Abstract：
BACKGROUND: To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine.
METHODS: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run.
RESULTS: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples.
CONCLUSIONS: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.
METHODS: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run.
RESULTS: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples.
CONCLUSIONS: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.
摘要：
背景：为了维护赛马和马术运动的完整性，马兴奋剂控制实验室必须开发一种全面的筛查方法，以覆盖马尿液中所需检测水平的多种目标物质。
方法：该程序涉及3mL尿液样品的酶促水解，然后使用HFBondElutC18柱进行固相萃取。然后将所得提取物在C18反相柱上分离,并使用液相色谱/高分辨率质谱(LC/HRMS)以电喷雾电离正模式和负模式在两个单独的注射中进行分析。在11分钟的LC运行中,以全扫描和产物离子扫描(PIS)模式获得分析数据。
结果：该方法可以检测1011种化合物（在正离子和负离子模式下）。超过95％的目标化合物的检测限（LOD）≤10ng/mL，超过50%的LODs≤0.5ng/mL。最低LOD可降至0.01ng/mL。通过成功检测国内外马尿样中的禁用物质，证明了该方法的适用性。
结论：我们已经成功开发了一种用于马尿液样本的常规筛查方法，该方法可以在正离子和负离子模式下通过全扫描和使用LC/HRMS的PIS检测1000多种亚ppb水平的化合物。此外，这种方法理论上可以扩展到在全扫描模式下容纳无限数量的违禁物质。
方法：该程序涉及3mL尿液样品的酶促水解，然后使用HFBondElutC18柱进行固相萃取。然后将所得提取物在C18反相柱上分离,并使用液相色谱/高分辨率质谱(LC/HRMS)以电喷雾电离正模式和负模式在两个单独的注射中进行分析。在11分钟的LC运行中,以全扫描和产物离子扫描(PIS)模式获得分析数据。
结果：该方法可以检测1011种化合物（在正离子和负离子模式下）。超过95％的目标化合物的检测限（LOD）≤10ng/mL，超过50%的LODs≤0.5ng/mL。最低LOD可降至0.01ng/mL。通过成功检测国内外马尿样中的禁用物质，证明了该方法的适用性。
结论：我们已经成功开发了一种用于马尿液样本的常规筛查方法，该方法可以在正离子和负离子模式下通过全扫描和使用LC/HRMS的PIS检测1000多种亚ppb水平的化合物。此外，这种方法理论上可以扩展到在全扫描模式下容纳无限数量的违禁物质。
