Analytical MethodsFast determination of isomeric triterpenic acids in Osmanthus fragrans (Thunb.) Lour. fruits by UHPLC coupled with triple quadrupole mass spectrometry
Graphical abstract
Introduction
Osmanthus fragrans (Thunb.) Lour., commonly named “Guihua” in Chinese, belongs to the Oleaceae family and as one of the ten traditional flowers in China, it provides high ornamental and economic value, and plays an important role in flower industry (Fan, Li, Song, Wu, & Qian, 2019). O. fragrans is well-known for its fragrance and color and is widely cultivated in Asia, especially in southern and central China (Liao, Hu, & Chen, 2018). And it plays a vital role in the field of cosmetics, food and medicines. The essential oil of the flowers is used in aromatherapy and to produce perfume (Wang et al., 2009), and flowers have been consumed for a long time as additives in food, tea, and other beverages to improve their taste (Morgan, Mohamed, Saophea, Park, & Kim, 2018).
On the contrary, limited attention was paid to the O. fragrans fruits, which are often considered as valueless agricultural waste and discarded. The fruits usually turn from green into atropurpureus when they are ripe in April and May (Liu & Min, 2017). O. fragrans fruits are composed of purple peel, green pulp and light brown seed. It has been recorded as a medicinal part in Compendium of Materia Medica for hundreds of years in China and has a wide range of biological effects, including anti-oxidation (Wang, Gan, Zhang, & Pan, 2010), platelet-aggregation inhibiting function(Tang, Cao, Zhang, & Zhao, 2015), antidepressant(Jawaid, Gupta, Rahman, & Kamal, 2005), anti inflammation and analgesia (Li, Zhao, Cheng, & Sun, 2013). In conclusion, O. fragrans fruits could be a great promising source and a good candidate of health food as well as medicines for the treatment of various diseases.
Abundant pharmacological activities of the O. fragrans fruits are mainly owed to the bioactive components. Over the past years, a few studies have reported the phytochemicals of the fruits, such as phenylethanoid glycosides (Liao et al., 2018), unsaturated fatty acids (Zhao, He, Li, & Dong, 2015), melanin (H. Wang, Pan, Tang, & Huang, 2005) and flavonoid (Lei, 2011). Pentacyclic triterpenes are gaining interest due to their beneficial health effects. Recently several triterpenic acids have been discovered and tentatively identified in the O. fragrans fruits by our group (Liao, Hu, & Chen, 2019). Triterpenic acids are arguably an important group of natural products. The literatures abound on their pharmacological properties such as hepatoprotective (Mankani et al., 2006), anti-tumor (Akihisa et al., 2010), anti-viral (Wu, Ouyang, Wang, Zhang, & Shen, 2007) and gastroprotective (Sánchez et al., 2006) effects. Therefore, they are being used as ingredients in medicines, dietary supplements and healthcare products (Pawar et al., 2013, Raskin et al., 2002).
The interest on natural products causes the present demand for development of fast and selective analysis methods. However, the quantitative researches on triterpenic acids in O. fragrans fruits have been scarcely reflected in literature (Hu et al., 2018, Liao et al., 2018). Triterpenic acids showed a huge structure diversity and similarity. It is challengeable to analyze the triterpennic acids entirely because of structure similarities and low concentrations of the isomers. The most challenge in existing approaches to the determination of triterpenic acids is the chromatographic separation of the isomers. Triterpenic acids fraction in plant matrices is quite complex, particularly because of the coexistence of many isometric compounds which will interfere each other. Thus it should ensure both the effective separation of the target components from the matrix and the sufficient resolution of isometric compounds. Therefore, the quantification is relatively difficult, making it mandatory to make the best use of a separation technique before their detection.
Methods reported for the analysis of triterpenic acids in other plant matrices and biological fluids. Gas chromatography coupled to the MS detector using silylation (Caligiani et al., 2013, Sanchez Avila et al., 2007) and pre-column derivatization HPLC-FLD method (Li et al., 2011) have been applied to separate analytes and interfering compounds after time-consuming pretreatment process. Although good separation could be generally achieved, low sensitivity by the UV (Lee et al., 2009) or ELSD (Guo et al., 2011) detectors as well as long analysis time was inevitable (Gimenez et al., 2015). Despite the high sensitivity of LC-MS/MS methods (Olmo-Garcia et al., 2016, Zheng et al., 2018), little attention has been paid to plenty of isomeric triterpenic acids at m/z 487. The existing literature only could simultaneous determine one to two triterpenic acids isomers at the m/z 487, 471 and 455 (Li et al., 2010, Sheng et al., 2010). However, many triterpenic acids isomers at m/z 487, 471 and 455 were found in O. fragrans fruits in our previous work (Liao et al., 2019). The analytes at m/z 471 and m/z 455 were separated well while three of triterpenic acids at m/z 487 could not achieve baseline separation even the effective PAH column reported in many literatures was applied.
Hence, the main aim of the present study is to develop a simple, fast and potent analytical method for the determination of triterpenic acids by LC-MS/MS, eliminating the need of a derivatization step and detecting in high sensitivity by tandem mass spectrometry. In addition, the applicability of the method is evaluated by the analysis of O. fragrans fruits collected from four regions. Some preliminary information on content differences of analytes will be provided.
Section snippets
Chemicals and reagents
Methanol (HPLC grade) and acetonitrile (LC-MS grade) were purchased from Fisher Scientific (Fairlawn, NJ, USA). Deionized water was prepared by using a Milli-Q system (MA, USA). Ammonium acetate (HPLC grade, 99%) was purchased from Aladdin (Shanghai, China) and ammonium hydroxide was provided by Beijing Chemical Works (Beijing, China). The pH value of mobile phase was measured by a pH meter from Mettler-Toledo (Zurich, Switzerland). Standards of euscaphic acid (PubChem CID: 471426, >98%),
Optimization of UHPLC conditions
Since the target triterpenic acids under study have structural similarity, the aim of establishing an appropriate chromatographic method was strengthened to assure their proper separation and accurate determination. Some important parameters influencing the separation behavior have been mainly discussed, including the columns and mobile phase system. A mixture of standards prepared in methanol was used. The experiments were processed on the basis of theory of the single factor optimization.
It
Conclusion
In view of the above exploration and investigation, a simple and sensitive approach has been established for the analysis of triterpenic acids in O. fragrans fruits by UHPLC-ESI-MS/MS system, which allows the separation of a series of isomeric compounds. Satisfactory linearity, precision, repeatability, and accuracy were obtained. According to the quantitative results, the variations of component contents in the samples from various habitats were analyzed. The validation confirmed that this
Funding sources
We acknowledge the support of the National Natural Science Foundation of China (Grant Nos. 81872828 and 81573384).
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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These authors have contributed equally to this work.