Analysis method for simultaneously analyzing various non-volatile compounds in tea leaves by using UPLC-MS / MS and application of analysis method

Abstract

The invention discloses an analysis method for simultaneously analyzing various non-volatile compounds in tea leaves by using UPLC-MS / MS. The method for simultaneously analyzing amino acid, alkaloid, nucleoside and nucleotide non-volatile compounds in tea leaves comprises the following steps: firstly, accurately weighing a tea leaf sample in a centrifugal tube, adding boiling water, uniformly mixing and soaking, and then, carrying out ultrasonic treatment; taking a proper amount of sample, passing through a microfiltration membrane, and putting into a sample injection bottle for detection; secondly, setting instrument conditions: an ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, gradient elution: a mobile phase A is chromatographic grade acetonitrile, a mobile phase B is 0.1% formic acid aqueous solution, the flow velocity is 0.200 mL / min, and positive ion scanning is carried out; monitoring multiple reaction ions; the method is high in accuracy, low in detection limit, stable and reliable, and the tea quality is judged by performing data analysis on 43 types of non-volatile compounds such as amino acids, alkaloids, nucleosides and nucleotides in commercially available special-grade Zunyi black tea and physical standard samples through the analysis method.

Description

Technical Field

[0001] The invention belongs to the field of food analysis, and specifically relates to an analysis method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS and an application thereof. Background Art

[0002] The color, aroma, taste and other quality characteristics of tea are affected by the combined effects of a variety of internal substances, especially the secondary metabolites such as catechins, caffeine and theanine, which are relatively abundant in the tea tree, as well as metabolites such as tea saponins, aroma substances, vitamins, etc. High-quality tea is not only more outstanding in appearance, aroma and taste, but also more reliable in terms of nutritional content and safety. The quality and safety of tea in my country is maintained at a high level, ensuring the safety of tea drinking and the safety of the industry. High-quality tea usually means that tea meets strict standards in growth, picking, processing and other links. The quality of tea is also directly related to the sustainable development of the tea industry and the reputation of the company. It fundamentally improves product competitiveness and credibility, meets market demand, and promotes the high-quality development of the tea industry.

[0003] Therefore, the importance of tea quality is indisputable and has a far-reaching impact on consumers, producers and the entire tea industry. Physical standard samples are the basis for formulating and supervising product quality, ensuring that the quality of commercially available tea meets the specified standards. In the tea market, physical standard samples can ensure that the appearance, aroma, taste and other characteristics of tea meet consumer expectations. Whether commercially available tea meets the requirements of physical standard samples directly affects consumers' trust and satisfaction with the product. If commercially available tea is consistent with the physical standard sample, consumers will be more assured when purchasing, increasing the possibility of purchase; conversely, if the commercially available tea is significantly different from the standard sample, consumers may have doubts about the product quality and reduce their trust in the brand. Therefore, companies that produce and sell tea should strictly abide by the provisions of physical standard samples to ensure stable product quality, enhance brand image, and win the trust of consumers. At the same time, there are many problems in the process of "Zunyi Red" entering the market, such as insufficient identification ability of consumers, irregular market, and many counterfeit products. Regulatory authorities also need to strengthen the quality sampling of commercially available tea to ensure that tea products on the market meet relevant standards, protect consumer rights, and promote the healthy development of the tea industry. There are many research methods for non-volatile compounds in black tea, such as high performance liquid chromatography and high performance liquid chromatography tandem mass spectrometry. Non-volatile substances contribute to the formation of the taste and health benefits of tea. Based on the preparation of "two green and one red" physical standards in our institute, the present invention establishes UPLC-MS / MS quantitative analysis technology for amino acids, alkaloids, nucleosides, nucleotides and other types of non-volatile compounds, and applies its analysis method to analyze the data of 43 types of non-volatile compounds such as amino acids, alkaloids, nucleosides, and nucleotides in the commercially available and physical standard special Zunyi Red black tea to judge the quality of tea. Summary of the invention

[0004] In view of the fact that there is no analytical method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves in the prior art, the present invention provides a method for simultaneously analyzing amino acids, alkaloids, nucleosides and nucleotides in tea leaves using UPLC-MS / MS, and applies the analytical method to perform data analysis on 43 types of non-volatile compounds such as amino acids, alkaloids, nucleosides and nucleotides in commercially available and physical standard premium Zunyi black tea to judge the quality of the tea leaves.

