Development and validation of an LC-ESI/MS/MS method with precolumn derivatization for the determination of betulin
Zhiwei Hu1, Na Guo, Ziming Wang
1Alkali Soil Natural Environmental Science Center, Northeast Forestry University, Key Laboratory of Saline-Alkali Vegetation Ecology Restoration in Oil Field, Ministry of Education, Harbin 150040, China.
Abstract:
Neutral pentacyclic triterpenes with only one or two hydroxyl groups, such as betulin, are not easily ionized by electrospray ionization (ESI). However, because betulin is reactive and neutral, derivatization may improve ionization efficiency. In the present study, the potency of different derivatization reagents was evaluated and p-toluenesulfonyl isocyanate (PTSI) was proven to be the optimal. The derivative generated by the reaction of betulin with PTSI was ionizable and fragmentable in the negative mode by liquid chromatography-electrospray ionization/tandem mass spectrometry (LC-ESI/MS/MS). Based on this chemical derivatization, an LC-ESI/MS/MS method was developed and validated for the quantification of betulin in rat plasma. The sample was extracted with ethyl acetate, derivatized with PTSI, separated on an ACQ UPLC BEH phenyl column, and analyzed in negative multiple reaction monitoring (MRM) mode. The calibration curve was linear over the betulin concentration range 2.5-200ng/mL. The lower limit of quantification was 2.5ng/mL. The inter- and intra-day accuracy and precision were within ±15%. Betulin recoveries were 86.7% or higher at three quality control levels (5, 50, and 160ng/mL). This validated method was subsequently applied to a pharmacokinetic study of betulin in rat plasma after oral administration.
