The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non‐peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UHPLC/HRMS). A simple mixed‐mode cation exchange solid‐phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS²). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin‐releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).

中文翻译：

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使用超高效液相色谱-高分辨率质谱 (UHPLC/HRMS) 检测马尿中包括促性腺激素释放因子 (GnRH) 在内的生物活性肽。

近年来，在人类和动物运动中使用生物活性肽作为兴奋剂越来越受欢迎。因此，控制在马运动中滥用生物活性肽的方法受到了关注。本文描述了一种使用超高效液相色谱-高分辨率质谱法检测马尿中低 pg/mL 水平的 40 种生物活性肽和两种非肽生长激素促分泌素 (< 2 kDa) 的灵敏准确质量方法。 UHPLC/HRMS）。一个简单的混合模式阳离子交换固相萃取 (SPE) 小柱用于提取 42 个目标和/或它们的体外来自马尿的代谢物。使用 UHPLC/HRMS 在正电喷雾电离 (ESI) 模式下在全扫描和数据独立采集（DIA，对于 MS ²）下分析最终提取物。大多数目标的估计检测限 (LoD) 在马尿中可低至 10 pg/mL。该方法经验证可用于定性检测。报告了验证数据，包括方法特异性、方法灵敏度、提取回收率、方法精密度和基质效应。还使用从马肝脏中分离的 S9 组分对四种促性腺激素释放因子 (GnRH)，即亮丙瑞林、布舍瑞林、戈舍瑞林和那法瑞林进行了彻底的体外研究。体外鉴定代谢物已被纳入控制 GnRH 滥用的方法中。该方法的适用性通过对纯种马（阉割马）肌肉注射亮丙瑞林后尿液中亮丙瑞林及其代谢物之一 Leu M4 的鉴定得到证明。