Simultaneous determination of oligosaccharides and iridoid glycosides in rat plasma using HILIC-MS/MS and its application to pharmacokinetic study of Rehmannia glutinosa extract

Quantitative bioanalysis of oligosaccharides by LC-MS/MS is challenging and rarely reported up to now. Simultaneous quantitative bioanalysis of oligosaccharides and iridoid glycosides by LC-MS/MS poses additional challenges due to their unique and different structural features. In the present study, a fast and sensitive analytical method based on hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) was developed for simultaneous determination of raffinose, manninotriose, stachyose, ajugol and catalpol in rat plasma. Multiple analytical challenges were encountered during method development, including very different retention behavior of oligosaccharides and iridoid glycosides, confusing split peaks for manninotriose, low ionization and extraction efficiency of oligosaccharides, thermal instability of catalpol and reduced column performance. The strategies to overcome these challenges were presented by optimizing chromatographic separation, mass spectrometric detection and sample preparation. The best separation was achieved on an Accucore-150-Amide-HILIC column (100 mm × 2.1 mm, 2.6 μm) at 50 °C with a gradient mobile phase containing acetonitril and 2.5 mM ammonium acetate. Ammonium adduct ions produced by positive electrospray ionization were chosen as precursor ions for multiple reaction monitoring transitions. The lower limits of quantification were 0.01-0.2 µg/mL using only 50 µL of plasma sample. The method was successfully applied to pharmacokinetic characterization of oligosaccharides and iridoid glycosides in normal and type 2 diabetic rats after intragastric administration of Radix Rehmanniae extract.