Mass spectrometric method for qualitative analysis of tea procyanidins and polymeric catechins

Abstract

The application provides a qualitative method for tea proanthocyanidin and polyester catechin, comprising the following steps: UPLC-HRMS analysis is performed on multiple pretreated tea standard samples and mixed standard samples QC respectively, RSD and FC values of the multiple pretreated tea standard samples and the mixed standard samples QC are calculated, and one-time screening information is obtained according to the RSD and FC values; the one-time screening information is screened again by using a precursor ion list to obtain potential mass spectrum peaks; MS 2 fragmentation pathways are summarized to obtain mass spectrum characteristic information; the above steps are used for detecting and analyzing the tea to be detected to obtain potential mass spectrum peaks, and the potential mass spectrum peaks of the tea sample to be detected are compared with the mass spectrum characteristic information to identify the types of proanthocyanidin and polyester catechin. The qualitative method can realize efficient identification and identification of proanthocyanidin and polyester catechin in tea.

Description

Technical Field

[0001] This invention relates to the field of tea detection and analysis, specifically to a mass spectrometry method for the qualitative analysis of proanthocyanidins and polyester catechins in tea. Background Technology

[0002] Flavan-3-ol oligomers are a class of chemical components in tea that possess important sensory qualities and various biological activities. They mainly include proanthocyanidins (PAs), polyester catechins (TSs), and theaflavins (TFs). PAs in tea not only significantly influence the taste and color of the tea infusion but also possess various biological activities, such as free radical scavenging and antioxidant capabilities. TSs are compounds linked by C2′→C2′ bonds, formed between catechin monomers through enzymatic oxidation reactions. Currently, research on tea proanthocyanidins and polyester catechins still has limitations. For example, most proanthocyanidin studies focus on dimers, with a few studies involving trimers, and the highest degree of polymerization of tea proanthocyanidins reported in the literature is 4. Furthermore, approximately 12 types of TSs have been reported in tea, but existing research mainly focuses on dimer TSs. Therefore, further comprehensive characterization of proanthocyanidins and polyester catechins in tea is essential.

[0003] Uplift-lower pressure chromatography-resonance microscopy (UPLC-HRMS) boasts advantages such as high resolution, high sensitivity, and high throughput, making it a powerful technique for qualitative and quantitative analysis of various compounds. It is widely used for the detection and identification of flavan-3-ol oligomers such as catechins, proanthocyanidins, polyester catechins, and theaflavins in tea. However, analyzing proanthocyanidins and polyester catechins in tea samples remains a valuable yet challenging task. On the one hand, the content of proanthocyanidins and polyester catechins in tea is much lower than that of other components, including monocatechins, flavonoids, and amino acids. For example, proanthocyanidin B1 accounts for only 5.92% of the EGCG content in raw Pu-erh tea. According to literature, the TSsA content in white tea is only about 2 mg / g. Due to its low content, flavan-3-ol oligomers always produce relatively low mass spectrometric responses, largely masked by other major ions or background noise. On the other hand, proanthocyanidins and polyester catechins exhibit structural diversity and multiple isomers. Many compounds in this class have the same molecular weight and the same monomers, but are linked by different polymerization mechanisms. For example, TSs A, TSs D, prodelphinidin B2-3,3'-di-O-gallate, and prodelphinidin B5-3,3'-di-O-gallate are all isomers formed by the polymerization of two EGCG molecules. Because proanthocyanidins and polyester-type catechins have multiple isomers that are difficult to distinguish using UPLC-HRMS, this presents a significant challenge for the UPLC-HRMS analysis of proanthocyanidins and polyester-type catechins.

