measurement of steroids in sports doping

An article by Alan D. Brailsford and WC Chang et al at the Drug Control Centre and two other institutions, published in August 2020 in the Journal of Chromatography A, described the development of a rapid GC-MS/MS study to measure anabolic androgenic steroids from Mitra^® microsampling devices based on VAMS^® technology. The paper is entitled “Determination of anabolic steroids in dried blood using microsampling and gas chromatography-tandem mass spectrometry: Application to a testosterone gel administration study.”

The study focused on simultaneously measuring nine anabolic androgenic steroids in 6.4 minutes and then applying this method to VAMS samples. The samples were collected from athlete volunteers to measure the detection of a testosterone gel. The authors concluded that VAMS DB could be employed for quantifying blood T [testosterone] level in agreement with using the serum specimen.

Sports Doping & the World Antidoping Agency

The World Antidoping Agency (WADA) was formed in 1999 after a major doping scandal hit the world of competitive cycling in 1998. Since the formation of WADA, the list of banned substances in competitive sports has expanded to include peptide hormones and related substances, beta-2-agonists, hormone and metabolic modulators, diuretics and masking ingredients, stimulants, narcotic cannabinoids, EPO (erythropoietin) and glucocorticoids.

The most famous class of banned substances are anabolic agents, where the vast majority are anabolic androgenic steroids (AAS). According to a WADA report published in 2018, misuse of AAS account for 44% of adverse analytical findings. Although AAS act to enhance performance, they also can cause long-term health issues for those who take them.

Indeed, they can adversely affect many areas of the body, potentially causing damage to the cardiovascular system, hormonal system, liver (e.g., tumor formation), and musculoskeletal system. Misuse of AAS also can cause psychiatric effects such as aggression, infections such as HIV from using shared needles, and skin problems such as severe acne.

Monitoring Androgenic Steroid Doping

In an attempt to avoid detection, some dopers use micro-dosing regimens whereby they take smaller doses of steroids and other banned substances that make detection more challenging. One micro-dosing strategy is to employ transdermal doping, such as applying hormone gels. For labs trying to effectively detect banned substances, whole blood, serum or plasma are preferred matrices for quantitative analysis. These are considered the best matrices for correlation with physiological responses. However, traditional venous blood collection using needle draws is invasive and requires phlebotomists to perform the sample collection.

On the other hand, urine sampling is non-invasive and samples can be self-collected. For these reasons, urine is currently the most popular method for sample collection in sports doping testing. However, urine collection poses its own challenges. Since an official needs to be present during sample collection, athletes find the process personally intrusive. Urine collection is also time consuming, and it can be challenging to efficiently store large volumes of liquid samples.

According to the study paper by Brailsford and Chang et al reviewed here, dried blood spots (DBS) offer a potential solution to overcome the challenges of venous blood sampling and urine sampling. However, traditional DBS cards also present some issues surrounding sample quality and hematocrit effects that limit their use in anti-doping screening. The study authors report in previous work that extraction recoveries of steroids from dried blood spots are low (~20%).

Due to the limitations of DBS, the research group chose to develop a method on Mitra-VAMS devices because they solve many of the drawbacks observed with DBS filter cards.

Sports Doping Study Methods and Findings

• The following steroids were used for the study: testosterone, nandrolone, boldenone, mesterolone, drostanolone, metenolone, metandienone, oxandrolone, and DHCMT.

• A rapid GC-MS/MS was developed, which improved upon the throughput of previous methods. Indeed, all nine targets were successfully identified in less than 6.4 minutes.
  
  
• For a control, steroid free blood was prepared by washing blood cells and introducing these to steroid-free plasma.
  
  
• Extraction from 20 µL VAMS tips employed a liquid-liquid extraction technique, using a water and MTBE mix using ultrasound, rotary mixing and centrifugation.
  
