Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high-resolution tandem mass spectrometry

IF 2.6 3区医学 Q2 BIOCHEMICAL RESEARCH METHODS

Drug Testing and Analysis Pub Date : 2023-11-02 DOI:10.1002/dta.3591

Katja Walpurgis, Aileen Gäde, Andreas Thomas, Soizic Gochard, Philippe Delahaut, Mario Thevis

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Abstract

The manipulation of blood and blood components in sports is prohibited at all times, and besides blood transfusions, also hemoglobin-based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. But while most drug candidates based on stabilized hemoglobin (Hb) were found to be characterized by serious side effects, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be another candidate for transfusion medicine and cheating athletes, as it was found to be well tolerated in preclinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration, tryptic digestion, and liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS). For the mass spectrometric identification of lugworm Hb, two precursor–product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T₆), hbB1 (T₃ and T₆), and the linker chain (T₁₆) were employed. The modified approach was comprehensively characterized and found to allow for the specific and sensitive detection of lugworm Hb down to concentrations of 10 μg/mL from 50 μL of serum/plasma. Therefore, it can serve as confirmation procedure for lugworm Hb following visual or electrophoretic screening. Moreover, a proof-of-concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 h (dose: 600 mg/kg) suggest that the approach can be readily employed to efficiently test in-competition doping control samples for the presence of the drug candidate.

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采用液相色谱法和高分辨率串联质谱法检测兴奋剂对照血清样品中的藻红细胞外血红蛋白。

在任何时候都禁止在运动中操纵血液和血液成分，除了输血外，还可以使用血红蛋白氧载体（HBOC）来人为提高血液的氧气输送能力。但是，尽管大多数基于稳定血红蛋白（Hb）的候选药物被发现具有严重副作用的特征，但来自海洋无脊椎动物海螺的天然巨大细胞外Hb可能是输血药物和作弊运动员的另一种候选药物，因为在临床前动物研究中发现它具有良好的耐受性。在该研究项目中，基于超滤、胰蛋白酶消化和液相色谱-高分辨率串联质谱（LC-HRMS/MS），将钩虫Hb纳入现有的牛HBOC兴奋剂控制检测方法中。为了对钩虫Hb进行质谱鉴定，使用了来源于亚基hbA2（T6）、hbB1（T3和T6）和连接链（T16）的总共四种胰蛋白酶肽的两个前体产物离子对。对改进的方法进行了全面的表征，发现可以特异而灵敏地检测浓度低至10 μg/mL从50 μL血清/血浆。因此，它可以作为在视觉或电泳筛查后对钩虫Hb的确认程序。此外，进行了概念验证大鼠给药研究，观察到至少4个（剂量：200 mg/kg）和8小时（剂量：600 mg/kg）表明该方法可以容易地用于在比赛中有效地测试兴奋剂控制样品中候选药物的存在。

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来源期刊

Drug Testing and Analysis BIOCHEMICAL RESEARCH METHODS-CHEMISTRY, ANALYTICAL

CiteScore

5.90

自引率

24.10%

发文量

191

审稿时长

2.3 months

期刊介绍： As the incidence of drugs escalates in 21st century living, their detection and analysis have become increasingly important. Sport, the workplace, crime investigation, homeland security, the pharmaceutical industry and the environment are just some of the high profile arenas in which analytical testing has provided an important investigative tool for uncovering the presence of extraneous substances. In addition to the usual publishing fare of primary research articles, case reports and letters, Drug Testing and Analysis offers a unique combination of; ‘How to’ material such as ‘Tutorials’ and ‘Reviews’, Speculative pieces (‘Commentaries’ and ‘Perspectives'', providing a broader scientific and social context to the aspects of analytical testing), ‘Annual banned substance reviews’ (delivering a critical evaluation of the methods used in the characterization of established and newly outlawed compounds). Rather than focus on the application of a single technique, Drug Testing and Analysis employs a unique multidisciplinary approach to the field of controversial compound determination. Papers discussing chromatography, mass spectrometry, immunological approaches, 1D/2D gel electrophoresis, to name just a few select methods, are welcomed where their application is related to any of the six key topics listed below.