Our research program aims to improve analytical techniques used in our sports drug testing laboratory (ASDTL) and to investigate new forms of doping which are currently undetectable. We receive funding from the World Anti-Doping Agency, the Department of Health and Ageing’s Anti-Doping Research Program and the Partnership for Clean Competition.
Erythropoietin (EPO) is a naturally occurring hormone that acts on the bone marrow to increase the production of red blood cells (erythrocytes), which carry oxygen. By raising the oxygen-carrying capacity of the blood, EPO improves endurance. In addition to EPO there are also now other erythropoiesis stimulating agents (ESAs) which also stimulate the production of more erythrocytes. These ESAs can be chemically modified forms of EPO such as darbepoietin and Mircera.
Detection of injected EPO is particularly challenging for two main reasons. First, the drug is rapidly broken down in the body after it is taken. This means that the levels rapidly become too low to detect even though the performance-enhancing benefit may last for weeks. Second, because EPO occurs naturally in the body, it is difficult to distinguish between injected EPO and the natural hormone. The EPO that is available for injection is produced in cell culture by recombinant DNA technology.
An alternative to testing for traces of EPO itself is to identify compounds in the blood that change significantly after the drug is taken. These compounds or blood parameters could act as indirect markers of EPO abuse. Testing for a combination of such indirect markers could prove sufficient evidence to indicate that the hormone has been injected.
From 1999 to 2000 ASDTL and the Australian Institute of Sport jointly conducted a series of EPO administration trials and collected samples from over 1000 elite athletes around the world. The results were used to produce a method to predict current or recent abuse of EPO using indirect blood markers. Samples from these trials helped another IOC-accredited laboratory in France to develop a test to distinguish between administered and natural EPO in urine. As a result of this work a test for EPO using both blood and urine testing was introduced just in time for the Sydney 2000 Olympic Games.
ASDTL continues to assist in the development and implementation of new techniques which are needed to detect the newly available ESAs such as darbepoietin and Mircera. These compounds last much longer in the body than does EPO which is a disadvantage for athletes seeking to avoid detection. Work is also proceeding on improving and simplifying the detection of EPO in urine so that the test can be applied to more samples.
Human growth hormone (hGH) is a banned substance which, like other endogenous hormones such as EPO, presents considerable analytical difficulties compared with doping agents which are not naturally found in humans. There are two main problems in detecting hGH abuse. Firstly, the hormone occurs naturally in the body, so that distinguishing an administered dose from the natural pool is difficult. Second, it is not practical to set a blood level of hGH that would be considered unnaturally high and indicative of doping, because levels of the naturally occurring hormone can vary by more than 100-fold in response to factors such as nutritional state, sleep and exercise.
Two approaches have adopted for the detection of GH abuse with one being based on direct detection of injected GH and the other being based on detecting other compounds whose concentration changes as a result of GH administration (indirect markers). The direct approach is simpler but the detection period is short whilst the indirect marker approach is more complex but offers much greater retrospectivity.
ASDTL was part of a WADA and Australian Government funded project conducted jointly by Garvan Institute of Medical Research, NMI, ANZAC Research Institute, Japan Institute of Sports Sciences and Kolling Institute of Medical Research. This project used samples collected from a worldwide population of elite athletes coupled with samples collected from subjects administered recombinant hGH and testosterone to develop and validate a method for the detection of the abuse of hGH using indirect markers. The markers selected were essentially those found by an earlier study which was funded by the International Olympic Committee. The additional data provided by our study should enable WADA to set legally defensible criteria for a test for hGH abuse based on indirect markers.
The direct marker approach focused on the differences between naturally occurring hGH and the recombinant hormone used for injection. Recombinant hGH contains a single form of the hormone, 22k hGH, the number indicating its size. The major natural form of hGH is also 22k hGH and cannot be distinguished from the recombinant form. However, natural hGH has several additional variants than can be distinguished from the recombinant form on the basis of size. A test was developed which compared the total GH concentration to the concentration of the 22k form. The test is based on the presumption that the natural variants of hGH are released into the blood in fixed proportions. Thus, if a sample was found to contain a high concentration of 22k hGH in relation to the total GH this would indicate hGH abuse. ASDTL has been involved in the validation of new tests for the measurement of GH isoforms in serum and is now routinely.
