ml). During GUM, negligible concentrations were found at baseline (0.3 ± 0.3 ng/ml), pre-exercise (1.3 ± 0.5 ng/ml) rising significantly post-exercise (3.2 ± 0.8 ng/ml). During PAT, concentrations were significantly elevated compared to GUM and PLA at baseline (34.2 ± 0.7 ng/ml), pre-exercise (38.3 ± 6.3 ng/ml) and post-exercise (43.5 ± 6.3 ng/ml) but did not change over time. 
On the basis of our results, i) it appears as though route and/or duration of administration affect absorption, ii) mass spectrometry is likely preferable over HPLC, where available, for sample analysis, and iii) we recommend future studies to incorporate urine analysis in addition to blood, and dose-dependent effects to be investigated.