[0004] Furthermore, the lack of standards makes the qualitative analysis of flavan-3-ol oligomers in tea leaves extremely difficult. When standards are unavailable, matching mass spectrometry information to databases is a common compound annotation method. However, reliable MS / MS spectra of proanthocyanidins and polyester catechins stored in public databases such as MassBank and GNPS are very limited. Therefore, the low abundance, lack of standards and reference MS / MS spectra, and the structural diversity of these compounds themselves pose significant challenges to UPLC-HRMS analysis of proanthocyanidins and polyester catechins. Summary of the Invention

[0005] To address the problems in existing technologies where the content of proanthocyanidins and polyester catechins in tea is relatively low, making them easily masked by high-signal compounds and difficult to distinguish, this invention proposes a qualitative method for identifying proanthocyanidins and polyester catechins in tea.

[0006] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows.

[0007] A qualitative method for identifying proanthocyanidins and polyester catechins in tea leaves includes the following steps:

[0008] Multiple teas were extracted separately to obtain multiple tea standard samples; multiple tea standard samples were mixed in equal volumes to obtain tea mixed standard samples; multiple tea standard samples and tea mixed standard samples were analyzed by UPLC-HRMS, and raw MS information was extracted using MZmine software; RSD and FC values ​​were calculated using raw MS information, and raw MS information was screened using RSD and FC values ​​to obtain first-screened MS information;

[0009] Calculate the predicted precursor ion values ​​for the n-mer compound and construct a list of precursor ions;

[0010] The formula for predicting precursor ion values ​​is as follows:

[0011] MW = M1 + M2 + ... + M n -(2n-2)×1.008, EW=MW-1.008;

[0012] Where MW is the predicted molecular weight, EW is the predicted molecular weight of the oligomer in negative ion mode, n is the number of catechin monomers participating in polymerization, Mn represents the molecular weight of the nth catechin monomer, and 1.008 represents the monoisotope atomic mass of element H.

[0013] The precursor ion values ​​in the MS information of the first screening are compared with the predicted precursor ion values. The precursor ion values ​​that are the same as the predicted precursor ion values ​​are retained, and the mass spectrum peaks corresponding to the precursor ion values ​​are the potential mass spectrum peaks.

[0014] MS of potential mass spectrometry peaks 2 The lysis pathway was analyzed to obtain mass spectrometry characteristics of multiple tea standard samples and mixed tea standard samples;

[0015] The tea sample to be tested was analyzed according to the above method to obtain the potential mass spectrometry peaks of the tea sample. The MS values ​​of the potential mass spectrometry peaks of the tea sample were then analyzed. 2 The fragmentation information was compared with the mass spectrometry characteristics to identify the types of proanthocyanidins and polyester catechins in the tea sample to be tested.

[0016] In a preferred embodiment, the specific process of the first filtering step is as follows:

[0017] Calculate the relative standard deviation (RSD) and the factor of difference (FC), and retain MS information with RSD ≤ 20% and FC ≥ 10% to obtain MS information for the first filtering stage;

[0018] RSD and difference factor FC are calculated according to the following equations (1) and (2);

[0019] The formula for calculating RSD is as follows:

[0020] RSD=IntSQC / IntMQC×100% (1)

[0021] Where IntSQC represents the standard deviation of the peak intensity of a specific chromatographic peak in the mixed standard sample QC, and IntMQC represents the average value of the peak intensity of a specific chromatographic peak in the mixed standard sample QC;

[0022] The formula for calculating the difference factor FC is as follows:

[0023] Difference factor FC = IntMQC / IntMBK(2)

[0024] Wherein, IntMQC represents the average peak intensity of a specific chromatographic peak in the mixed standard sample QC, and IntMBK represents the average peak intensity of a specific chromatographic peak in the blank sample.

[0025] In a preferred embodiment, the formula for calculating the predicted precursor ion value is derived based on the known chemical structural rules of proanthocyanidins and known polyester catechins.

[0026] In a preferred embodiment, the specific chemical structural regularity is as follows:

[0027] The known proanthocyanidins and the known polyester catechins are formed by linking monomeric catechins through C–C bonds. When two monomeric catechins are linked through C–C bonds, hydrogen is removed. The amount of hydrogen lost is twice the amount of the monomeric catechins minus 2.

