Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, Würzburg, Germany.
Central Laboratory, Core Unit Clinical Mass Spectrometry, University Hospital Würzburg, Würzburg, Germany.
Clin Chem Lab Med. 2023 Nov 28;62(5):919-928. doi: 10.1515/cclm-2023-0250. Print 2024 Apr 25.
Primary aldosteronism is the most common cause of endocrine hypertension and is associated with significant cardiovascular morbidities. The diagnostic workup depends on determinations of plasma aldosterone and renin which are highly variable and associated with false-positive and false-negative results. Quantification of aldosterone in 24 h urine may provide more reliable results, but the methodology is not well established. We aimed to establish an assay for urinary aldosterone and related steroids with suitability for clinical routine implementation.
Here, we report on the development and validation of a quantitative LC-MS/MS method for six urinary steroids: aldosterone, cortisol, 18-hydroxycorticosterone, 18-hydroxycortisol, 18-oxocortisol, tetrahydroaldosterone. After enzymatic deconjugation, total steroids were extracted using SepPak tC18 plates and quantified in positive electrospray ionization mode on a QTRAP 6500+ mass spectrometer.
Excellent linearity was demonstrated with R>0.998 for all analytes. Extraction recoveries were 89.8-98.4 % and intra- and inter-day coefficients of variations were <6.4 and <9.0 %, establishing superb precision. Patients with primary aldosteronism (n=10) had higher mean 24 h excretions of aldosterone-related metabolites than normotensive volunteers (n=20): 3.91 (95 % CI 2.27-5.55) vs. 1.92 (1.16-2.68) µmol/mol for aldosterone/creatinine, 2.57 (1.49-3.66) vs. 0.79 (0.48-1.10) µmol/mol for 18-hydroxycorticosterone/creatinine, 37.4 (13.59-61.2) vs. 11.61 (10.24-12.98) µmol/mol for 18-hydroxycortisol/creatinine, 1.56 (0.34-2.78) vs. 0.13 (0.09-0.17) µmol/mol for 18-oxocortisol/creatinine, and 21.5 (13.4-29.6) vs. 7.21 (4.88-9.54) µmol/mol for tetrahydroaldosterone/creatinine.
The reported assay is robust and suitable for routine clinical use. First results in patient samples, though promising, require clinical validation in a larger sample set.
原发性醛固酮增多症是内分泌性高血压最常见的病因，与显著的心血管发病率相关。诊断工作依赖于血浆醛固酮和肾素的测定，而这些指标的变异性较大，且与假阳性和假阴性结果有关。24 小时尿液中醛固酮的定量可能提供更可靠的结果，但该方法尚未得到很好的确立。我们旨在建立一种适合临床常规应用的用于尿液醛固酮和相关类固醇的测定方法。
在此，我们报告了一种用于六种尿液类固醇：醛固酮、皮质醇、18-羟皮质酮、18-羟皮质醇、18-氧皮质醇、四氢醛固酮的 LC-MS/MS 定量方法的开发和验证。在酶去共轭后，使用 SepPak tC18 板提取总类固醇，并在 QTRAP 6500+质谱仪上以正电喷雾电离模式定量。
所有分析物的线性均极好，R>0.998。提取回收率为 89.8-98.4%，日内和日间变异系数均<6.4%和<9.0%，表明精密度极好。与 20 名血压正常的志愿者相比，原发性醛固酮增多症患者（n=10）的 24 小时尿液中相关代谢物的平均排泄量更高：醛固酮/肌酐为 3.91（95%CI 2.27-5.55）vs. 1.92（1.16-2.68）µmol/mol，18-羟皮质酮/肌酐为 2.57（1.49-3.66）vs. 0.79（0.48-1.10）µmol/mol，18-羟皮质醇/肌酐为 37.4（13.59-61.2）vs. 11.61（10.24-12.98）µmol/mol，18-氧皮质醇/肌酐为 1.56（0.34-2.78）vs. 0.13（0.09-0.17）µmol/mol，四氢醛固酮/肌酐为 21.5（13.4-29.6）vs. 7.21（4.88-9.54）µmol/mol。
所报道的方法稳健，适合临床常规使用。尽管初步结果很有前景，但在更大的样本量中仍需要进行临床验证。
