Abstract: 【Objective】The fast method based on ultra performance liquid chromatograph (UPLC) for the analysis of main phenolic acids and flavonoids in citrus was developed in order to provide technological supports for exploitation of phenolic substance of citrus.【Method】 First, the instrument condition optimized the detection wavelength selection based on full-scan (190-400 nm), all material have maximum absorption spectrum at around 283 nm. The optimization of column temperature, mobile phase and flow rate refer to the relevant literature. In order to make the baseline separation using a gradient elution; to optimize the citrus sample processing method, the extracting agent type, extraction times and time comparing by single-factor comparison test. Then citrus samples were extracted with ethyl acetate for 30 min, evaporated and concentrated. The residue was redissolved in methanol for UPLC analysis, peel samples were extracted for 3 times and pulp for 4 times. 【Result】 The chromatographic separation was carried on a ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7 μm) in gradient elution with a mobile phase of acetic acid aqueous solution (0.3%, w/w) (A) and methanol (B) as follows: 95%-80% (0-3 min)A, 80%-80% (3-8 min)A, 80%-70% (8-12 min)A, 70%-20% (12-17 min)A and 20%-95% (17-20 min)A. The column temperature was 35℃, the injection volume was 3 μL and the flow rate was 0.3 mL·min^-1. DAD spectra was recorded and 283 nm was the detection wavelength of phenolic compounds. A total of 19 analytes were separated to the baseline within 18 min with the correlation coefficients higher than 0.999 and linear range from 0.01 to 500 mg·L^-1. The precision, reproducibility and stability were satisfactory with the relative standard deviations (RSDs) less than 5%. Peel samples were extracted for 3 times and pulp for 4 times. Recoveries achieved from the spiked peel and pulp at two concentrations ranged from 85.8% to 109.4% (RSDs, 0.86%-6.06%) and 88.4% to 112.7% (RSDs, 1.05%-5.23%), respectively. The limits of detection (LODs, S/N=3) ranged from 0.001 to 0.09 mg·kg^-1. The proposed method was applied to the determination of phenolic compounds in citrus samples including five categories, which were mandarin orange (sugar orange and ponkan), sweet orange (Newhallnavel orange), pomelo grapefruit (cocktail), citron lemon (lemon) and kumquat. There were different flavonoids and phenolic acids and their different contents in various types of citrus. The total contents of the phenolic compounds in ponkan and cocktail grapefruit peels were the highest, ponkan was about 1.5 times of other species, 5 times of kumquat and the peel was about 3-5 times of the pulp. The content of the flavonoids in cocktail peel with 1 813.22 mg·kg^-1 was the highest, followed by ponkan, Newhallnavel orange, sugar orange, lemon and kumquat. The variation trend of the flavonoid content in the pulp was similar to the peel with the peel significantly higher than the pulp. Flavanones including eriocitrin, naringin, hesperidin, neohesperidin, didymin, narirutin, naringenin and hesperetin are the main flavonoids in citrus. The content of the flavanones in cocktail peel with 1 491.8 mg·kg^-1 was the highest, followed by lemon and Newhallnavel orange and kumquat was the least with the pulp significantly lower than the peel. The total content of the phenolic acids in sugar orange peel with 515.21 mg·kg^-1 was the highest, followed by ponkan, lemon, cocktail and Newhall navel orange and kumquat was the least. The phenolic acid contents of the pulp and peel had a consistent trend and the content of peel was about 3-5 times of pulp. Chlorogenic and ferulic acids were the main acid in citrus.【Conclusion】 A rapid method for the simultaneous detection of the major phenolic and flavonoid substances by UPLC was established. This method was efficient, accurate, low-loss, environment friendly and authenticated by the actual citrus samples including five categories. The flavonoids and phenolic acids were detected in different varieties of citrus and their contents and types. This method can be used for the conventional analysis of the fast and simultaneous detection of flavonoids and phenolic acids in citrus.

Key words: citrus, ultra performance liquid chromatography, simultaneous determination, phenolic acids, flavonoids