  
• The analytical method was validated in accordance with international guidelines from the Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). Highlights are outlined below:
  
  
  • Most analytes passed precision and accuracy except for drostanolone (23.1% RSD @ the low level) and DHCMT (2.25% RSD @ the high level) during inter-day precision.
    
  • All analytes give calibration curves of r² = >0.999 within the concentration ranges with acceptable biases.
    
    
  • The method could confidentially measure T in healthy men (plasma range = 2.8-13.2ng mL^-1) but was not sensitive enough to measure normal female levels (plasma range = 0.1-07 ng mL^-1).
    
    
  • Extraction recoveries were between 43% to 89%.
    
    
  • Comparison of wet serum to dried wet blood was conducted from 40 venous blood samples. Using hematocrit values, the group were able to adjust for the hematocrit portion. Good agreement was shown by Deming regression and Bland-Altman analysis, so the group concluded that VAMS extracts were as suitable as serum for T determination.
    
    
• Healthy male volunteers were chosen to pilot the assay. They were screened to ensure they were not participating in competitive sporting events (or testing pools), were not taking any substances, and did not have any health conditions that might affect the results of the study.
  
  
• The cohorts were split into 3 groups and then capillary blood was collected using Mitra devices with 20 µL VAMS tips.
  
  
• Study volunteers collected samples over 6 timepoints (0, 9, 24, 48, 72 and 96 hr). The volunteers could take part several times in more than one cohort, as long as there was a week’s washout between tests.
  
  
  • Group 1 – One dose of a placebo gel (n = 7)
    • Samples were at normal T levels (<30 ng mL^-1).
      
      
  • Group 2 – One dose of testosterone (Tostran®) gel (50 mg, n=7)
    • Peak T was at 9h (mean C_max = 91.2 ng mL⁻¹) although some barely hit 30 ng mL^-1 at this timepoint, which showed a high inter-subject variability. It was concluded that this could be due to differences in rate of absorption and metabolism from individual to individual.
      
      
  • Group 3- One dose of Tostran^® gel (100 mg, n=7)
    • Peak T was also at 9h (mean C_max = (234.9 ng mL⁻¹). Also some subjects did not fully return to baseline even after 96h. If a cut-off of 30 ng mL⁻¹ would be applied, then a detection window could be up to 48h after dosing.

Study Authors’ Conclusions

• The study validated a reliable and promising approach to measuring 9 AAS from VAMS samples.
  
  
• The new GC-MS/MS method rapidly (6.4 min runtime) detects as low as 0.10 ng mL⁻¹ in 20 μL VAMS samples.
  
  
• Extraction recoveries from VAMS are superior to DBS.
  
  
• Good agreement seen between serum and VAMS samples.
  
  
• The method is a sensitive one for measuring microdosing of pseudo-endogenous T.
  
  
• The assay is being expanded to a large cohort study.

Neoteryx Comments

In 2021, the WADA executive committee approved a technical document for the employment of innovative dried blood spot (DBS) testing in sports. This was as a result of the WADA technical document, which was based on the findings of several studies that found dried blood could detect doping, WADA approved the method for preliminary use at the Tokyo Olympics.

As evidenced by the paper reviewed here, the quantitative nature of Mitra devices with VAMS and their ease of use compared to DBS, as well as their excellent extraction recovery demonstrate their applicability in sports doping testing. A related study we reviewed in a recent blog shows that not only is VAMS suitable for analysis of small molecules but also for large molecules, such as transgene DNA in sports doping.

Where tamper resistant sampling is desired, the hemaPEN^® is a good option. This portable device rapidly collects 4 homogenous DBS samples from a single source, and locks shut after sampling, making it ideal for remote dried blood sampling in sports. A recent study was successful in using hemaPEN to measure metabolomic biomarkers from athletes, further demonstrating the utility of this sampler in sports testing.

This article was summarized for our readers by James Rudge, PhD, Neoteryx Technical Director. This is curated content. To learn more about the important research outlined in this blog, visit the original article in the Journal of Chromatography A.

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