[0028] In a preferred embodiment, the MS of the potential mass spectrometry peak 2 The specific process for analyzing the pyrolysis pathway is as follows:

[0029] MS based on the known proanthocyanidins in the potential mass spectrometry peaks 2 Fracturing information, MS of known proanthocyanidins 2 The fragmentation pathways were analyzed and summarized to obtain the mass spectrometry characteristics of known proanthocyanidins;

[0030] MS based on the known polyester-type catechins in the potential mass spectrometry peaks 2 Pyrolysis information, MS of known polyester catechins 2 The fragmentation pathways were analyzed and summarized to obtain the mass spectrometry characteristics of known polyester catechins.

[0031] In a preferred embodiment, the mass spectrometry feature information includes feature fragment information and neutral loss information; the MS values ​​of the potential mass spectrometry peaks of the tea leaves to be detected... 2 Fragmentation information includes characteristic fragment information and neutral loss information.

[0032] In a preferred embodiment, the MS of the known proanthocyanidins 2 The specific process of fragmentation pathway analysis is as follows:

[0033] When the proanthocyanidin structure is known to contain at least one catechol-type catechin, it is known that the proanthocyanidin can produce a characteristic fragment m / z 407.08 and a neutral loss of 152.05 Da;

[0034] When the proanthocyanidin structure is known to contain at least one pyrogallol-type catechin, it is known that the proanthocyanidin can produce a characteristic fragment m / z of 423.07 and a neutral loss of 168.04 Da;

[0035] When the precursor ions in the proanthocyanidin structure are known to be continuously cleaved, proanthocyanidins are known to produce fragment ions of the corresponding monomeric flavan-3-ol with m / z 289.07, m / z 305.07, m / z 441.09, and m / z 457.08. In this case, when proanthocyanidins contain compounds linked by C4→C8 bonds, the relative response intensity of the proanthocyanidin fragment ions with m / z 423.07 or m / z 407.08 is known to be lower than that of the monomeric flavan-3-ol fragment ions. When proanthocyanidins contain compounds linked by C4→C6 bonds, the relative response intensity of the proanthocyanidin fragment ions with m / z 423.07 or m / z 407.08 is known to be higher than that of the monomeric flavan-3-ol fragment ions.

[0036] In a preferred embodiment, the MS of the known polyester-type catechin 2 The specific process of fragmentation pathway analysis is as follows:

[0037] When the parent nucleus structure of polyester-type catechins is known to be two pyrogallol-type catechins, it is known that polyester-type catechins can produce characteristic fragment ions with m / z 609.13, m / z 591.12, m / z 453.08, m / z 471.10, and m / z 333.06.

[0038] When the parent structure of polyester-type catechins is known to be a pyrogallol-type catechin and a dihydroxyapatol-type catechin, it is known that polyester-type catechins can produce characteristic fragments at m / z 593.13, m / z 575.12, m / z 437.09, m / z 455.10, and m / z 317.07.

[0039] In a preferred embodiment, the raw MS information includes precursor ion value, retention time, MS information, and MS... 2 Information, peak area.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) This invention calculates the RSD and FC values ​​based on the original MS information of tea leaves, and uses the RSD and FC values ​​to perform a first screening of the original MS information. Then, it performs a second screening of the precursor ion values ​​in the MS information after the first screening based on the constructed precursor ion list. This allows for the rapid and targeted extraction of the mass spectrometry information of proanthocyanidins and polyester catechins in tea samples. The MS values ​​of the proanthocyanidins and polyester catechins corresponding to the extracted second-screen information are then analyzed. 2 The fragmentation pathway was analyzed and summarized, and mass spectrometry (MS) characteristic information was obtained. MS peaks in the characteristic information were annotated, ultimately leading to the comprehensive discovery and annotation of 7 monomeric catechins and 52 oligomers from tea leaves. The 52 oligomers were 2-6 polymers, and for the first time, proanthocyanidins and polyester-type catechins with a degree of polymerization of 5-6 were detected in tea leaves. The method of this invention can rapidly and effectively extract masked proanthocyanidins and polyester-type catechins from MS peaks with high response intensity. Furthermore, the 7 monomeric catechins and 52 oligomers annotated using this method can serve as standards, providing assistance for the qualitative analysis of flavan-3-ol oligomers in tea leaves.

