Extraction-free in situ derivatisation of timosaponin AIII using direct analysis in real time TOF/MS.

Introduction: Direct analysis in real time (DART) TOF/MS has been used for mass information of various non-polar phytochemicals in raw material with no sample preparation. However, low ionisation efficiency for polar compounds including glycosides limits its extensive use in the field of phytochemical analysis.

Objective: In order to develop a direct analysis method for polar glycosides using in situ derivatisation, which improves ionisation efficiency of hydrophilic glycosides.

Method: Anemarrhena Rhizoma was used as a model plant targeting on Timosaponin AIII utilising a Dip-It module. Permethylation was applied to the powdered raw material with tetramethylammonium hydroxide in front of a DART ion source. Also, DART TOF/MS combined with permethylation was applied to timosaponin AIII standard solution to obtain the limit of detection (LOD).

Results: In situ methylation of timosaponin AIII and Anemarrhena Rhizoma raw material were successfully used to ionise the glycoside. The LOD was found to be in the range of 2.4-4.8 ng for permethylated timosaponin AIII and this level is four times higher than the range of the underivatisation analysis. Direct analysis of permethylated timosaponin from Anemarrhena Rhizoma was also successfully performed.

Conclusion: A simple and quick derivatisation method with tetramethylammonium hydroxide was developed for the direct identification of a hydrophilic saponin from the plant tissue. Better ionisation efficiency conferred by in situ permethylation enabled ionisation of whole molecules of timosaponin AIII from the plant tissue. This simple analytical method will provide a solution to reduce tedious sample preparation steps, not only for non-polar but also hydrophilic natural products directly from the tissue.