Huang Weili, Kalhorn Thomas F, Baillie Mark, Shen Danny D, Thummel Kenneth E
Department of Pharmaceutics, University of Washington, Seattle, Washington, USA.
Ther Drug Monit. 2007 Apr;29(2):215-24. doi: 10.1097/FTD.0b013e31803d14c0.
Cortisol is an important adrenal steroid hormone involved in the regulation of metabolic homeostasis. A new liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) multiple reactant monitoring (MRM) procedure for the measurement of cortisol concentration in plasma ultrafiltrate, whole plasma, and urine was developed and validated. Plasma, plasma ultrafiltrate, or urine was extracted by ethyl acetate. The extract was subjected to liquid chromatography with an Inertsil ODS-3 column with an aqueous NH4Cl (1 mM, pH 9.0):methanol mobile phase. The presence of NH4Cl in the mobile phase induced the formation of [M+Cl] in the first quadrupole at m/z 397 and 409 for cortisol and 6alpha-methylprednisolone (internal standard), respectively. In the collision cell, the complex dissociated to the neutral parent and the chloride ion at m/z 35; the latter ion was used for quantification. The calibration curve was linear from 0.5 to 100 ng/mL. The lower limit of quantification was 0.50 ng/mL and the limit of detection was 0.25 ng/mL. For quality control samples prepared in water, the intrabatch assay precision was 5.6%, 9.6%, and 9.9% at 50, 10, and 1 ng/mL, respectively. The interbatch assay precision was 4.2%, 6.3%, and 7.5% at 50, 10, and 1 ng/mL, respectively. For measurement of endogenous cortisol in plasma and urine samples, the intra-assay and interassay precision was 10.8% and 4.8% for total plasma cortisol, 13.1% and 5.2% for free plasma cortisol, 10.9% and 13.1% for cortisol protein-binding free fraction, and 8.9% and 14.4% for urine cortisol, respectively. A simple procedure of ultrafiltration coupled with the highly sensitive LC-MS/MS quantification offered a rapid and reproducible assay for plasma free cortisol, which may be useful in the assessment of adrenal function in patients, especially critically ill patients with abnormal protein binding. It may also be useful for plasma and urinary cortisol measurements in pharmacodynamic studies of adrenocorticoid response.
皮质醇是一种重要的肾上腺类固醇激素，参与代谢稳态的调节。开发并验证了一种新的液相色谱 - 质谱/质谱（LC-MS/MS）多反应监测（MRM）方法，用于测量血浆超滤液、全血和尿液中的皮质醇浓度。血浆、血浆超滤液或尿液用乙酸乙酯萃取。萃取物在配备Inertsil ODS-3柱的液相色谱仪上进行分析，流动相为含NH4Cl（1 mM，pH 9.0）的水溶液：甲醇。流动相中NH4Cl的存在分别在第一四极杆中诱导皮质醇和6α-甲基泼尼松龙（内标）在m/z 397和409处形成[M + Cl]。在碰撞池中，该络合物解离为中性母体和m/z 35处的氯离子；后者用于定量。校准曲线在0.5至100 ng/mL范围内呈线性。定量下限为0.50 ng/mL，检测限为0.25 ng/mL。对于在水中制备的质量控制样品，批内分析精密度在50、10和1 ng/mL时分别为5.6%、9.6%和9.9%。批间分析精密度在50、10和1 ng/mL时分别为4.2%、6.3%和7.5%。对于血浆和尿液样品中内源性皮质醇的测量，总血浆皮质醇的批内和批间精密度分别为10.8%和4.8%，游离血浆皮质醇分别为13.1%和5.2%，皮质醇蛋白结合游离部分分别为10.9%和13.1%，尿液皮质醇分别为8.9%和14.4%。超滤与高灵敏度LC-MS/MS定量相结合的简单方法为血浆游离皮质醇提供了一种快速且可重复的检测方法，这可能有助于评估患者的肾上腺功能，尤其是蛋白结合异常的重症患者。它也可能有助于肾上腺皮质激素反应药效学研究中的血浆和尿液皮质醇测量。
