Zheng Liyun, Luo Xi, Zhu Lijun, Xie Wenzhao, Liu Shikun, Cheng Zeneng
Research Institute of Drug Metabolism and Pharmacokinetics, School of Pharmaceutical Sciences, Central South University, 410013 Changsha, Hunan, P.R. China.
The Third Xiangya Hospital, Central South University, 410013 Changsha, Hunan, P.R. China.
J Chromatogr Sci. 2015 Apr;53(4):451-5. doi: 10.1093/chromsci/bmt034. Epub 2015 Jan 26.
A specific and sensitive method based on high-performance liquid chromatography with ultraviolet absorbance detection (HPLC-UV) was developed for the simultaneous determination of urinary cortisol (F), cortisone (E), 6β-hydroxycortisol (6β-OHF) and 6β-hydroxycortisone (6β-OHE) using dexamethasone as the internal standard. The method involved solid-phase extraction of the five compounds from urine using Oasis HLB Waters cartridges with an elution solvent of ethyl acetate-diethyl ether (5 mL; 4:1, v/v), followed by 1 mol/L of NaOH (1 mL) and 1.0% acetic acid (1 mL). Separation of the five analytes was achieved within 31 min by using a reversed-phase C18 analytical column (200 × 4.6 mm, 5 µm, Agilent). A UV detector operated at 245 nm was used. According to the method validation, inter-run and intra-run precision was below 9.45% and accuracy ranged from 98.16 to 115.50%. The lower limits of quantitation were 5 ng/mL for four analytes. This is the first HPLC method that can simultaneously determine F, E, 6β-OHF and 6β-OHE in human urine. The assay was applied to research the ratio of (6β-OHF + 6β-OHE)/(F + E) as a non-invasive biomarker for the metabolism of tacrolimus.
建立了一种基于高效液相色谱-紫外吸收检测（HPLC-UV）的特异且灵敏的方法，以地塞米松为内标，同时测定尿中皮质醇（F）、可的松（E）、6β-羟基皮质醇（6β-OHF）和6β-羟基可的松（6β-OHE）。该方法包括使用Waters Oasis HLB固相萃取柱从尿液中萃取这五种化合物，洗脱溶剂为乙酸乙酯-乙醚（5 mL；4:1，v/v），随后加入1 mol/L氢氧化钠（1 mL）和1.0%乙酸（1 mL）。使用反相C18分析柱（200×4.6 mm，5 µm，安捷伦）在31分钟内实现了这五种分析物的分离。使用紫外检测器，检测波长为245 nm。根据方法验证，批间和批内精密度均低于9.45%，准确度在98.16%至115.50%之间。四种分析物的定量下限均为5 ng/mL。这是首个能同时测定人尿中F、E、6β-OHF和6β-OHE的HPLC方法。该检测方法用于研究（6β-OHF + 6β-OHE）/（F + E）比值，作为他克莫司代谢的非侵入性生物标志物。
