Screening and confirmation of recombinant human follistatin in equine plasma for doping control purposes,Drug Testing and Analysis

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Screening and confirmation of recombinant human follistatin in equine plasma for doping control purposes
Drug Testing and Analysis ( IF 2.6 ) Pub Date : 2023-07-04 , DOI: 10.1002/dta.3540
Kin-Sing Wong ₁ , Hiu Wing Cheung ₁ , Yung-Ching Choi ₁ , Ning-Sum To ₁ , Terence S M Wan ₁ , Emmie N M Ho ₁

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Recombinant human follistatin (rhFST) is a potential performance-enhancing agent owing to its stimulating effect on muscle growth. Administration of rhFST to athletes is prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horseracing Authorities (IFHA). For effective control of the potential misuse of rhFST in flat racing, methods for screening and confirmatory analysis are required. This paper describes the development and validation of a complete solution for detecting rhFST and confirming its presence in plasma samples collected from racehorses. A high-throughput analysis of rhFST with a commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for the screening of equine plasma samples. Any suspicious finding would then be subjected to a confirmatory analysis using immunocapture, followed by nano-liquid chromatography/high-resolution tandem mass spectrometry (nanoLC-MS/HRMS). The confirmation of rhFST by nanoLC-MS/HRMS was achieved by comparing the retention times and relative abundances of three characteristic product-ions with those from the reference standard in accordance with the industry criteria published by the Association of Official Racing Chemists. The two methods achieved comparable limit of detection (~2.5–5 ng/mL) and limit of confirmation (2.5 ng/mL or below), as well as adequate specificity, precision and reproducibility. To our knowledge, this is the first report of the screening and confirmation methods for rhFST in equine samples.

中文翻译：

用于兴奋剂控制的马血浆中重组人卵泡抑素的筛选和确认

重组人卵泡抑素（rhFST）由于其对肌肉生长的刺激作用而成为一种潜在的性能增强剂。根据国际赛马机构联合会 (IFHA) 发布的《国际育种、赛马和投注协议》第 6 条，世界反兴奋剂机构 (WADA) 禁止在人类体育运动中对运动员使用 rhFST，并禁止在赛马运动中对运动员使用 rhFST。为了有效控制 rhFST 在平地赛车中潜在的滥用，需要筛选和验证分析的方法。本文描述了用于检测 rhFST 并确认其在赛马血浆样本中存在的完整解决方案的开发和验证。使用市售酶联免疫吸附测定 (ELISA) 对 rhFST 进行高通量分析，以筛选马血浆样本。然后，任何可疑的发现都将使用免疫捕获进行验证性分析，然后进行纳米液相色谱/高分辨率串联质谱（nanoLC-MS/HRMS）。根据官方赛车化学家协会发布的行业标准，通过将三种特征产物离子的保留时间和相对丰度与参考标准的保留时间和相对丰度进行比较，通过 nanoLC-MS/HRMS 对 rhFST 进行确认。这两种方法实现了相当的检测限（~2.5–5 ng/mL）和确认限（2.5 ng/mL 或更低），以及足够的特异性、精密度和重现性。据我们所知，这是马样本中 rhFST 筛选和确认方法的第一份报告。

更新日期：2023-07-04

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