Large-scale qualitative and quantitative characterization of components in Shenfu injection by integrating hydrophilic interaction chromatography, reversed phase liquid chromatography, and tandem mass spectrometry

It is of great importance to clarify in depth the chemical composition, including qualitative and quantitative aspects, of traditional Chinese medicine (TCM) injection that contains a great number of hydrophilic and hydrophobic ingredients to guarantee its safe medication in clinic. Column-switching hydrophilic interaction liquid chromatography-reversed phase liquid chromatography coupled with tandem mass spectrometry (HILIC-RPLC-MS/MS) has been revealed to be advantageous at simultaneous measurement of compounds covering a broad polarity range. Previous studies have profiled the hydrophobic components, mainly aconite alkaloids and ginsenosides, in Shenfu Injection (SFI); however, the hydrophilic substances haven't been taken into account. In the present study, we aim to holistically characterize the hydrophilic constituents and to simultaneously quantitate both hydrophilic and hydrophobic components in SFI. A strategy integrating predefined multiple reaction monitoring, step-wise multiple ion monitoring, and enhanced product ion scans was proposed to universally screen the hydrophilic substances using a hybrid triple quadrupole-linear ion trap mass spectrometer. Structural identification was carried out by comparing with authentic compounds, analyzing MS(2) spectra, and referring to accessible databases (e.g., MassBank, METLIN and HMDB). A total of 157 hydrophilic compounds were detected from SFI, and 154 ones were identified as amino acids, nucleosides, organic acid, carbohydrates, etc. A column-switching HILIC-RPLC-MS/MS system was developed and validated for simultaneously quantitative analysis of 40 primary hydrophilic and hydrophobic ingredients in SFI, including eleven amino acids, nine nucleosides, nine aconite alkaloids, and eleven ginsenosides. Taken together, the findings obtained could provide meaningful information for comprehensively understanding the chemical composition and offer a reliable approach for the quality control of SFI.