Analytical Challenges in Doping Detection

As new compounds and masking agents continue to emerge, analytical methods must evolve to enable their detection

Michelle Dotzert, PhD

The First World Conference on Doping in Sport was held in Lausanne, Switzerland in 1999. The Lausanne Declaration on Doping in Sport was produced and subsequently, the World Anti-Doping Agency (WADA) was established. WADA’s mission is to “lead a collaborative worldwide movement for doping-free sport”.

The World Anti-Doping Code International Standard Prohibited List

Prohibited Substances: Performance Effects and Health Risks

Anabolic androgenic steroids 

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives. They may be administered orally, as intramuscular injections, or topically as gels or creams. AAS contribute to increased muscle mass, an advantage for athletes in sports that require strength and high levels of peak power, such as weightlifting, throwing, and sprinting.  AAS also increase red blood cell mass and hematocrit, improving oxygen delivery to working muscle, an advantage for endurance athletes. 

Potential health risks:

  • Elevated LDL and reduced HDL-C
  • Increased blood pressure
  • Left ventricular hypertrophy
  • Altered liver function
  • Mental health effects

Peptide hormones, growth factors, and related substances

Erythropoietin (EPO) promotes erythrocyte production for enhanced oxygen delivery to working muscles, and subsequently, improved VO2max. Recombinant rhEPO is difficult to detect, as it has the same amino acid sequence as endogenous EPO.

Potential health risks:

  • Increased blood viscosity as a result of increased red blood cell mass may increase the risk of hypertension, headache, and thrombosis.

Human chorionic gonadotropin (hCG) and luteinizing hormone (LH) act on Leydig cells to stimulate testosterone production in males. Unlike exogenous testosterone, hCG and LH increase the production of testosterone and epitestosterone, preserving the naturally occurring ratio. In females, endogenous hCG and LH stimulate progesterone and estradiol production by the ovaries, but do not increase testosterone levels and are therefore not part of routine testing. 

Growth hormone (GH) and insulin-like growth factor 1 (IGF-1) influence cell division, amino acid uptake, and protein synthesis. GH and IGF-1 are often used by bodybuilders, weightlifters, or athletes in sports that require strength and power. Use of GH and IGF-1 by endurance athletes is thought to enhance performance through glycogen-sparing effects.  

Potential health risks:

  • Fluid retention
  • Edema
  • Headache
  • Hypertension
  • Aspects of acromegaly
  • Increased risk of cardiomyopathy
  • Arrhythmia
  • Insulin resistance
  • Renal failure

Hormonal modulators

Aromatase inhibitors and selective estrogen receptor modulators (SERMs) have different effects in males and females. In males, estradiol is produced by testosterone aromatization, and is crucial for regulating testosterone production via the hypothalamic-pituitary-thyroid axis. Estrogen blocking drugs may increase pituitary gonadotropin secretion, contributing to increased testosterone and a potential increase in muscle mass or strength. In females, such compounds are unlikely to increase testosterone. 

Potential health risks:

  • Arrhythmia
  • Dizziness
  • Joint pain

Metabolic modulators

Peroxisome proliferator activated receptor δ (PPAR- δ) agonists have been shown to increase myocardial contractility, and regulate fatty acid transport and oxidation in slow-twitch muscle fibers, thereby improving endurance. The long-term health risks have not yet been determined. 

Masking agents

Masking agents do not improve performance, but are often used to hide other doping agents. Diuretics are used to increase urine production and sodium excretion, thereby lowering the concentration of doping agents in a sample. Diuretics may also alter urinary pH and prevent certain drugs from being excreted in urine. Plasma expanders exert similar effects in the blood, especially to mask the use of EPO, by diluting blood to reduce elevated hematocrit/hemoglobin.

Analytical Challenges in Doping Detection and Improved Methodology

Sensitivity

Doping strategies continue to evolve in attempts to elude detection. A major analytical challenge is increasing the sensitivity of analytical methods without compromising the specificity. 

Coupling gas or liquid chromatography to mass spectrometry creates a highly sensitive technique. Triple-quadrupole (QqQ) systems further improve capabilities, and those with time-of-flight high-resolution mass spectrometers (TOF-MS) or high-resolution orbitrap mass analyzers enable detection of long-term metabolites of various steroids. 

The identification of long-term metabolites of steroids has significantly prolonged detection time. Oxymesterone, for example is a synthetic anabolic androgenic steroid. The identification of two metabolites (17α-methyl-3,17β-dihydroxy-5α-androstane-4-one and 17α-methyl-3α,4β,17β-trihydroxy-5α-androstane) increased detection time to 4 days compared to 2.25 days for the parent compound.

Time constraints and multiple compounds

With a wide variety of target compounds, there is a need for a rapid method that is capable of detecting many different compounds in a single experiment. A liquid chromatography coupled to quadrupole Orbitrap mass spectrometry (LC/Q/Orbitrap MS) platform is being validated for screening urine samples. The system enables targeted and untargeted analyses in a single, rapid, chromatographic run. 

Proteases

Proteases have been used to degrade and subsequently mask detection of several drugs such as EPO and peptide hormones in biological samples. New approaches aim to identify the degradation products that result from exogenous proteases.  A combination of SDS-PAGE and capillary liquid chromatography-Orbitrap (tandem) MS may be employed to identify these products in urine samples. 

Isoforms

Human growth hormone (hGH) is present mainly as a 22 kDa isoform and a 20 kDa isoform. Recombinant GH (rGH) is also a 22 kDa protein with an identical amino acid sequence as circulating GH, posing challenges for detection. There are two approaches to detecting GH abuse, the isoform method and the biomarker method.

Isoform method: one immunoassay is used to detect the 22 kDa isoform, and another is used to detect a combination of isoforms. Since rGH is the 22 kDa isoform, it will disrupt the ratio of this isoform to all others.

Biomarker method: this approach quantifies downstream effects, including IGF-1 and pro-collagen type III amino-terminal pro-peptide (P-III-NP), and the results are entered into a formula factoring in age and gender.


Michelle Dotzert, PhD

Michelle obtained her PhD in Kinesiology from the University of Western Ontario. Her research examined the effects of exercise training on skeletal muscle lipid metabolism and insulin resistance in the context of Type 1 Diabetes.