通过超高效液相色谱—四极杆-静电场轨道阱高分辨质谱联用仪对人尿中42种小肽类禁用物质进行兴奋剂检测

Detection of 42 Prohibited Small Peptides in Human Urine via Ultra-high Performance Liquid Chromatography Coupled with Hybrid Quadrupole-Orbitrap Mass Spectrometer in Doping Control

目的:通过采用超高效液相色谱—四极杆-静电场轨道阱高分辨质谱联用仪(Q Exactive Plus UHPLC/HRMS),建立对人尿中42种小肽类禁用物质的检测方法。被检测的小肽类禁用物质涵盖了世界反兴奋剂机构(WADA)禁用清单中的生长激素促分泌剂类(GHS)、生长激素释放肽类(GHRP)、促性腺激素释放因子(GnRHs)、人类生长激素(h GH)、生长因子以及生长因子调节剂等分类的定性物质、片段及代谢物,还包括了两种利尿剂去氨加压素(Desmopressin)和苯赖加压素(Felypressin)及其代谢物。方法:使用直接进样方法对尿液样本进行预处理,取待测样品加入内标溶液涡旋混匀,以10000 r/min离心10分钟后。取上清液与稀释溶液混匀,在超高效液相色谱-Q Exactive Plus组合型四极杆Orbitrap质谱仪上进行分析。结果:采用全扫描(Full Scan)和平行反应监测(PRM)模式对含有42种小肽类禁用物质及内标的阳性质量控制样品(QCP)进行定性分析。在最低要求检测浓度(MRPL)下,该方法均可以正确检出尿样中的小肽类目标化合物(其信噪比S/N>3),可满足WADA的相关技术文件的要求。结论:该方法通过直接进样的前处理方法,使得样品前处理程序变得简单、高效,同时因为采用了Q Exactive Plus UHPLC/HRMS,尽可能多地保留了样品检测的灵敏度和特异性,从而使得该方法的灵敏度、特异性以及检出限等均可满足WADA对此类药品检测能力的要求。

ObjectiveTo establish a method to detect 42 prohibited small peptides in human urine for doping control using the ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap mass spectrometer(Q Exactive Plus UHPLC/HRMS). The prohibited small peptides to be detected cover the qualitative substances, fragments and metabolites of the growth hormone secretagogues, growth hormone-release peptides, gonadotrophin-releasing factors, human growth hormone,growth factors and growth factor modulators in the World Anti-Doping Agency(WADA) prohibited list,in addition to two diuretics of desmopressin and felypressin, as well as their metabolites.Methods The urine samples were pretreated by"dilute-and-shoot"method,and the sample to be tested was added with the internal standard solution before vortex mixing,and centrifuged at 10000 r/min for 10 minutes. The supernatant was then mixed with diluted solution and analyzed by using the Q Exactive Plus UHPLC/HRMS.ResultsPositive quality control samples(QCP) containing 42 prohibited small peptides and internal standard were conducted a qualitative analysis in the full scan and parallel reaction monitoring(PRM) mode. At the concentration of minimum required performance levels, the target small peptide compounds could be detected correctly in human urine with the signal-to-noise ratio greater than 3, which met the requirements of relevant technical documents of WADA.Conclusion The pretreatment of"dilute-and-shoot"method is simple and efficient. Meanwhile,by using Q Exactive Plus UHPLC/HRMS,the sensitivity and specificity of the assay are maintained as much as possible. Therefore,the sensitivity,specificity and limit of detection of this assay can meet the WADA detection requirements for these prohibited substances.