Rapid Analysis of 18 Flavonoids in Tea by Ultrahigh-Performance Liquid Chromatography Coupled with Quadrupole-Time of Flight Mass Spectrometry

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Introduction
Tea is a popular natural plant beverage [1,2]. Due to the fact that tea contains tea polyphenols, cafeine, amino acids, tea polysaccharides, vitamins, and other nutritional functional components [3,4], its good antiaging [5], anticancer [6], bactericidal [7], and lowering blood sugar [8] efects have always been valued by researchers. Tea polyphenols in tea mainly include catechins, favonoids, phenolic acids, anthocyanins, and other substances, and their content accounts for about 20% of the dry weight of tea [9]. Our research team has established a detection method for catechins in tea in the early stage and analyzed the content of catechins in tea [10]. Flavonoids are another important tea polyphenol in tea. According to the diferent modifcations of the central C ring, favonoids can be divided into favonols, favanols, isofavones, favanones, and anthocyanins. Nearly one-third of the favonoids are favonols [11,12]. Flavonols have a hydroxyl group in position 3 of the C ring, which is easy to be glycosylated ( Figure 1) [13,14]. Te glycosylation makes favonols commonly present in plants as glycosides and 3% to 4% of the dry matters of tea are favonol glycosides. Flavonoids have the functions of strengthening the heart, lowering blood pressure, promoting cell proliferation, reducing the accumulation of sugar alcohols, and helping to prevent cataracts. Tey are also natural food antioxidants [15][16][17][18][19]. Te main mechanism of healthcare efect of favonoids comes from enzyme regulation, improved blood circulation, cell apoptosis regulation, and stem cell signal transduction and reduction [20,21].
Flavonoids play an important role in the organoleptic quality and physiological function of tea. Accurate determination of favonoids in tea is of great signifcance for tea sensory taste research, raw material control, and tea production process improvement [22,23]. How to accurately determine the content of favonoids in tea is a key concern of tea researchers. Detection methods of favonoids include ultraviolet spectrophotometry [24,25], thin-layer chromatography (TLC) [26], high-performance liquid chromatography-diode-array detector (HPLC-DAD) [27,28], and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/ MS) [29][30][31]. Ultraviolet spectrophotometry is generally used to determine the content of total favonoids. TLC sufers from a low accuracy and fnds it difcult to distinguish similar compounds. It is difcult to separate various favonoids using HPLC, and it has a long analysis time and poor specifcity. Te UPLC-MS/MS method has a high sensitivity and selectivity [32,33], but the reference materials must constantly be purchased and renewed because of the limitation of their period of validity. Tese factors increase the detection time and costs [34,35]. Te development of the mass spectrometry technology makes Q-TOF/MS which has a high resolution and fast acquisition rate. UPLC-Q-TOF/MS has the characteristics of high resolution, high sensitivity, high accuracy, and wide scanning range. Moreover, it can also enable possession of the capability of nontarget matters screening and data tracing through the accurate mass, isotope distribution, and characteristic mass spectrum of MS/MS [36][37][38][39]. Researchers have made systematic studies and attempts on the IDA MS/MS mode using UPLC-Q-TOF/MS. IDA MS/MS is a mode in which the primary full scan is performed frst, and then the highest ones are selected for the secondary mass spectrometry scan based on the abundance of ions in the primary full scan. Previously, we performed the targeted and nontargeted preliminary identifcation of favonoids in tea using the Triple TOF ™ 6600 + MS system. Te compounds were rapidly selected by the targeted and nontargeted peak fnding approaches and then tentatively identifed by comparing with the TCM MS/MS database, online ChemSpider database, or inferred through mass spectrometry fragment ion analysis and literature data. Second, based on the published literature [40,41], we summarized and sorted out the contained and possibly contained favonoids in tea. Finally, we identifed the types of favonoids to be studied, purchased standards of favonoids, and quantifed them accurately. In this paper, UPLC-Q-TOF/MS was frst used for the fast and accurate determination of 18 favonoids in tea. It can obtain accurate test results and provide reliable technical support for enterprises and regulatory authorities.

Reagents and Standards.
Methanol was of HPLC grade and purchased from Fisher Scientifc (Loughborough, UK). Formic acid was of HPLC grade supplied by Sigma-Aldrich (St. Louis, Missouri, USA). Ultrapure water (resistivity 18.2 M·Ω·cm) was generated by a Milli-Q system (Millipore, USA). All tea samples were purchased from local tea shops in China.

Sample Preparation.
1.00 g of crushed tea (dry leaves) was weighed into a 50 mL centrifuge tube. After spiking of 20 mL of 70% methanol-water solution, it was vortexed for 1 min and then sonicated for 30 min at room temperature. Te sample was centrifuged at 8000 ×g for 3 min at 4°C. 1 mL of supernatant was fltered through a 0.22 μm flter membrane and analyzed by UPLC-Q-TOF/MS.

Chromatographic Conditions.
Te chromatographic separation was performed on a Kinetex F5 column (2.1 mm × 100 mm, 2.6 μm). Te column temperature was 35°C, and the injection volume was 5 μL. Te chromatographic analysis was performed by a gradient elution with a mobile phase A (0.1% formic acid solution) and a mobile phase B (methanol). Te gradient profle started from 10% B and linearly increased to 90% B within 11 mins and kept at 90% B for 5 mins before being returned to 10% B for the next injection. Te postrun equilibrium time was 4 min, and the fow rate was 0.3 mL/min.

Mass Spectrometry Conditions. For the MS analysis, the
Triple TOF ™ 6600 + equipped with a DuoSpray ™ ion source was used. In this study, an electrospray ionization source mode was used for detection and an atmospheric pressure chemical ionization (APCI) source was used for calibration. Te spray voltage (ISVF) was set at 5500 V/4500 V and the source temperature at 400°C under the positive mode (ESI + ). Te curtain gas pressure was 0.24 MPa, the nebulizer gas pressure was 0.34 MPa, and the auxiliary gas pressure was 0.38 MPa. Te TOF MS data were collected with a duration time of 30 min (scan 100-100 Da), and the accumulation time was 0.15 s. Te information-dependent acquisition (IDA)-MS/MS conditions were as follows: accumulation time was 0.05 s, high sensitivity mode, switch criteria 100 cps, isotopes within 4 Da were excluded, declustering potential was 80 V, and the collision energy was 40 ± 20 V. An automatic batch calibration was performed to ensure the accuracy and reproducibility.

Method Validation.
In order to determine the specifcity of the method, the chromatograms were compared between the 18 favonoids and the tea samples were spiked with standards. 18 favonoids mixed with standard intermediate solutions were accurately pipetted, and standard series solutions were prepared with methanol. Te linear regression equation was established with the quantifcation of the peak area of the target primary excimer as the ordinate (Y) and the concentration of each component as the ordinate (X). A series of spiked samples were prepared to determine the limits of detection (LOD) and the limits of quantifcation (LOQ) when the signal-to-noise ratios of 18 favonoids were about 3 and 10, respectively. Te reproducibility and stability of the method were investigated by measuring the intraday and interday precision (n � 3) [42]. Two diferent concentrations were added to the standard samples at 0.2 and 2.0 ppm. Each additive concentration was measured 3 times in parallel, and the relative standard deviation of the results of the 3 replicates was evaluated for analysis.
Te recovery and precision experiments were carried out by adding 18 favonoids' standard solutions to tea samples at three concentration levels. Te recovery and RSDs were calculated at six repetitions for each level. Te recovery was calculated according to the following formula: where A is the measured sample concentration and B is the actual sample concentration.

Optimization of Extraction Solvents.
Te type of extraction solvent used directly afects the extraction efect of the target compound, so the choice of the extraction solvent is crucial. Tis experiment investigated the extraction efect of water with various concentrations of methanol (30%, 50%, 70%, and 90%). As shown in Figure 2, 70% of the methanolwater solution yielded the best recovery of the 18 favonoids. Terefore, 70% methanol-water solution was selected as the extraction solvent.  Figure 3 shows the extraction ion chromatograms of the 18 favonoids.  Journal of Food Quality 5  Journal of Food Quality 7 curtain gas (CUR), pressure of the nebulizer gas (GS1), pressure of the auxiliary gas (GS2), declustering potential (DP), collision energy (CE), and dynamic energy of collision (CES) parameters were optimized to achieve an efective isolation and response of all target compounds. CES is an important parameter to improve the sensitivity of the target compounds and to reduce the ion information loss of secondary fragments. CES was set at 20 eV which made the rich second-order fragment ion information of EPI scan maps at 20, 40, and 60 eV. Diferent from the quantitative means of triple quadrupole, Triple TOF ™ 6600 + high-resolution mass spectrometry carried out the frst-order full scan and data-dependent second-order mass spectrometry simultaneously in the TOF-MS-IDA-MS/ MS mode. Te parent ion was used as the quantitative ion, and it simplifed the optimization process of mass spectrum parameters and improved efciency. It is more conducive to rapid screening and quantitative analysis. Electrospray ionization and positive/negative ion scanning mode were used in the experiment to obtain the frst-order full-scan mass spectrograms.  [43]. Figure 4 shows the second-order characteristic maps of 18 favonoids. Te accuracy of the qualitative analysis can be improved through fragment ion information.

Specifcity of the Method.
Both standards and the spiked tea samples showed a sharp and symmetric peak in the chromatograms, and there were no peaks in the samples at the same retention time. Figure 2 shows that there are no interferences from the matrix components on the retention time of the 18 favonoids.

Linearity, Sensitivity, Intraday, or Interday Precision.
From the determination results in Table 2, it can be seen that the linearity of 18 favonoids in the concentration range of 0.10-200 ng/mL was good, the R 2 ≥ 0.998, LOD was 0.0010-0.040 ppm, and LOQ was 0.0020-0.10 ppm. Te intraday precision ranges from 0.141% to 8.32%, and the interday precision ranges from 1.32% to 9.29%. Huang et al. established a method for the detection of nine favonoids with LOD ranging from 10 to 66 μg/kg [19]. Te LOD and LOQ of this method are lower than those of the published methods, indicating that the method has a high sensitivity. Table 3, when the supplemental level was 0.0020 ppm-1.00 ppm, the recoveries ranged from 73.8% to 107%, and RSD ranged from 0.135% to 8.05%. Te results were able to meet the requirements of the "Criterion on quality control of laboratories-Chemical testing of food" [44], that is, when the content of the measured component is less than 0.1 mg/kg, the recovery is in the range of 60-120% with RSD ≤ 15%. Te recoveries were considered acceptable to the method, and the results indicated that the precision was reasonable.

Application to Tea Samples.
In order to investigate the content of the 18 favonoids in tea, 10 tea samples from local tea shops were analyzed by using the established method in this research. Qualitative screening of the 18 favonoids was conducted by using an accurate mass number and retention time. Te results were confrmed by secondary characteristic fragment ions. Te compositions and contents of favonoid substances were diferent in ten tea samples. Isoliquiritigenin, silymarin, trilobatin, baimaside, and camellianin A were not detected. Te determination results of tea samples are shown in Table 4. Te content of plantagoside and luteoloside in green tea was higher than that in black tea,  3.003 ± 3.5 1.334 ± 4.  which may be due to the fermentation process. Tis may be due to the fact that favonoids are unstable in the natural state and are transformed and degraded under the action of polyphenol oxidase, heat, microorganisms, and their secreted enzymes and oxidative substances during the fermentation process [45]. Correlation analysis was performed on the 18 favonoids of 10 tea samples, and the results are shown in Supporting Information Table S1. As shown in the supplementary Table S1, there was an extremely signifcant correlation (P < 0.01) between the content of quercitrin and the content of tiliroside. Tere was an extremely signifcant correlation (P < 0.01) between the content of myricetin and the content of afzelin, rutinum, and quercetin. Tere was an extremely signifcant correlation (P < 0.01) between the content of isovitexin and the content of vitexin, schaftoside, and plantagoside. Tere was an extremely signifcant correlation (P < 0.01) between the content of vitexin and the content of schaftoside. Tere was an extremely signifcant correlation (P < 0.01) between the content of rutinum and the content of quercetin. Tere was an extremely signifcant correlation (P < 0.01) between the content of kaempferol and the content of quercetin.

Conclusions
At present, the imperfection of the relevant testing system and the lack of testing methods make tea quality supervision not in place, which restricts the improvement of tea product quality. In this study, a UPLC-Q-TOF/MS method was successfully established for the determination of 18 favonoids in tea. Te analytes were determined by Q-TOF/MS in the TOF MS and IDA-MS/MS modes. In the TOF MS mode, the target compounds are qualifed by the retention time, accurate mass, isotope distribution, and isotope abundance ratio of the target, and quantifed by the peak area of the excimer ion peak. In the IDA-MS/MS mode, the target compounds were further confrmed by the ion fragment information under the corresponding collision energy. In addition, the high-resolution mass spectrometry efectively reduced the matrix efect of tea. In this study, the determination conditions of favonoids in tea were optimized by liquid chromatography and mass spectrometry (LC/MS), and the methodology was confrmed. Te limits of detection were 0.0010-0.040 ppm, and the limits of quantifcation were 0.0020-0.10 ppm. Te recoveries ranged from 73.8% to 107% at spiked levels of 0.0020-1.0 ppm, with RSDs less than 10%. Te method was simple, sensitive, precise, and reproducible. It is suitable for the rapid determination of favonoids in tea. Tis study provides a reference for quality evaluation of tea and its products, and promotes sustainable development of the industry. Te quantitative detection of active ingredients in tea, the dose-efect relationship between active ingredients and body function, and the development of tea-functional products are expected to become the technology and market trend of industrial development.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.