Abstract：
24-hour urinary free cortisol (UFC) is considered as the first-line test for screening and diagnosis of Cushing\'s syndrome. Although 24-hour UFC assay has been extensively studied by liquid chromatography-tandem mass spectrometry (LC-MS/MS), an accurate assay coupled with a reliable sample preparation procedure and a method-specific reference interval would be very important for reasonable diagnosis. In this study, a simple dilute and shoot method has been proposed for UFC determination by LC-MS/MS. Namely, 50 µL of urine sample was mixed with 200 µL of a 50 % methanol/water solution containing the internal standard cortisol-13C3. The mixture was centrifuged and the supernatant was used for direct analysis by LC-MS/MS. This method was validated with wide linear range from 0.625 to 500 ng/ml with coefficients of variation (CVs) ≤ 3.64 %, excellent precision (intra-day CVs ≤ 5.70 % and inter-day CVs ≤ 5.33 %) and good recovery in the range of 93.3-109 %. The preservatives were further evaluated for urine storage. It was recommended that no preservatives could be used in collection of 24-hour urine for good detecting peaks. The investigation of reference interval and diagnostic performance finally confirmed the potential usage of this LC-MS/MS assay in routing clinical testing.
摘要：
24小时尿游离皮质醇（UFC）被认为是筛查和诊断库欣综合征的一线测试。尽管已通过液相色谱-串联质谱（LC-MS/MS）对24小时UFC测定进行了广泛研究，准确的分析加上可靠的样品制备程序和特定方法的参考间隔对于合理的诊断非常重要.在这项研究中,提出了一种简单的稀释和射击方法，用于通过LC-MS/MS测定UFC。即,将50微升尿样与200微升含有内标皮质醇-13C3的50%甲醇/水溶液混合。将混合物离心,上清液用于LC-MS/MS直接分析。该方法在0.625至500ng/ml的宽线性范围内进行了验证，变异系数（CV）≤3.64％，良好的精度（日内CV≤5.70％和日间CV≤5.33％）和良好的恢复范围内93.3-109％。进一步评估防腐剂的尿液储存。建议在收集24小时尿液时不使用防腐剂以良好地检测峰。参考间隔和诊断性能的研究最终证实了该LC-MS/MS测定在路由临床测试中的潜在用途。
