2-(2-(Pyren-1-yl)-1H-benzo[d]imidazol-1-yl)-ethyl-4-methyl benzenesulfonate (PBITS) and its application for determination of bile acids by HPLC-FLD-MS

Abstract

A method of high performance liquid chromatography-fluorescence detection-mass spectrometry identification (HPLC-FLD-MS) coupled with pre-column derivatization to determine bile acids (BAs) from bio-sample has been developed here. The labeling reagent of 2-(2-(pyren-1-yl)-1H-benzo[d]imidazol-1-yl)-ethyl-4-methyl benzenesulfonate (PBITS) contains six benzene rings and two weak basic nitrogens in the structure, resulting in a higher degree of conjugation and perfect ionization efficiency. The PBITS could easily and quickly label BAs at the optimized derivatization conditions and the labeling yield was close to 100%. Separation of the derivatized BAs exhibited a good baseline resolution in combination with a gradient elution on a reversed-phase Hypersil BDS C₁₈ column. Calculated detection limits for HPLC-FLD, at a signal-to-noise ratio of 3, were 10.09–16.33 fmol. Excellent linear responses were observed with correlation coefficients >0.9995. The mean inter-day precision for all standards was <3.63% and the experimental recoveries were 89.9–106.3%. Good compositional data were obtained from the analysis of the extracted BAs from pig bile sample. Therefore, the facile PBITS derivatization coupled with HPLC-FLD-MS analysis allowed the development of a highly sensitive method for the quantization of trace levels of BAs from biological samples.