Multiplexed adrenal steroid measurement provides critical diagnostic information for patients with congenital adrenal hyperplasia (CAH) as confirmation of newborn screening (NBS) or as initial diagnosis. This study reports the implementation of an adrenal steroid profiling method with a turnaround time (TAT) of less than 24 h using liquid chromatography and tandem-mass spectrometry (LC-MS/MS). A lab-developed multiplexed LC-MS/MS assay was used to quantify levels of 11-deoxycortisol, cortisol, 17-hydroxy-progesterone (17-OHP), androstenedione, and testosterone. Intra and interassay imprecision were found to be <10%. Comparison with a reference laboratory revealed <20% bias for all 5 analytes and Deming correlation coefficients >0.990. Linearity ranges were established from the lowest to upper limit calibrator concentrations with 100- to 800-fold maximum dilution. Run to run carryover was <0.1%, and acceptable matrix effect was observed (i.e., ion suppression enhancement <15%). Compared to serum samples, ethylenediaminetetraacetic acid (EDTA) and heparin plasma had large positive bias in the measurement of 11-deoxycortisol (62.2% and 60.2%, respectively) and androstenedione (43.8% and 33.2%, respectively), while cortisol, 17-OHP and testosterone showed less than 20% bias between sample types. Hemoglobin, bilirubin, or triglyceride interference decreased 11-deoxycortisol measurement in EDTA plasma (−19.3%, −25.6%, and −25.0%, respectively). Lipemia increased the measurement of testosterone by 28.9%. In summary, our multiplexed LC-MS/MS method provided highly sensitive and specific measurement of adrenal steroids. EDTA, heparin, hemolysis, icterus and/or lipemia may significantly impact assay results and should be avoided. This method provides an effective strategy for improving TAT in CAH testing and confirmation of NBS results.

多重肾上腺类固醇测量为先天性肾上腺增生 (CAH) 患者提供重要的诊断信息，作为新生儿筛查 (NBS) 的确认或初步诊断。本研究报告了使用液相色谱和串联质谱 (LC-MS/MS) 实现的肾上腺类固醇分析方法，周转时间 (TAT) 小于 24 小时。使用实验室开发的多重 LC-MS/MS 测定来定量 11-脱氧皮质醇、皮质醇、17-羟基孕酮 (17-OHP)、雄烯二酮和睾酮的水平。测定内和测定间不精确度为 <10%。与参考实验室的比较显示，所有 5 种分析物均存在 <20% 偏差，戴明相关系数为 >0.990。以 100 至 800 倍最大稀释度建立从最低到上限校准浓度的线性范围。运行间残留为<0.1%，观察到可接受的基质效应（即离子抑制增强<15%）。与血清样本相比，乙二胺四乙酸（EDTA）和肝素血浆在11-脱氧皮质醇（分别为62.2％和60.2％）和雄烯二酮（分别为43.8％和33.2％）的测量中具有较大的正偏差，而皮质醇，17- OHP 和睾酮在样本类型之间的偏差小于 20%。血红蛋白、胆红素或甘油三酯干扰降低了 EDTA 血浆中 11-脱氧皮质醇的测量值（分别为 -19.3%、-25.6% 和 -25.0%）。脂血症使睾酮测量值增加 28.9%。总之，我们的多重 LC-MS/MS 方法提供了肾上腺类固醇的高度灵敏和特异性测量。 EDTA、肝素、溶血、黄疸和/或血脂可能会显着影响测定结果，应避免。 该方法为提高CAH测试中的TAT和NBS结果的确认提供了有效的策略。