Simultaneous determination of six flavonoids in Wuyao leaves by ultra-high performance liquid chromatography tandem mass spectrometry
Lindera aggregata (Sims) Kosterm, a plant belonging to the Camphor family and the Piper genus, has anti-inflammatory, analgesic, antioxidant, and anti-tumor effects. It is mainly produced in southeastern coastal provinces of China, with Tiantai County in Zhejiang Province being the most famous, hence it is called Tiantai Wuyao. In 2018, it was selected as one of the new “Zhejiang Eight Flavors” in Zhejiang. Modern pharmacological research has shown that black medicine also has immunomodulatory effects on the central nervous system and cardiovascular system, making its market prospects broad. However, the medicinal parts of Wuyao grow slowly and often take 8-10 years to meet the requirements for quality medicinal herb processing. During the growth cycle, its stem and leaf parts are often discarded, resulting in low resource utilization. In the folk areas of Zhejiang, Wuyao leaves are often used to treat acute cellulitis, rheumatoid arthritis and other inflammations, and are also often made into tea drinks for lipid-lowering, liver protection and other health benefits. Previous studies have shown that Wuyao leaf extracts have a certain lipid-lowering effect. The research team conducted a pharmacological correlation study on Wuyao leaf extracts and found that they have a certain mechanism of action against hyperlipidemia and osteoporosis. At the same time, systematic chemical component separation and extraction were also carried out on Wuyao leaves in the early stage. Six flavonoids were isolated for the first time from the ethanol extract of Wuyao leaves, which were identified as: kaempferol-3-O – α – L-rhamnoside (1), quercetin (2), kaempferol-3-O – β – D-xyloside (3), kaempferol-3-O – α – L-arabinofuranoside (4), kaempferol-3-O – (2 ″ -3-O-trans-3-O-p-3-O-coumaroyl) – α – L-rhamnoside (5), kaempferol-3-O – (2 ″, 4 ″ -3-O-di-3-3-O-3-3-3-O-di-3-glucoside) O-trans-3-O-p-3-O-coumaroyl) – α – L-rhamnoside (6), chemical structure shown in Figure 1.

At present, there are relevant research reports on the quality evaluation of Wuyao leaves. Yi et al. used HPLC to determine the content of four flavonoids in Wuyao leaves from different origins in Hunan Province. Sun et al. used UV spectrophotometry to determine the total flavonoid content in Wuyao leaf extracts and investigated the relevant factors for extracting total flavonoids, in order to optimize the extraction process. At the same time, Sun et al. used HPLC to determine the content of four sesquiterpenoid lactones in Wuyao leaves from different origins. However, these are traditional detection methods, which lack high efficiency and accuracy in content determination. With the advancement of mass spectrometry technology, ultra high performance liquid chromatography tandem mass spectrometry technology with high efficiency, high specificity, and high sensitivity has been developed. Chromatography tandem mass spectrometry (UPLC-MS/MS) has become an important analytical method for detecting the quality of traditional Chinese medicinal materials. Therefore, this study will use UPLC-MS/MS technology to quantitatively study the six main flavonoids in Wuyao leaves from different origins, and combine the entropy weight – α – L-approximate ideal solution ranking method (TOPSIS) for analysis and evaluation, in order to provide scientific basis for the internal quality control of Wuyao leaves.

 