[0005] The technical scheme of the present invention comprises: an analysis method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS, and simultaneously analyzing amino acids, alkaloids, nucleosides and nucleotides in tea leaves using UPLC-MS / MS, comprising the following steps:

[0006] (1) Accurately weigh 0.1 g of tea sample into a 50 mL centrifuge tube, add 10 mL of boiling water, mix well, soak for 15 min, take an appropriate amount of sample through a 0.2 μm microporous filter membrane and place in a 2 mL injection bottle for testing;

[0007] (2) Instrument conditions: Ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer (Agilent 1290 infinity II, chromatographic column Agilent Eclipse Plus C18 RRHD 1.8 μm 2.1 × 100 mm) Gradient elution: Mobile phase A: chromatographic grade acetonitrile, B: 0.1% formic acid in water, 0-5.5 min 2% A + 98% B, 5.5-7 min 3% A + 97% B, 7-8 min 5% A + 95% B, 8-9 min 55% A + 45% B, 9-10 min 95% A + 5% B, 10-12 min 95% A + 5% B, 12-12.1 min 2% A + 98% B, 12.1-14 min 2% A + 98% B flow rate 0.200 mL / min, maximum pressure limit: 1300 bar, mass spectrometry conditions: source gas parameters: ion source: electrospray ion source; scanning mode: positive ion scanning; detection mode: multiple reaction ion monitoring (MRM); spray voltage: 3500 V; ion source temperature: 550°C; auxiliary gas 1: 50 psi.

[0008] Non-volatile compounds of amino acids, alkaloids, nucleosides and nucleotides include: histidine, serine, glycine, arginine, aspartic acid, homoserine, threonine, alanine, lysine, glutamine, glutamic acid, γ-aminobutyric acid, cysteine, cytosine, proline, cytosine nucleotide, uracil nucleotide, valine, cytosine nucleoside, theanine, adenine nucleotide, inosine nucleotide, adenine, isoleucine, uridine, uracil, asparagine, leucine, tyrosine, xanthine, adenine nucleoside, inosine nucleoside, inosine, guanosine, guanine, methionine, phenylalanine, xanthine nucleoside, thymidine nucleoside, tryptophan, theobromine, theophylline, and caffeine.

[0009] The mass spectrometry parameters are shown in Table 1:

[0010] Table 1 Mass spectrometry parameters of compounds

[0011]

[0012]

[0013]

[0014] Preferably the tea used in the analysis is black tea.

[0015] The application of this analytical method is to judge the quality of tea by comparing the data of non-volatile compounds such as amino acids, alkaloids, nucleosides, and nucleotides in commercially available tea and physical standard samples.

[0016] The beneficial effects achieved by the present invention compared with the prior art are as follows: there is no analysis method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves in the prior art. The present invention provides a method for simultaneously analyzing amino acids, alkaloids, nucleosides and nucleotides in tea leaves using UPLC-MS / MS. The method of the present invention has high accuracy, low detection limit, stability and reliability. The analysis method of the present invention is used to analyze the data of 43 types of non-volatile compounds of amino acids, alkaloids, nucleosides and nucleotides in commercially available and physical standard samples of special Zunyi red black tea. Among them, the median values ​​of 15 components have a content deviation greater than or equal to 1 from the special standard sample, proving that the above components can reach the content standard of the special Zunyi red physical standard sample. In addition, the median content deviation of serine (Ser), arginine (Arg), homoserine (Hser), threonine (Thr), etc. is about 0.8-0.9, which is not much different from the content deviation of the special physical standard sample. The median value of the remaining ingredients in commercially available Zunyi black tea deviates from the content of the special-grade standard by less than 1, ranging from about 0.4 to 0.7. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Attached Figure 1 : Total ion chromatogram of nonvolatile compounds in tea leaves. DETAILED DESCRIPTION

[0018] Example 1

[0019] 1 Detection limit, repeatability, linearity and recovery

[0020] The detection limit / quantification limit is calculated using S / N (h sample / h noise), where the qualitative detection limit of chromatographic analysis is defined as the sample amount required when the response value is three times the baseline noise; the quantitative detection limit of chromatographic analysis is defined as the sample amount required when the response value is ten times the baseline noise. Repeatability is measured using the relative standard deviation of 6 times at three concentrations of 50 mg / kg, 250 mg / kg, and 500 mg / kg. The spiked recovery rate was calculated after three concentrations of 25.00 μg / g, 100 μg / g, and 150 μg / g were added for measurement. The detection limit, quantification limit, repeatability, linearity, and recovery rate of the method of the present invention were judged for compliance with GB / T 27404-2008 "Laboratory Quality Control Specifications for Physical and Chemical Testing of Foods", and all met the requirements of the national standard. The results are shown in Table 2, and the total ion flow diagram is attached. Figure 1 As shown:

[0021] Table 2 Detection limit, quantification limit, repeatability, linearity and recovery of the method

[0022]

[0023]

[0024] Example 2

[0025] 1 Materials and reagents

[0026] 1.1 Experimental Materials

[0027] The physical standard sample of the special grade Zunyi black tea prepared by our institute; the information of the commercially available "Zunyi black" black tea is shown in Table 3:

[0028] Table 3 Information of commercially available "Zunyi Red" black tea

[0029]

[0030] 2.2 Reagents and instruments

[0031] Standard solution and reagents: standard substances (histidine, serine, glycine, arginine, aspartic acid, homoserine, threonine, alanine, lysine, glutamine, glutamic acid, γ-aminobutyric acid, cysteine, cytosine, proline, cytosine nucleotide, uracil nucleotide, valine, cytosine nucleoside, theanine, adenine nucleotide, inosine nucleotide, adenine, isoleucine, uridine, uracil, asparagine, leucine, tyrosine, xanthine, adenine nucleoside, inosine nucleotide, inosine, guanosine, guanine, methionine, phenylalanine, xanthine nucleoside, thymidine, tryptophan, theobromine, theophylline, caffeine), first-grade water, chromatographic grade acetonitrile, chromatographic grade formic acid.

[0032] Instruments and equipment used for inspection: ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer (Agilent1290infinityII, chromatographic column Agilent EclipsePlus C18 RRHD 1.8μm 2.1×100mm), centrifuge (Beijing Bolixing Instrument Co., Ltd.), vortex mixer (Multi-Tube Vortexer), electronic balance (METTLER TOLEDO, 1 / 10,000), induction cooker (Joyoung Co., Ltd.), digital display constant temperature water bath (Changzhou Zhiborui Instrument Manufacturing Co., Ltd.).

[0033] 2 Pre-treatment steps and inspection methods

[0034] 2.1 Experimental procedures

[0035] Accurately weigh 0.1 g of sample into a 50 mL centrifuge tube, add 10 mL of boiling water, mix well, soak for 15 min, take an appropriate amount of sample through a 0.2 μm microporous filter membrane and place in a 2 mL injection bottle for testing.

[0036] 2.2 Inspection methods

[0037] 2.2.1 Gradient elution conditions are shown in Table 4:

[0038] Table 4 Gradient elution conditions

[0039]

[0040] 2.2.2 LC-MS / MS parameters

[0041] The mass spectrometry parameters of the compounds are shown in Table 5 below:

[0042] Table 5 Mass spectrometry parameters of compounds

[0043]

[0044]

[0045]

[0046] 3. Analysis of results of method application

[0047] The present invention uses UPLC-MS / MS to perform data analysis on non-volatile compounds such as amino acids, alkaloids, nucleosides, and nucleotides in commercially available and physical standard special-grade Zunyi red tea. The content ranges of 43 components in commercially available special-grade Zunyi red tea are shown in Table 6. Among them, the median values ​​of 15 components have a content deviation greater than or equal to 1 from the special-grade standard sample, proving that the above components can reach the content standard of the special-grade Zunyi red physical standard sample. In addition, the median content deviations of serine (Ser), arginine (Arg), homoserine (Hser), threonine (Thr), etc. are about 0.8-0.9, which is not much different from the content deviation of the special-grade physical standard sample. The median values ​​of the remaining components in commercially available Zunyi red tea have a content deviation less than 1 from the special-grade standard sample, which is about 0.4-0.7.

[0048] Table 6 Content of physical standard sample and commercially available special grade Zunyi black tea (μg / mL)

[0049]

[0050]

Claims

1. A method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS, characterized in that: The simultaneous analysis of amino acids, alkaloids, nucleosides and nucleotides in tea leaves using UPLC-MS / MS includes the following steps: (1) Accurately weigh 0.1 g of tea sample into a 50 mL centrifuge tube, add 10 mL of boiling water, mix well, soak for 15 min, take an appropriate amount of sample through a 0.2 μm microporous filter membrane and place in a 2 mL injection bottle for testing; (2) Instrument conditions: Ultra-high performance liquid chromatography triple quadrupole tandem mass spectrometer, gradient elution: mobile phase A: chromatographic grade acetonitrile, B: 0.1% formic acid in water, 0-5.5 min 2% A + 98% B, 5.5-7 min 3% A+97% B, 7-8min 5% A+95% B, 8-9min 55% A+45% B, 9-10min 95% A+5% B, 10-12min 95% A+5% B, 12-12.1min 2% A+98% B, 12.1-14min 2% A+98% B flow rate 0.200mL / min, maximum pressure limit: 1300bar, mass spectrometry conditions: source gas parameters: ion source: electrospray ion source; scanning mode: positive ion scanning; detection mode: multiple reaction ion monitoring; spray voltage: 3500V; ion source temperature: 550℃; auxiliary gas 1: 50psi.

