Advanced drug testing methods in sport

Materials World magazine
,
1 May 2014

Mass spectroscopy and gas chromatography may be the grey beards of sports drug testing, but their evolved forms can still snare you a cheat. Eoin Redahan reports.

The cheating archer will use a beta-blocker to lower the heart rate and quieten trembling fingers. The scheming cyclist will transfuse erythropoietin (EPO) to boost oxygen delivery to the muscles and increase endurance. And the sprinter with relaxed morals will swallow amphetamines to burn fat and increase adrenaline production.

Cheating, you see, is a complicated business. A complicated, lucrative business. In a matter of decades, athletic doping has evolved to a frightening degree. While athletes are still nabbed by way of ointment and dietary supplement, anti-doping authorities must now also contend with gene doping and designer drugs that cannot be traced. But what about the wholesome side of the moral divide? Are testing methods keeping pace with crooked peddlers?

Undoubtedly, drug-testing technology has improved, but it has been a subtle evolution. Mass spectrometry and liquid and gas chromatography are still used to scrutinise samples, several decades after their first use. With liquid and gas chromatography, compounds can be separated for analysis without decomposition, while mass spectrometry separates compounds by weight or mass for a more detailed look at a urine or blood sample’s chemical structure.

So why have these techniques endured for so long? According to Olivier Rabin, Science Director of the World Anti Doping Authority (WADA), ‘A separation method by gas or liquid chromatography, combined with a detection method such as spectrometry, offers a versatile and highly sensitive analytical approach that covers a wide range of chemical structures. Significant progress is being made in this field, with macromolecules such as hormones and peptides becoming more and more detectable using these techniques.’

Easy PIESI
Many of the latest advances build on chromatography and spectroscopy foundations. One example is the paired ion electrospray ionisation (PIESI) tool recently developed at the University of Texas Arlington, USA, which is a variant of mass spectrometry that can be used on existing test instruments.

Lead researcher Daniel Armstrong explains, ‘We apply a high voltage to a capillary tube (made from fused silica or steel) through a fluid sample to produce a very fine spray, which is known as the electrospray. Spray droplets containing a small amount of the analysed material quickly evaporate. [You are then left] with the charged molecules that enter the mass spectrometer to be analysed.’

One of the key aspects of the PIESI method is the addition of chemical agents that bundle tiny traces of a given drug. The polyionic pairing reagents enable the researchers to pair anions and analyse negatively charged molecules in the more readily detectable positive mode.

According to Armstrong, the method is excellent at picking up the metabolites of moderate-sized drugs such as steroids, stimulants and depressants. In fact, the researchers say the method could be up to 1,000 times more sensitive than existing mass spectrometry methods. By increasing a test’s sensitivity, drug residue will be visible in smaller concentrations. This should widen the detection window and improve the effectiveness of random drug tests.

But while this test is useful for seeing steroids in sprinters’ bodies, it does not work for larger protein or peptide hormones such as EPO and human growth hormone (HGH). The emergence of HGH abuse, in particular, is a major concern for anti-doping bodies.

Studies have shown that HGH can lower body fat, boost muscle growth and increase sprint speed by 10%. The World Anti-Doping Agency estimates that 5% of US high school students have abused HGH to improve athletic performance or benefit from its performance-enhancing results. However, relatively few professional athletes have been caught taking HGH.

The problem with developing tests for blood and gene doping is that these substances already exist within the body. As a result, analysis focuses on studying biomarkers to find evidence that is consistent with doping. For example, a stark rise in a cyclist’s red blood cell count is indicative of EPO abuse. As such, professional cyclists are required to carry biological passports to keep an eye on the proportion of red blood cells in their blood.

In response to this growing worry, WADA has teamed up with several antidoping bodies to develop an advanced HGH biomarkers test. The liquid chromatographytandem mass spectrometry (LC-MS/MS) technique is based on a mass spectroscopy method used at the 2012 Olympics. WADA’s Rabin explains, ‘The biomarkers test is based on the detection of proteins that are specifically affected by the use of HGH and offers an extended window of detection.’

Unlike the existing immunoassays test – which measures macromolecules in a solution using antibodies – the LC-MS/ MS method can identify and quantify the presence of insulin-like growth factor, allowing for a detection window of weeks.

And yet, people will always be people
Despite the strides being made, the sporting establishment will probably always be swimming against the current. As Lance Armstrong and his alleged doping consigliere Dr Michele Ferrari have shown, millions of pounds can be made from cheating – riches that remain even after positive tests and admissions of guilt.

Anti-doping bodies will continue to foster close ties with pharmaceutical companies to stay wise to the latest illicit performanceenhancing substances. They will also rely on increasingly sensitive testing technology. As Nichols notes, many experts didn’t think an effective gene doping detection test could ever be developed. The HGH biomarkers test is testament to the progress made.

However, even if gene doping or illicit supplements are at risk of detection, the athlete has one last option – hard liquor. In 1998, testers accused Irish swimmer Michelle Smith de Bruin of handing them a tainted sample. But what gave this away? Did they need an advanced combination of gas chromatography and mass spectroscopy to figure it out? Perhaps specific immunoassays turned up something suspicious? Apparently, not. The sample smelled of whiskey.

How do gas chromatography and mass spectrometry work?
In gas chromatography, a urine or blood sample is vapourised in the presence of a gaseous solvent before being sent through the chromatographic column. Each substance within the sample dissolves differently and stays in the gaseous phase for a set time, which is known as the retention time. As each substance slips from the gaseous phase, it is absorbed into a solid or liquid for analysis. Gas chromatography can also be used for polymer analysis and in forensic science for paint chip inspection and arson investigations.

In mass spectroscopy, electron beams are used to break a sample into fragments. These fragments are accelerated through a magnetic tube to the detector, in which they are separated according to their mass. This technique enables researchers to identify and quantify the presence of drugs in a sample. It is also used in industry to assess oil composition and water quality.