[0042] (2) This invention utilizes UPLC-HRMS technology to first screen the obtained raw MS information using RSD and FC values, and then screen it again using a precursor ion list. This allows for the rapid and efficient extraction of the masked MS peaks of proanthocyanidins and polyester catechins, thereby enabling accurate and rapid annotation of proanthocyanidins and polyester catechins in tea. This achieves efficient identification and characterization of proanthocyanidins and polyester catechins, laying the foundation for research on the bioactivity and flavor material basis of tea. Attached Figure Description

[0043] Figure 1 The TIC plots are for four types of tea, m / z 120-1200, and the EIC plots are for proanthocyanidins and polyester catechins identified and annotated using this method.

[0044] Figure 2 The TIC plots are for four types of tea, m / z 1200-2600, and the EIC plots are for proanthocyanidins and polyester catechins identified and annotated using this method.

[0045] Figure 3 To compare the number of mass spectrometry peaks in the original UPLC dataset of four types of tea with the number of mass spectrometry peaks identified and annotated using this method.

[0046] Figure 4 The diagram shows the characteristic neutral loss, secondary fragment ions, fragmentation pathways, and summary of proanthocyanidins and polyester-type catechins.

[0047] Figure 5MS of four standards for proanthocyanidins and polyester catechins 2 Figure and pyrolysis pathway. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0049] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the materials and equipment used in the embodiments of the present invention can be purchased on the market.

[0051] In the following embodiments of the present invention, since many proanthocyanidins and polyester catechins in tea are masked by MS peaks with high response intensity and are difficult to distinguish, the main approach is to mine and annotate potential target components from complex UPLC-HRMS raw datasets through appropriate and effective data processing to quickly and efficiently extract the masked MS peaks. Specifically, prior information based on certain characteristics, such as the known mass-to-charge ratio of precursor ions, key component ions, and characteristic neutral loss, can improve the efficiency of data mining, which is highly beneficial for quickly eliminating interfering MS information and identifying target components. For proanthocyanidins and polyester catechins, the catechin monomers constituting them are well-defined. Therefore, the theoretical molecular formula and molecular weight of the corresponding polymers can be calculated based on the polymerization method and degree of polymerization, and the chromatographic-mass spectrometric information of these components can be used for targeted extraction. It should be noted that the information extracted through targeted extraction requires further annotation to further clarify the corresponding chemical structure and determine whether it truly belongs to proanthocyanidins, polyester catechins, or any class of flavan-3-ol oligomers. In mass spectrometry analysis, each class or compound undergoes characteristic fragmentation, producing characteristic fragment ions and neutral loss. These characteristic MS... 2 Information can provide crucial evidence for the structural annotation of compounds. Therefore, it is essential to analyze the MS of known proanthocyanidins and polyester-type catechins. 2 The fragmentation patterns were analyzed and summarized, and MS characteristics that can be used for the qualitative analysis of proanthocyanidins and polyester catechins were extracted. Therefore, establishing a rapid, efficient, and accurate method for the chemical composition analysis of proanthocyanidins and polyester catechins is of great significance for providing a methodological basis for the identification of tea quality, production processes, and product quality stability.