Selection of extraction conditions. In this study, UPLC-MS/MS method was used to simultaneously determine the contents of six indicator compounds, namely kaempferol-3-O – α – L-rhamnoside (1), quercetin (2), kaempferol-3-O – β – D-xyloside (3), kaempferol-3-O – α – L-arabinofuranoside (4), kaempferol-3-O – (2 “- trans – p-coumaroyl) – α – L-rhamnoside (5), and kaempferol-3-O – (2”, 4 “- di trans – p-coumaroyl) – α – L-rhamnoside (6), in Wuyao leaves within 10 minutes. The methanol concentrations were 30%, 50%, and 70%, respectively. Based on the analysis of the spectrum, 70% methanol was selected for the best extraction effect. At the same time, a comparison was made between heating reflux and ultrasonic extraction methods. Based on the content results, the extraction rates obtained were comparable. Considering time and consumable costs, ultrasonic extraction was adopted.
Chromatography mass spectrometry conditions. This study investigated three mobile phase systems: acetonitrile water, acetonitrile-0.1% formic acid water, and acetonitrile-0.1% ammonium formate water. The peak area and peak shape of six flavonoids were used as evaluation indicators to determine the appropriate mobile phase system. It was found that when acetonitrile water was selected as the mobile phase, there was a tailing phenomenon in the compounds of kaempferol-3-O – (2 “- trans-pcoumaroyl) – α – L-rhamnoside and kaempferol-3-O – (2”, 4 “- di-trans-pcoumaroyl) – α – L-rhamnoside; When acetonitrile 0.1% ammonium formate water is selected as the mobile phase, quercetin 3-O – α – L-arabinofuranoside exhibits slight tailing; When acetonitrile 0.1% formic acid water was chosen as the mobile phase, the peak shapes of all six compounds were relatively symmetrical. Therefore, acetonitrile 0.1% formic acid water was chosen as the mobile phase system. In the selection of mass spectrometry conditions, the study used positive and negative ion modes to determine the content of six target compounds. In order to ensure that the target compounds have better stability, higher response values, and better peak shapes, multiple repeated experiments have shown that using negative ion scanning mode for detection can make the six flavonoid compounds more stable, with better peak shapes and response values under the detection conditions. At the same time, during the investigation of chromatography-mass spectrometry conditions, the retention times of flavonoids 2 and 5 extracted from the ion chromatogram in Figure 2 were basically the same. Compared with traditional HPLC analysis techniques, the chromatography-mass spectrometry based on mass to charge ratio analysis does not rely on peak to peak separation, and has short signal collection time and high sensitivity. Therefore, in this study, UPLC-MS/MS analysis technology was used to simultaneously determine six flavonoids in Wuyao leaves, which has more advantages.
Result analysis. This study collected medicinal samples from 10 Wuyao production areas in China, and used UPLC-MS/MS technology to simultaneously determine the content of six chemical components in Wuyao leaves. The entropy weight TOPSIS method was used to comprehensively analyze and evaluate the six compounds from the 10 production areas. The comprehensive ranking was as follows: Tiantai, Zhejiang>Jinhua, Zhejiang>Changsha, Hunan>Ningde, Fujian>Kunming, Yunnan>Tonglu, Zhejiang>Guilin, Guangxi>Yingtan, Jiangxi. The results showed that the comprehensive quality of Wuyao leaves in Tiantai, Zhejiang was relatively high, which is related to the traditional belief that Tiantai, Zhejiang is a genuine Wuyao medicinal material production area. At the same time, it also confirms the record in the Compendium of Materia Medica that for Wuyao, Tiantai is the winner; The Dictionary of Traditional Chinese Medicine records that the production of Wuyao is the largest in Zhejiang, and the quality of the products produced in Tiantai, Zhejiang is the best, hence it is called “Tiantai Wuyao” or “Tai Wuyao”. At the same time, the evaluation results also reflect that there are significant differences in the content of six main flavonoids in Wuyao leaves from different origins, and there are also significant differences in Wuyao leaves from different cities in the same province. This may be related to the sources of Wuyao introduction in different planting bases and different ecological environments.
At present, Wuyao Ye has been approved as a new resource food, so its product development will continue to be explored. This study has established a method that is easy to operate, has good precision, stability, and repeatability, and can simultaneously determine the content of six flavonoids in Wuyao Ye. It can be used for quantitative analysis and comprehensive evaluation of the main flavonoids in Wuyao Ye, and the entropy weight TOPSIS method used also provides a theoretical basis for evaluating the quality of other medicinal herbs with medicinal value.