Chiara Francesco, Allegra Sarah, Liuzzi Simona, Puccinelli Maria Paola, Mengozzi Giulio, De Francia Silvia
Department of Physics, University of Trento, 38123 Povo, Italy.
Department of Clinical and Biological Sciences, University of Turin, 10043 Orbassano, Italy.
Life (Basel). 2025 May 22;15(6):829. doi: 10.3390/life15060829.
Urinary steroid profiling plays a key role in the diagnosis of inherited and acquired endocrine disorders. Despite the proven diagnostic value of gas chromatography-mass spectrometry (GC-MS), standardized and clinically validated protocols for extended steroid panels remain limited. We developed and validated a GC-MS method for the quantification of 32 urinary steroid metabolites, including androgens, estrogens, progestins, glucocorticoids, and mineralocorticoids. Sample preparation involved solid-phase extraction, enzymatic hydrolysis, and dual derivatization, followed by chromatographic separation and mass detection under full scan mode. Validation followed ICH M10 guidelines. The method demonstrated high selectivity, accuracy (within ±15%), and precision (CV% < 15%) across three QC levels. Limits of Quantification were estimated using the Hubaux-Vos approach and were suitable for detecting both physiological and pathological steroid concentrations. Robustness and matrix effect tests confirmed the method's reliability and reproducibility. This GC-MS protocol enables comprehensive urinary steroid profiling and calculation of diagnostic ratios for inborn errors of steroid metabolism and endocrine disorders. The method is suitable for clinical application and future integration into personalized medicine workflows.
尿甾体谱分析在遗传性和获得性内分泌疾病的诊断中起着关键作用。尽管气相色谱 - 质谱联用（GC-MS）已被证明具有诊断价值，但用于扩展甾体检测组的标准化且经过临床验证的方案仍然有限。我们开发并验证了一种GC-MS方法，用于定量32种尿甾体代谢物，包括雄激素、雌激素、孕激素、糖皮质激素和盐皮质激素。样品制备包括固相萃取、酶水解和双重衍生化，然后在全扫描模式下进行色谱分离和质谱检测。验证遵循ICH M10指南。该方法在三个质量控制水平上均表现出高选择性、准确性（±15%以内）和精密度（CV% < 15%）。使用Hubaux-Vos方法估计定量限，其适用于检测生理和病理甾体浓度。稳健性和基质效应测试证实了该方法的可靠性和可重复性。这种GC-MS方案能够实现全面的尿甾体谱分析，并计算甾体代谢先天性缺陷和内分泌疾病的诊断比率。该方法适用于临床应用以及未来整合到个性化医疗工作流程中。
