Mazzarino Monica, Perretti Ilaria, Stacchini Carlotta, Comunità Fabio, de la Torre Xavier, Botrè Francesco
Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, Rome 00197, Italy.
Dipartimento di Chimica e Tecnologia del Farmaco, 'Sapienza' Università di Roma, Piazzale Aldo Moro 5, Rome 11085, Italy.
J Anal Toxicol. 2021 Feb 13;45(2):184-194. doi: 10.1093/jat/bkaa055.
This article presents newly developed screening and confirmation analytical procedures to detect the misuse of nine prolyl-hydroxylase inhibitors of the hypoxia-inducible factor: daprodustat, desidustat, FG2216, IOX2, IOX4, JNJ-42041935, molidustat, roxadustat and vadadustat, targeting either the parent drugs and/or their main metabolite(s). For the sample pretreatment, different extraction protocols and technologies were evaluated. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. The chromatographic separation was performed on a C18 column, employing water and acetonitrile, both containing 0.1% formic acid, as mobile phase. Detection was achieved using as analyzer either a triple quadrupole or an Orbitrap, with positive and negative electrospray ionization and different acquisition modes. Validation of the procedures was performed according to the ISO 17025 and World Anti-Doping Agency guidelines. The methods do not show any significant interference at the retention times of the analytes of interest. The extraction efficiency was estimated to be greater than 75% for all analytes and the matrix effect smaller than 35%. Detection capability was determined in the range of 0.25-2.0 for the screening procedure and in the range of 0.5-2.0 ng/mL for the confirmation procedure, that is, in a range of concentration small enough to reveal the abuse of the compounds considered, in case they are used as performance-enhancing agents. The repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the selected ion transitions, considered only in the case of triple quadrupole (CV% < 15), was confirmed to be fit for purpose to ensure the unambiguous identification of all the target analytes in human urine. The applicability of the newly developed methods was verified by the analysis of urine samples containing molidustat, roxadustat or daprodustat. The developed procedures enabled to detect the compounds under investigation and their main metabolites.
本文介绍了新开发的筛查和确证分析程序，用于检测九种缺氧诱导因子脯氨酰羟化酶抑制剂的滥用情况，这些抑制剂包括达普司他、地西司他、FG2216、IOX2、IOX4、JNJ - 42041935、莫利司他、罗沙司他和伐达司他，目标是母体药物和/或其主要代谢物。对于样品预处理，评估了不同的提取方案和技术。仪器分析通过超高效液相色谱与高分辨率或低分辨率质谱联用进行。色谱分离在C18柱上进行，使用均含有0.1%甲酸的水和乙腈作为流动相。使用三重四极杆或Orbitrap作为分析仪进行检测，采用正离子和负离子电喷雾电离以及不同的采集模式。根据ISO 17025和世界反兴奋剂机构的指南对程序进行了验证。这些方法在目标分析物的保留时间处未显示任何明显干扰。所有分析物的提取效率估计大于75%，基质效应小于35%。筛查程序的检测能力在0.25 - 2.0范围内，确证程序的检测能力在0.5 - 2.0 ng/mL范围内，即浓度范围足够小，足以揭示所考虑化合物作为性能增强剂被滥用的情况。仅在三重四极杆的情况下，相对保留时间（CV% < 0.5）和所选离子跃迁相对丰度的重复性（CV% < 15）被确认为符合目的，以确保在人尿中明确鉴定所有目标分析物。通过分析含有莫利司他、罗沙司他或达普司他的尿液样本，验证了新开发方法的适用性。所开发的程序能够检测所研究的化合物及其主要代谢物。