Accredited laboratories are required to detect certain anabolic steroids at levels of 2 ng/mL or lower. Detection at such low levels has until recently required dedicated high-resolution mass spectrometry (HRMS) or tandem mass spectrometry (MS/MS) instruments, both of which are more sensitive than conventional mass spectrometry. The main advantage of these sensitive techniques is that steroids can be detected for a much longer time after administration — abuse of steroids can now be identified for weeks longer than was possible a few years ago. The disadvantage from the laboratory’s perspective is that these additional techniques add significantly to the cost and complexity of testing.
ASDTL has been at the forefront of developing multi residue methods in an attempt to control and even reduce the cost of laboratory drug testing. The WADA List of Prohibited Compounds expands each year however the funds for drug testing do not. Since 2000 the number of compounds that must be tested for has increased by approximately 50%. ASDTL has met this challenge by developing new automated extraction procedures and combining more compounds (multi residue) into each instrumental method. Recent developments in liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) have meant that it may be possible to replace the GC/HRMS steroid method with a multi residue method that includes low level steroids, diuretics, corticosteroids and narcotics in one sample extraction and analysis procedure. Research funds have been provided by WADA and the method is showing great promise.
The usual technique for the detection of the abuse of synthetic versions of naturally occurring steroids such as testosterone is to compare its concentration to that of a related compound, epitestosterone, in the urine (T/E ratio). A T/E ratio greater than 4 may indicate abuse of testosterone. However, there is a wide variation in natural T/E ratios between individuals, so that in some cases the T/E ratio may be above 4 even though the individual has not taken steroids, whereas in others the value may stay below 4 despite steroid abuse. It has recently been established that those with very low natural T/E ratios have a genetic variation which means that urinary testosterone excretion is low. The natural T/E ratio, measured over a period of time, tends to be constant, and any variation in an individual's T/E ratio over time may indicate doping. However at present laboratories are unaware of the T/E values from previous samples and only samples with T/E values above 4 are investigated.
One technique that can complement the measurement of T/E ratios is the use of gas chromatography coupled to isotope ratio mass spectrometry (GC-IRMS). This technique utilises the fact that natural and administered substances, such as testosterone, have small but measurable differences in the ratio of carbon-12 to carbon-13 isotopes (because of the different pathways used in the preparation of the natural and synthetic forms).
By measuring the C12:C13 ratio of steroids detected in urine, GC-IRMS can distinguish between administered and naturally occurring steroids and can identify steroid abuse in cases that would have previously gone undetected. The application of this technique is not simple — the instrumentation is expensive because of the high precision required, and larger sample sizes are needed, which increases the amount of sample preparation required before analysis. Because the GC-IRMS analysis procedure is so complex and expensive it is only applied to suspicious samples which effectively means those with a T/E value above 4. This means that subjects who have the genetically predetermined low excretion of testosterone may abuse testosterone with little risk of detection.
Results from a study funded by the Department of Health and Ageing have shown that measurement of luteinising hormone (LH) in urine can be used as a marker for testosterone abuse in all male subjects including those who are low testosterone excretors. ASDTL now measures LH in all samples from male athletes. A WADA-funded study to improve the measurement of LH in urine is underway.
With both elevated T/E values and suppressed urinary LH being used to initiate the application of GC-IRMS there is now more chance of catching those who abuse synthetic forms of endogenous steroids.
NMI has produced a freeze-dried urine reference material certified for the concentration of the endogenous steroids testosterone and epitestosterone (T and E) to allow laboratories to reliably compare their measurements of these steroids. Screening of T/E ratios gives an initial indication of testosterone abuse. Work is underway to certify the carbon isotope ratio of this material to allow interlaboratory comparison of measurements made to assist in determining if abnormal T/E ratios are a result of doping.
The certification of this material and another previously certified for the concentration of nandrolone is being extended to include concentrations of additional endogenous steroids. These materials will provide an international benchmark for these compounds at their respective concentrations and ratios in human urine. The confidence to compare measurements of this range of steroids made in different laboratories or at different times will allow longitudinal studies to effectively provide evidence of doping with endogenous steroids. This type of study has the additional benefit of being capable of detecting steroid abuse by individuals who are genetically disposed to have low T/E ratios even when doping.