An effective integrated method for comprehensive identification of eighty-five compounds in Zhi-Zi-Da-Huang decoction by HPLC-DAD-ESI-MS (TOF) and HPLC-DAD-ESI-MS/MS (QqQ) without the help of reference standards

Zhi-Zi-Da-Huang decoction (ZZDHD) is an effective formula to treat alcoholic liver disease in China for nearly 2000 years. However, compounds in the formula are still not clear due to the complexity of the coexisting constituents. In this study, a sensitive integrated method based on HPLC-DAD-ESI-MS (TOF) and HPLC-DAD-ESI-MS/MS (QqQ) was developed and 85 chemical compounds in ZZDHD were identified. By TOF/MS, the quasi-molecular ions of the constituents were deduced according to the accurate mass of corresponding positive and negative ions, and product ion scan was then carried out using QqQ/MS. The fragments of each compound were observed by TOF/MS and/or QqQ/MS. Compounds with different chemical structures and concentration levels were tentatively identified according to their chromatographic retention time, UV absorption and MS data. Fragmentation patterns of six types were proposed to verify the identification results. It was found that at TOF/MS mode, a retro diels-alder (RDA) reaction occurred at bonds 1 and 3 in C-ring of flavanone 7-O-diglycosides when 4′-OH exists at B-ring. While the reaction was not observed if a methoxyl group appeared at the same site of flavanone skeleton. Another important phenomenon was noticed that 3-sinapoyl-5-caffeoylquinic acid, 4-sinapoyl-3-caffeoylquinic acid, and sinapylglucoside were detected only in ZZDHD while they were not found in four component herb extracts. The phenomenon revealed that changes of compounds happened when the component herbs were decocted together. The results in the paper are favorable of the clinic dosage and have laid a foundation for the subsequent research of ZZDHD in vivo.