Unexpected retention behavior of baicalin: Hydrophilic interaction like properties of a reversed-phase column

Abstract

The original aim of this study was to develop a method for the determination of baicalin from membrane vesicles. The unconventional chromatographic separation (“inverse gradient elution” on a reversed phase column) was due to a lucky chance, which is detailed and discussed in this study. The validated liquid chromatography-tandem mass spectrometry (LC–MS/MS) method is proved to be sensitive, rapid and selective. Chromatographic separation was performed on a Zorbax SB-C8 column (250mm×4.6mm, i.d.; 5μm) with 0.1% formic acid in water and methanol by linear gradient elution. Quantification of baicalin was determined by multiple reaction monitoring (MRM) mode using electrospray ionization (ESI). The calibration curve was linear (r=0.9987) over the concentration range from 1 to 1000nM. The coefficient of variation and relative error of baicalin for intra- and inter-assay at three quality control (QC) levels were 2.0–10.2% and −6.1 to 6.7%, respectively. The lower limit of quantification (LLOQ) for baicalin was 1nM (0.446ng/ml), without preconcentration of the sample. This method was subsequently applied to vesicular transport assays of baicalin in membrane vesicles successfully. The developed method can open up new area of research in the chromatographic separation of flavonoids and their glucuronides.