Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), and School of Pharmaceutical Sciences, Wuhan University, Wuhan, 430071, China.
Department of Thyroid & Breast Surgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China.
J Chromatogr A. 2018 Dec 14;1580:12-21. doi: 10.1016/j.chroma.2018.10.041. Epub 2018 Oct 25.
A hexafluroisopropanol (HFIP)-alkyl carboxylic acid high-density supramolecular solvent (SUPRAS) was proposed and combined with dispersive liquid-liquid microextraction (DLLME) for extraction and preconcentration of steroid sex hormones in human urine prior to analysis by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Octanoic acid was used as extraction agent and HFIP, with the properties of high density, strong hydrogen bond donor and high hydrophobicity, was used as dispersing agent of DLLME as well as coacervation-inducing agent and density-regulating agent of octanoic acid. HFIP-octanoic acid SUPRAS was spontaneously formed in the process of DLLME and located in the bottom of a urine solution due to its density higher than water. Thus, the SUPRAS phase at trace volume was easy to be collected after centrifugation using normal centrifuge tubes. Five hormones (four estrogens and progesterone) were chosen as analytes. Urine hydrolysis time, extraction conditions and derivatization conditions for estrogens were optimized. Under the optimal conditions, only 0.07 mL of organic solvent (HFIP) was consumed. An external standard calibration method was used for quantification, and a linear relationship was obtained with correlation coefficients higher than 0.9957 and p values for linear trend less than 0.0001. Limits of detection of the method based on a signal-to-noise ratio of 3 were 0.01-0.10 μg L. The recoveries ranged from 82.7% to 120.2%, and intra-day and inter-day relative standard deviations of the method were less than 15%. The matrix effects of 84.0%-105.7% showed no urine matrix interferences with MS detection, which was mainly due to the restricted access property of HFIP-octanoic acid SUPRAS with reverse micelle aggregates. The absence of matrix effects contributed to the reliable quantitation by external calibration, avoiding the use of expensive isotopically labeled internal standards. The novel SUPRAS-based DLLME method, coupled with HPLC-MS/MS, was applied for determination of target hormones in real urine.
提出了一种六氟异丙醇（HFIP）-烷基羧酸高密度超分子溶剂（SUPRAS），并将其与分散液液微萃取（DLLME）结合，用于人尿中甾体性激素的提取和预浓缩，然后通过高效液相色谱-串联质谱（HPLC-MS/MS）进行分析。辛酸用作萃取剂，HFIP 具有高密度、强氢键供体和高疏水性的性质，用作 DLLME 的分散剂以及辛酸的凝聚诱导剂和密度调节剂。HFIP-辛酸 SUPRAS 在 DLLME 过程中自发形成，由于其密度高于水，位于尿液溶液的底部。因此，使用普通离心管在离心后很容易收集痕量体积的 SUPRAS 相。选择了 5 种激素（4 种雌激素和孕激素）作为分析物。优化了尿液水解时间、萃取条件和雌激素衍生化条件。在最佳条件下，仅消耗 0.07mL 有机溶剂（HFIP）。采用外标校准法进行定量，线性关系良好，相关系数均大于 0.9957，线性趋势 p 值均小于 0.0001。该方法的检出限基于信噪比为 3，为 0.01-0.10μg/L。方法的回收率为 82.7%-120.2%，日内和日间相对标准偏差均小于 15%。84.0%-105.7%的基质效应表明，MS 检测没有尿液基质干扰，这主要是由于 HFIP-辛酸 SUPRAS 具有反向胶束聚集体的受限进入特性。基质效应的不存在有助于通过外标校准进行可靠的定量，避免使用昂贵的同位素标记内标。该新型基于 SUPRAS 的 DLLME 方法与 HPLC-MS/MS 结合，用于测定实际尿液中的目标激素。
