基质辅助激光解吸电离成像质谱法可视化分析制川乌炮制过程生物碱空间分布的研究

Visualization Analysis of Spatial Distribution of Alkaloids in Aconiti Radix Cocta During Processing Process by Matrix⁃Assisted Laser Desorption Ionization Mass Spectrometry Imaging

目的采用质谱成像显微镜可视化分析制川乌炮制过程中6个生物碱的变化情况。方法准备不同炮制时间(9个时间点)的制川乌样品,用明胶溶液包埋后冷冻,制备30μm厚度的切片样品进行实验。根据乌头碱、次乌头碱、新乌头碱、苯甲酰新乌头原碱、苯甲酰次乌头原碱和苯甲酰乌头原碱共6种标准品的质谱信号,选取α⁃氰基⁃4⁃羟基肉桂酸为基质,采用升华法涂覆基质后再进行喷雾。质谱数据采集质量范围为m/z 200~800,使用DHB溶液在m/z 100~900进行质量校正,以1000 Hz频率的正离子模式进行基质辅助激光解吸⁃质谱成像(MALDI⁃MSI)。通过超高效液相色谱(UHPLC)测定不同炮制时间样品中6个生物碱的含量,用含量测定结果验证质谱成像结果的准确性。结果质谱成像技术可以区分制川乌不同炮制程度的样品,并且可以实现生物碱空间分布的可视化分析。结论质谱成像技术可运用于制川乌炮制过程质量研究,为中药炮制研究提供一种新思路。

OBJECTIVE To visually analyze the changes of six alkaloids in Aconiti Radix Cocta during the processing process by matrix⁃assisted laser desorption ionization mass spectrometry imaging(MALDI⁃MSI).METHODS The samples of Aconiti Radix Cocta at different processing times(9 time points)were embedded in gelatin solution and frozen to prepare 30μm thick slice samples by fro⁃zen slicing.According to the mass spectrum signals of aconitine,hypaconitine,mesaconitine,benzoylmesaconine and benzoylaconine,benzoylhypaconine,α⁃cyano⁃4⁃hydroxycinnamic acid was selected as substrate.The substrate was then sublimated and then sprayed.The data acquisition quality range was m/z 200~800.DHB solution was used for mass/charge ratio calibration at m/z 100~900.Ma⁃trix assisted laser desorption mass spectrometry imaging(MALDI⁃MSI)was performed in 1000 Hz positive ion mode.The contents of six alkaloids in the samples were determined by UPLC.The accuracy of MS imaging microscope was verified by the results of content determination and acute toxicity test.RESULTS The MS imaging microscope could distinguish differently processed samples and re⁃alize the visualization analysis of spatial distribution of alkaloids in Aconiti Radix Cocta.CONCLUSION MALDI⁃MSI can be used to study the processing quality of Aconiti Radix Cocta and this method provides a new way of thinking for the processing of traditional Chi⁃nese medicine.