2. The method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS according to claim 1, characterized in that: Non-volatile compounds of amino acids, alkaloids, nucleosides and nucleotides include: histidine, serine, glycine, arginine, aspartic acid, homoserine, threonine, alanine, lysine, glutamine, glutamic acid, γ-aminobutyric acid, cysteine, cytosine, proline, cytosine nucleotide, uracil nucleotide, valine, cytosine nucleoside, theanine, adenine nucleotide, inosine nucleotide, adenine, isoleucine, uridine, uracil, asparagine, leucine, tyrosine, xanthine, adenine nucleoside, inosine nucleoside, inosine, guanosine, guanine, methionine, phenylalanine, xanthine nucleoside, thymidine nucleoside, tryptophan, theobromine, theophylline, and caffeine.

3. The method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS according to claim 2, characterized in that: Mass spectrometry parameters: hypoxanthine nucleotide: parent ion 349, quantitative ion 136.8, collision voltage 17V, qualitative ion 110, collision voltage 48V, capillary outlet voltage 5V; adenine nucleotide: parent ion 348.1, quantitative ion 135.9, collision voltage 33V, qualitative ion 97.05, collision voltage 15V, capillary outlet voltage 5V; uracil nucleotide: parent ion 325.2, quantitative ion 97.05, collision voltage 15V, qualitative ion 41 .15, collision voltage 50V, capillary outlet voltage 5V; cytosine nucleotide: parent ion 324, quantitative ion 112.05, collision voltage 16V, qualitative ion 94.55, collision voltage 50V, capillary outlet voltage 5V; xanthine nucleoside: parent ion 285.25, quantitative ion 152.7, collision voltage 12V, qualitative ion 133.1, collision voltage 11V, capillary outlet voltage 5V; guanosine nucleoside: parent ion 284.25, quantitative ion 1 52.1, collision voltage 13V, qualifier ion 135.05, collision voltage 42V, capillary outlet voltage 5V; inosine: parent ion 269.25, quantifier ion 137.05, collision voltage 12V, qualifier ion 110.1, collision voltage 41V, capillary outlet voltage 5V; adenine: parent ion 268.25, quantifier ion 136.05, collision voltage 19V, qualifier ion 119.05, collision voltage 49V, capillary outlet voltage Pressure 5V; Uridine: parent ion 245.2, quantitative ion 113, collision voltage 11V, capillary outlet voltage 5V; Cytosine: parent ion 244.2, quantitative ion 112.1, collision voltage 13V, qualitative ion 95, collision voltage 44V, capillary outlet voltage 5V; Thymidine: parent ion 243.25, quantitative ion 127.1, collision voltage 25V, qualitative ion 60.05, collision voltage 15V, capillary outlet voltage 5V; Tryptophan: parent ion 205.25, quantitative ion 188.1, collision voltage 10V, qualifier ion 118.15, collision voltage 25V, capillary outlet voltage 5V; Caffeine: parent ion 195, quantitative ion 138.1, collision voltage 22V, qualifier ion 110.15, collision voltage 42V, capillary outlet voltage 5V; Tyrosine: parent ion 182.2, quantitative ion 135.8, collision voltage 14V, qualitative ion 91.2, collision voltage 29V, capillary outlet voltage 5V; Theobromine: parent ion 180.95, quantitative ion 138.05, collision voltage 21V, qualitative ion 110.1, collision voltage 25V, capillary outlet voltage 5V; Theophylline: parent ion 180.95, quantitative ion 124.1, collision voltage 21V, qualitative ion 69.1, collision voltage 34V, capillary outlet voltage 5V; Arginine: parent ion 175.2, quantitative ion 70.25, collision voltage 22V, qualifier ion 60.15, collision voltage 16V, capillary outlet voltage 5V.

4. The method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS according to claim 1, characterized in that: The tea is black tea.

5. The use of the method for simultaneously analyzing multiple types of non-volatile compounds in tea leaves using UPLC-MS / MS according to claims 1-4, characterized in that: The quality of tea is judged by comparing the data of amino acids, alkaloids, nucleosides and nucleotides in commercially available tea and physical standard samples through UPLC-MS / MS.

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