Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
Dipartimento di Medicina Sperimentale, "Sapienza" Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy.
Anal Bioanal Chem. 2019 Dec;411(30):8153-8162. doi: 10.1007/s00216-019-02203-4. Epub 2019 Dec 3.
This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra, Humalog, Levemir, NovoRapid, and Tresiba) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba and/or Humalog, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.
本工作描述了一种分析程序，基于自动化亲和纯化，随后进行液相色谱-电喷雾串联质谱分析，采用常规三重四极杆分析器，以检测人尿中的合成胰岛素（Apidra、Humalog、Levemir、NovoRapid 和 Tresiba）。样品制备包括超滤，随后在整体式微柱上进行免疫亲和纯化。色谱分离采用 C18 微柱进行，而分析物的质谱鉴定则通过三重四极杆质谱仪在正离子电喷雾电离和选择反应监测采集模式下实现。通过为每个分析物选择至少两个特征离子跃迁来进行合成胰岛素的鉴定。新开发的方法在特异性、回收率、基质效应、灵敏度、稳健性和保留时间以及相对离子跃迁丰度的重复性方面进行了验证。证实了新方法的特异性和相对保留时间以及所选特征离子跃迁的相对丰度的重现性适用于确保所有目标分析物的明确鉴定，包括在法医领域。提取产率估计大于 60%，基质效应小于 35%。检测限下限在 0.02-0.05 ng/mL 范围内，证明该方法足够灵敏，可用于检测作为运动中增强表现的药物滥用胰岛素。通过分析接受 Tresiba 和/或 Humalog 治疗的糖尿病患者的尿液样本，评估了所开发方法的适用性，在给予治疗剂量后收集的尿液样本中证实了这些胰岛素的存在。
