Growth hormone-releasing hormone and its analogues sermorelin, tesamorelin and CJC-1295 are included in the prohibited list of the World Antidoping Agency. These target peptides are found at very low concentrations in urine (at the pg/mL level). For this reason, hyphenated enrichment and purification steps prior to mass spectrometric detection are required. Among different strategies, immunopurification based on magnetic beads is an excellent alternative, as it offers improved selectivity when the immunoreactivity and orientation of the antibody are optimum and non-specific adsorption is minimized. However, choosing the magnetic bead surface functionalities that provide the best recoveries is not so straightforward. In this work, we have evaluated the suitability of magnetic beads with different supports, binding capacities and affinity chemistries prior analysis of human urine samples by liquid chromatography coupled to high resolution mass spectrometry using a Quadrupole-Orbitrap instrument. After optimization of the immunopurification protocol with the magnetic beads that provided better recoveries, the method was fully validated and found to be adequate considering the parameters specificity, intra- and inter-day precision (lower than 15 and 25%, respectively), matrix effect, limit of detection (0.2 ng/mL) and limit of identification (0.5 ng/mL).

生长激素释放激素及其类似物sermorelin、tesamorelin和CJC-1295被列入世界反兴奋剂机构禁用名单。这些目标肽在尿液中的浓度非常低（pg/mL 水平）。因此，需要在质谱检测之前进行连字符富集和纯化步骤。在不同的策略中，基于磁珠的免疫纯化是一个很好的选择，因为当抗体的免疫反应性和方向最佳且非特异性吸附最小化时，它提供了更高的选择性。然而，选择提供最佳回收率的磁珠表面功能并不是那么简单。在这项工作中，我们评估了具有不同载体的磁珠的适用性，结合能力和亲和化学分析之前，使用 Quadrupole-Orbitrap 仪器通过液相色谱与高分辨率质谱联用对人尿样品进行分析。在使用提供更好回收率的磁珠优化免疫纯化方案后，该方法得到了充分验证，并发现考虑到参数特异性、日内和日间精密度（分别低于 15% 和 25%）、基质效应，该方法是足够的、检测限 (0.2 ng/mL) 和鉴定限 (0.5 ng/mL)。