Enhancement of atmospheric pressure chemical ionization for the determination of free and glycine‐conjugated bile acids in human serum

Abstract

A highly sensitive and accurate method based on the precolumn derivatization of bile acids (BA) with a high ionization efficiency labeling reagent 1,2-benzo-3,4-dihydrocarbazole-9-ethyl-benzenesulfonate (BDEBS) coupled with LC/MS has been developed. After derivatization, BA molecules introduced a weak basic nitrogen atom into the molecular core structure that was readily ionized in commonly used acidic HPLC mobile phases. Derivatives were sufficiently stable to be efficiently analyzed by atmospheric pressure chemical ionization (APCI)-MS/MS in positive-ion mode. The MS/MS spectra of BA derivatives showed an intense protonated molecular ion at m/z [M + H]+. The collision-induced dissociation of the molecular ion produced fragment ions at [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+. The characteristic fragment ions were at m/z 320.8, 262.8, and 243.7 corresponding to a cleavage of N-CO, O-CO, and C-OCO, respectively, and bonds of derivatized molecules. The selected reaction monitoring, based on the m/z [M+H]+ --> [MH-H2O]+, [MH-2H2O]+, [MH-3H2O]+, 320.8, 262.8, and 243.7 transitions, was highly specific for the BA derivatives. The LODs for APCI in a positive-ion mode, at an S/N of 5, were 44.36-153.6 fmol. The validation results showed high accuracy in the range of 93-107% and the mean interday precision for all standards was <15% at broad linear dynamic ranges (0.0244-25 nmol/mL). Good linear responses were observed with coefficients of > 0.9935 in APCI/MS detection. Therefore, the facile BDEBS derivatization coupled with mass spectrometric analysis allowed the development of a highly sensitive and specific method for the quantitation of trace levels of the free and glycine-conjugated BA from human serum samples.