Qualitative and quantitative characterization of secondary metabolites and carbohydrates in Bai-Hu-Tang using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector

Wei-Fang Zhong ^a,b,1, Wing-Sum Tong ^a,1, Shan-Shan Zhou ^a,c,1, Ka-Man Yip ^a, Song-Lin Li ^c, Zhong-Zhen Zhao ^a, Jun Xu ^a,*, Hu-Biao Chen ^a,*

^a School of Chinese Medicine, Hong Kong Baptist University, Hong Kong

^b Ocean College, Agricultural University of Hebei, Qinhuangdao, China

^c Department of Pharmaceutical Analysis and Metabolomics, Jiangsu Province Academy of Traditional Chinese

Medicine and Jiangsu Branch of China Academy of Chinese Medical Sciences, Nanjing, China

Bai-Hu-Tang (BHT), a classic traditional Chinese medicine (TCM) formula used for clearing heat and promoting body fluid, consists of four traditional Chinese medicines, i.e., Gypsum Fibrosum (Shigao), Anemarrhenae Rhizoma (Zhimu), Glycyrrhizae Radix et Rhizoma Praeparata cum Melle (Zhigancao), and nonglutinous rice (Jingmi). The chemical composition of BHT still remains largely elusive thus far. To qualitatively and quantitatively characterize secondary metabolites and carbohydrates in BHT, here a combination of analytical approaches using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry and ultraperformance liquid chromatography coupled with photodiode array detector was developed and validated. A total of 42 secondary metabolites in BHT were tentatively or definitely identified, of which 10 major chemicals were quantified by the extracting ion mode of quadrupole time-of-flight mass spectrometry. Meanwhile, polysaccharides, oligosaccharides, and monosaccharides in BHT were also characterized via sample pretreatment followed by sugar composition analysis. The quantitative results indicated that the determined chemicals accounted for 35.76% of the total extract of BHT, which demonstrated that the study could be instrumental in chemical dissection and quality control of BHT. The research deliverables not only laid the root for further chemical and biological evaluation of BHT, but also provided a comprehensive analytical strategy for chemical characterization of secondary metabolites and carbohydrates in traditional Chinese medicine formulas.

Keywords: Bai-Hu-Tang, carbohydrate, secondary metabolite, ultraperformance liquid chromatography coupled with photodiode array detector, ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry