Jian Li, Junmei M Yan Zhang*, Lei Zheng*

a School of Food and Biological Engineering, Hefei University of Technology, Hefei 230009, China

b College of Applied Arts and Science, Beijing Union University, Beijing 100191, China

c Hebei Key Laboratory of Forensic Medicine, College of Forensic Medicine, Hebei Medical University, Shijiazhuang 050017, China

d Hebei Food Safety Key Laboratory, Hebei Food Inspection and Research Institute, Shijiazhuang 050227, China

Keywords:

Ultra-high performance liquid chromatography

Quadrupole-time of flight mass spectrometry

Tea

Polyphenols

A B S T R A C T

A rapid method was presented for the determination of 19 polyphenols in tea by ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS).Tea samples were extracted by 50% (V/V) ethanol, then separated by Waters Acquity BEH C18 column using a binary solvent system composed of acetonitrile and water (0.1% formic acid) by gradient elution.The analytes were determined by Q-TOF MS in TOF MS and information dependent acquisition (IDA)-MS/MS mode.The results showed that mass accuracy error of the 19 polyphenols were lower than 5.0 × 10–6, good linear relationship was got in range of 0.2-500 μg/L and correlation coefficient was higher than 0.999 0.The LOD was in the range of 0.002–0.100 mg/kg and the LOQ was in the range of 0.004–0.200 mg/kg.Recovery of the method was in range of 78.4%–109.2% with spike levels of 0.004–2.000 mg/kg, relative standard deviations were lower than 10%.The method was simple, rapid and accurate.It could be used for the rapid screening and quantitative analysis of 19 polyphenols in tea.

1.Introduction

Tea is the most popular functional beverage in the world and has been gaining more and more attention for its health beneficial poperties [1,2].The chemical components contained in tea are the material basis that determines the characteristics of tea and the specific characterization of the sensory properties of tea [3].There are a variety of chemical components affecting the special flavor and quality of tea, including polyphenols, amino acids, caffeine and so on [4].Among them, the polyphenols are the main chemical components in tea [5,6], and many recent studies have indicated that polyphenols in tea have many health promoting activities due to their diverse biological actions including anti-inflammatory,anti-bacterial, anti-cancer, anti-oxidant and metabolic regulatory effects [7,8]that could lead to cancer prevention and protect against metabolic, cardiovascular and inflammatory diseases [9-11].Therefore, it is feasible to use the content of polyphenols as the chemical index of tea quality evaluation in the process of tea quality analysis [12].

Selecting suitable and efficient detection methods to achieve the quality identification and classification of tea is significant important for the further development and application of tea polyphenols [13].Various analytical methods have been proposed for detecting sulfonamides, such as capillary electrophoresis [14,15], thin layer chromatography [16],gas chromatography [17], high-performance liquid chromatography [18-21]and liquid chromatography-tandem mass spectrometry [22-27].Among these methods, capillary electrophoresis and thin layer chromatography are cumbersome and time-consuming; gas chromatography requires derivation and determination, and the process is relatively cumbersome;high performance liquid chromatography has poor antiinterference ability.Liquid chromatography-tandem mass spectrometry has a limited ability to identify isomers, and the ion residence time is limited due to the low scan rate, which limits the number of compounds in one scan.Ultra-high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF MS) has the characteristics of high resolution, high sensitivity, high accuracy, short period, and wide scanning range.It is an important tool for studying the chemical constituents and quantitative analysis of complex samples [28,29],and it has been widely used in quantitative analysis of pesticide residues, veterinary drug residues and other research fields.

In this study, UPLC-Q-TOF MS technology was first used to determine the contents of 19 polyphenols in the tea.The analytes were determined by Q-TOF MS in TOF MS and IDA-MS/MS mode.In TOF MS mode, the target compounds qualified by the retention time, accurate mass, isotope distribution and isotope abundance ratio of the target, and quantitated by the peak area of the excimer ion peak.In IDA-MS/MS mode, the target compounds were further confirmed by the ion fragment information under the corresponding collision energy.It was suitable for the identification and quantitative research of polyphenols in the tea.It provided a reference for comprehensive and rapid analysis of tea ingredients.

2.Materials and methods

2.1 Reagents and standards

Methanol, acetonitrile, ethanol and formic acid were HPLC grade and were purchased from Merck (Darmstadt, Germany).Water was purified using a Milli-Q-System (Millipore, Guyancourt, France).p-Hydroxybenzoic acid (99.0%), ethyl gallate (98.0%), protocatechuic acid (98.0%), gallic acid (98.0%), pyrogallol (98.0%), catechin(98.0%), epicatechin (98.0%), chlorogenic acid (98.0%), caffeic acid(98.0%), quercetin (98.0%), kaempferol (98.0%) were purchased from Yuanye Biotechnology (Shanghai, China).Ferulic acid (99.3%),luteolin (96.0%) were purchased from Zhenxiang Technology(Beijing, China).Epigallocatechin gallate (96.0%), epigallocatechin(99.90%), epicatechin gallate (98.58%) were purchased from Dr.Ehrenstorfer (Augsburg, Germany).Gallocatechin (98.2%), catechin gallate (99.0%), gallocatechin gallate (98.4%) were purchased from ANPEL laboratory technologies (Shanghai, China).Black tea,anji white tea, green sword tea, Maojian tea, dandelion, yacca tea,matricariarecutita were collected from supermarkets in China.

2.2 Instruments

The high-speed refrigerated centrifuge (CR22N, Hitachi,Germany), the vortex mixer (Vortex Genius 3, IKA, Germany), and the ultrasonic cleaner (Elmasonic P300H, Elma, Germany) were used in the procedure of extraction.Quantitative analysis of target compounds were conducted on a TripleTOFTM5600+quadrupole/time of flight mass spectrometry (AB Sciex, USA).The separation of compounds were carried out on a LC-30AD UPLC system equipped with binary solvent manager, sample manager, and column manager(Shimadzu, Japan).

2.3 Sample preparation

The tea was pulverized uniformly on a small pulverizer, 1 g of pulverized sample was weighed and transferred to a 50 mL centrifuge tube.Following spiking 20 mL of 50% ethanol, vortexed for 1 min,and sonicated for 30 min.The sample was centrifuged at 8 000 r/min for 5 min.After centrifugation, the supernatant was filtered through a 0.22 μm nylon membrane before UPLC-Q-TOF MS detection.

2.4 Chromatographic conditions

The chromatographic separation was performed on an Acquity BEH C18column (100 mm × 2.1 mm, 2.5 μm; Waters, USA).The column temperature was 40 °C, the injection volume was 5.0 μL,The flow rate was 300 nL/min.Water containing 0.1% (V/V) formic acid (phase A) and acetonitrile (phase B) were used as mobile phase.0-3.00 min, 10% B; 3.0-13.0 min, 10% to 60% B; 13.0-17.0 min,60% B; 17.0–18.0 min, 60% to 10% B; 18.0–20.0 min, 10% B.

2.5 Mass spectrometry conditions

The MS analysis was performed using an electrospray ion source(ESI) in negative ionization mode.The optimized parameters of ion source were as follows: the ionization voltage was -4.5 kV,the source temperature was 500 °C, pressure of curtain gas was 30 psi, pressure of nebulizer gas was 50 psi, pressure of auxiliary gas was 55 psi; TOF MS conditions as follows: scan range wasm/z50-500, duration time was 20 min, accumulation time was 0.15 s.Only the ions that reached the IDA threshold can trigger the fragmentation and the product ion scan, IDA-MS/MS conditions as follows: accumulation time was 0.05 s, high sensitivity mode was set, exclude isotopes within 4 Da.Declustering potential was 80 V, collision energy was 35 V, dynamic energy of collision was 15 eV.Before each experiment, the instrument performed mass accuracy calibration through the Chromatography Data System(CDS).During the experiment, every 5 samples were run, the mass accuracy calibration was automatically performed.The mass spectrum information of 19 polyphenols were shown in Table 1.

Table 1Mass parameters for the 19 polyphenols.

3.Results and discussion

3.1 Optimization of extraction solvents

The type of extraction solvent directly affects the extraction effect of the target compound, so the choice of extraction solvent is crucial.This experiment investigated the extraction effect of 19 polyphenols when methanol, ethanol, and water were used as the extraction solvent.When methanol was used as the extraction solvent, the recovery of ethyl gallate was low.When water was used as the extraction solvent, the recovery of chlorogenic acid was low.Therefore, in the next step of the experiment, the extraction effect of water with various concentrations of ethanol (20%, 40%, 50%, 60%, 70%) were tested.As Fig.1 showed, 50% ethanol-water solution yielded the best reproducibility and recovery of the 19 polyphenols.Therefore, 50% ethanol-water solution was selected as the extraction solvent.

Fig.1 Comparison of extraction efficiency of ethanol-water in different proportions.

3.2 Optimization of extraction times

Optimization of extraction time was aimed to obtain maximum extraction efficiency.The extraction time of 10, 20, 30, 40 and 50 min were compared when 50% ethanol-water solution was used as the extraction solvent.As Fig.2 showed, within 30 min, the extraction efficiency of polyphenols gradually increase with the time, and the growth trend was large.After 30 min, the growth trend decelerates, and the extraction efficiency of polyphenols change little.Considering the extraction efficiency of the experiment and time cost, 30 min was chosen as extraction time.

Fig.2 Comparison of extraction efficiency at different extraction times.

3.3 Optimization of liquid chromatography conditions

Optimization of the liquid chromatographic conditions was investigated for the optimum separation of all the compounds.Different mobile phase systems acetonitrile-water, acetonitrile-0.1% (V/V)formic acid water, methanol-water and methanol-0.1% (V/V) formic acid water were used, methanol and acetonitrile as mobile phases have little effect on the signal intensity, but when methanol was used as the mobile phase for gradient elution, column pressure varies widely and equilibration takes longer.Acetonitrile-0.1%formic acid water as the mobile phase showed better separation and elution capabilities than the acetonitrile-water system, which can achieve effective separation of target substances.Therefore,acetonitrile-0.1% formic acid water solution was selected as the mobile phase for subsequent experiments.

3.4 Optimization of mass spectrometry parameters

The TripleTOFTM5600+instrument has a CDS automatic calibration infusion system.The reference spray of the DuoSprayTMion source was used to input the calibration solution for automatic system calibration.The DuoSprayTMion source has two types: electron spray ionization (ESI) and atmospheric pressure chemical ionization source (APCI).In this experiment, the ESI was selected as the detection ion source, and the APCI was used as the calibration ion source.Through the automatic calibration system, automatic batch calibration was performed to ensure the accurate mass of the system was stable for a long time.

Syringe injection was used to inject 19 polyphenols directly into the mass spectrometer in the positive and negative ionization mode.The results showed that compounds have higher response in negative ionization mode, so the negative ionization mode was used for detection.For this method, TOF MS and IDA MS/MS modes were adopted for detection of target analytes.Under TOF MS mode, the experiment investigated the response of target compounds under different declustering potentials (50-300 V), with the findings that at the fragmentor voltage of 80 V, compound response was the highest; relatively low fragmentor voltage was unfavorable for ion transmission,and overly high fragmentor voltage would cause the compound to fragment within the source.The accurate mass, retention time, isotope ratio was obtained.Fig.3 showed the extracted ion chromatograms of 19 polyphenols (100 ng/mL).The accurate mass deviations of target compounds were less than 5.0 × 10–6(Table 1), which meets the requirements of high resolution mass spectrometry detection.The MS/MS scan was run in an IDA MS/MS mode.The MS/MS spectra of the target was used for the final confirmation of the initial screening results of the accurate mass (Fig.4).

Fig.3 Extracted ion chromatograms of the 19 polyphenols.A.p-hydroxybenzoic acid; B.ethyl gallate; C.protocatechuic acid; D.gallic acid; E.pyrogallol;F.catechin; G.epicatechin; H.chlorogenic acid; I.caffeic acid; J.ferulic acid; K.quercetin; L.luteolin; M.kaempferol; N.epigallocatechin gallate; O.epigallocatechin; P.epicatechin gallate; Q.gallocatechin; R.catechin gallate; S.gallocatechin gallate.Same as in Fig.4.

Fig.4 MS/MS spectra of 19 polyphenols.

Fig.4 (Continued)

3.5 Linearity and sensitivity

The linearity of the method was determined by constructing calibration curves with different concentrations of 19 polyphenols.As Table 2 showed, the linear range was studied by preparing a calibration curve with a concentration range of 0.2-500 μg/L for each compound, and a good linear relationship with correlation coefficient (R2) between 0.999 5 and 0.999 9 was achieved for 19 polyphenols in their respective linear range.Limits of detection (LODs) and limits of quantification (LOQs)were evaluated using the spiked samples, LODs and LOQs of the 19 polyphenols in tea samples were calculated by signalto-noise ratio of 3 and 10 (the ratio between intensity of signal of each compound obtained under TOF MS mode and intensity of noise in a spiked sample).The LODs of 19 polyphenolic compounds were in range of 0.002–0.100 mg/kg, The LOQs of 19 polyphenolic compounds were in range of 0.004–0.200 mg/kg.LOD and LOQ for the methods on determination of 19 polyphenols in the tea were shown in Table 2.

Table 2Linearity, LOD and LOQ of the 19 polyphenols.

3.6 Recovery and precision

The recoveries of analytes were calculated using the samples spiked at 3 levels (1 × LOQ, 3 × LOQ, 10 × LOQ), and each level was repeated 6 times.Precision of the method was expressed by relative standard deviation (RSD).The data of recovery and precision were given in Table 3, the average recoveries of 19 polyphenols were in range between 78.4% and 109.2%.The RSDs were in range of 1.33%-9.72%.

Table 3Spiked recoveries and RSDs of the 19 polyphenols.

Table 3 (Continued)

3.7 Application to actual samples

In order to investigate the content of 19 polyphenols in commercially available tea leaves, 7 tea samples from local supermarket were analyzed using the developed method in this study.Their detection results were shown in Table 4, the compositions and contents of polyphenols were different in 7 tea samples.The contents of epigallocatechin gallate in black tea, anji white tea, green sword tea, mao jian tea and yacca tea were the highest, which were 1 493,4 759, 4 657, 5 608 and 6 250 mg/kg, respectively.The contents of chlorogenic acid in dandelion and matricariarecutita were the highest, which were 432.7 and 2 981 mg/kg, respectively.The order of total contents of the 19 polyphenols from highest to lowest were anji white tea, mao jian tea, green sword tea, yacca tea, black tea,matricariarecutita and dandelion.

Table 4Content of the 19 polyphenols in actual samples.

4.Conclusions

In this experiment, a rapid and sensitive UPLC-Q-TOF MS method was developed to analyze 19 polyphenols in tea sample.The linearity, sensitivity, accuracy and precision of the method were investigated.This method can meet the needs of rapid and accurate quantification of 19 polyphenols in tea samples, has great significance for tea quality control, and is a powerful supplement to the existing national standards for detecting polyphenols in food.

Conflict of interest

The authors declared that they have no conflicts of interest to this work.

Acknowledgements

This work was supported by National Key Research and Development Program of China (2018YFC1603400).