Hexafluoroisopropanol-alkyl carboxylic acid high-density supramolecular solvent based dispersive liquid-liquid microextraction of steroid sex hormones in human urine

Abstract 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 −1 . 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.