The feasibility of taking the ratio of 6β-hydroxycortisol (6β-OHCOR) to cortisol (COR) in plasma as a biomarker to reflect CYP3A4 activity needs to be verified, but the low concentration of 6β-OHCOR which is an endogenous substance in plasma presents a challenge for determination. In this study, a Liquid chromatography with tandem mass spectrometry (LC–MS/MS) method was established to simultaneously quantify the COR and 6β-OHCOR in plasma with COR-d4 and 6β-OHCOR-d4 as internal standards (ISs). Plasma samples were treated by protein precipitation using acetonitrile. Separation with a gradient elution within 5 min was achieved on C18+ column utilizing 5 mM ammonium formate and methanol. An API 4,000 MS in multiple reaction monitoring mode with transitions of 407.1 → 361.1 and 423.1 → 347.1 was utilized. Albumin solution was used as a surrogate matrix, with good linearities over the concentration of 1.20–300 ng/mL for COR and 0.0400–10.0 ng/mL for 6β-OHCOR. The precisions for intrarun and interrun were < 6.8%, and the accuracy was fell in the interval of −5.2 to 3.5%. Matrix effect was not found. Recovery was close to 100.0%. Stability was confirmed under the storage and processing conditions. The validated method was applied to evaluate the inhibitory effect of voriconazole to CYP3A by the ratio of 6β-OHCOR to COR.

中文翻译：

---

液相色谱-串联质谱法同时测定人血浆中皮质醇和 6β-羟基皮质醇

以血浆中6β-羟基皮质醇（6β-OHCOR）与皮质醇（COR）的比值作为反映CYP3A4活性的生物标志物的可行性有待验证，但血浆内源性物质6β-OHCOR的低浓度呈现决心的挑战。本研究建立了液相色谱串联质谱 (LC–MS/MS) 方法，以 COR-d4 和 6β-OHCOR-d4 为内标 (ISs) 同时定量血浆中的 COR 和 6β-OHCOR。使用乙腈通过蛋白质沉淀处理血浆样品。使用 5 mM 甲酸铵和甲醇在 C18+ 柱上实现了 5 分钟内梯度洗脱的分离。使用多反应监测模式的 API 4,000 MS，离子对为 407.1 → 361.1 和 423.1 → 347.1。白蛋白溶液用作替代基质，在 1.20–300 ng/mL 的 COR 浓度和 0.0400–10.0 ng/mL 的 6β-OHCOR 浓度范围内具有良好的线性。批内和批间的精确度≤ 6.8%，准确率下降到-5.2%到3.5%之间。未发现基质效应。回收率接近 100.0%。在储存和加工条件下确认了稳定性。采用经过验证的方法，通过 6β-OHCOR 与 COR 的比值来评估伏立康唑对 CYP3A 的抑制作用。在储存和加工条件下确认了稳定性。采用经过验证的方法，通过 6β-OHCOR 与 COR 的比值来评估伏立康唑对 CYP3A 的抑制作用。在储存和加工条件下确认了稳定性。采用经过验证的方法，通过 6β-OHCOR 与 COR 的比值来评估伏立康唑对 CYP3A 的抑制作用。