Development of a Sensitive Reagent, 1,2-Benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate, for Determination of Bile Acids in Serum by HPLC with Fluorescence Detection, and Identification by Mass Spectrometry with an APCI Source

Abstract

A pre-column derivatization method with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-p-toluenesulfonate (BDETS) as labeling reagent followed by high-performance liquid chromatography with fluorescence detection has been developed for sensitive determination of bile acids (BA). Derivatives were sufficiently stable to be efficiently analyzed by high-performance liquid chromatography. The derivatives also formed an intense protonated molecular ion corresponding to m/z (M + H)⁺, and fragment ions at (MH⁺ – H₂O)⁺, (MH⁺ – 2H₂O)⁺, and (MH⁺ – 3H₂O)⁺, in positive-ion mass spectrometry with an APCI source. Collision-induced dissociation of the protonated molecular ion produced fragment products at m/z 319.1 and 246.1 corresponding to cleavage of the C-O and N-CO bonds of derivative molecules. Maximum yields close to 100% were observed when a 10 to 15-fold molar excess of the reagent was used in the presence of potassium citrate as catalyst. The derivatives fluoresced strongly, which enabled the direct injection with no significant disturbance from the main by-products from reagent degradation, for example 1,2-benzo-3,4-dihydrocarbazole-9-ethanol (BDCE-OH). The limit of detection, at a signal-to-noise ratio of 3, was 12.94–21.94 fmol. Results from validation showed the method to be highly accurate and precise (<6.4%). Excellent linear responses were observed with correlation coefficients >0.9996.