[0052] The following section describes how to use the UPLC-HRMS method and appropriate and effective data processing to mine and annotate potential target components from complex UPLC-HRMS raw datasets of four tea varieties collected from major tea-producing areas in Yunnan Province, in order to quickly and efficiently extract masked MS peaks. The specific method is as follows:

[0053] I. Material Preparation:

[0054] 1.1 Main Materials and Instrument Parameters

[0055] 1.1.1 Main Materials

[0056] The tea samples were collected from 24 locations in Yunnan's major tea-producing areas, including Dali, Baoshan, Dehong, Lincang, Pu'er, and Xishuangbanna. Among them, there were 6 samples each of black tea, green tea, dark tea, and white tea.

[0057] 1.1.2 Instrument Parameters

[0058] UPLC-HRMS was performed using an instrument platform consisting of a Dionex U3000 UHPLC system and a Q-Exactive mass spectrometer connected in series.

[0059] 1) Chromatographic conditions:

[0060] 120-1200 m / z chromatographic columns are Acquity HSS C18, 100mm × 2.1mm, 1.8μm, Waters analytical column. Acquity columns (1200-2600 m / z) are also used. CSH C18, 100mm×2.1mm, 1.7μm, Waters.

[0061] The mobile phase consists of A-acetonitrile and B-an aqueous solution containing 0.1% formic acid.

[0062] The column oven temperature was 35℃, the injection volume was 1μL, and the chromatographic separation was performed at a flow rate of 0.3mL / min.

[0063] The elution gradients are as follows: 0-3.0 min, 10%-13% A; 3.0-5.0 min, 13%-40% A; 5.0-9.0 min, 40%-55% A; 9.0-11.0 min, 55%-75% A; 11.0-12.0 min, 75%-10% A; 12.0-15.0 min, 10% A.

[0064] In addition, before detection, the column was first flushed with 10% A for 10 minutes to equilibrate the column.

[0065] 2) Mass spectrometry conditions:

[0066] HRMS data from 120 to 3000 m / z were acquired using full-range mass spectrometry scanning in negative ion mode. MS scan windows were segmented as follows: 120-300 m / z; 300-500 m / z; 500-700 m / z; 700-900 m / z; 900-1200 m / z; 1000-1400 m / z; 1400-1800 m / z; 1800-2200 m / z; 2200-2600 m / z.

[0067] Spray voltage: 3.2kV; capillary temperature: 320℃; sheath flow rate: 15L / min; auxiliary flow rate: 8L / min; capillary temperature: 350℃; S-Lens RF level: 50; impact energy: 120-1200m / z: 25eV, 35eV, 45eV; 1000-1400m / z: 35eV, 45eV, 55eV; 1400-2200m / z: 45eV, 55eV, 65eV; 2200-2600m / z: 55eV, 65eV, 75eV.

[0068] II. Qualitative methods for identifying proanthocyanidins and polyester-type catechins in tea leaves.

[0069] 2.1 Sample Preparation

[0070] 2.1.1 Preparation of tea standard samples:

[0071] Extracts were extracted from four types of tea: black tea, green tea, dark tea, and white tea, to prepare standard samples of different teas. The specific methods are as follows:

[0072] Four tea samples were ground separately using a grinder and passed through a 60-mesh sieve to obtain tea powder. Then, 0.1 g of each tea powder was weighed into a 5 mL centrifuge tube, and 4 mL of 70% methanol was added for ultrasonic extraction at 50℃ (200 W, 35 kHz). The tubes were centrifuged at 10000 rpm for 10 min. The supernatant was collected to obtain different tea extracts. 1 mL of each tea extract was used to prepare different tea standard samples. Six replicates were performed for each group.

[0073] 2.1.2 Preparation of mixed tea standard samples:

[0074] Take 200 μL of each of the different tea extracts and mix them in equal volumes to obtain different tea mixed extracts. Use the different tea mixed extracts as mixed standard samples QC for multiple pretreated tea standard samples.

[0075] 2.2 Obtaining preliminary filtered MS information

[0076] 2.2.1 Obtaining raw MS information:

[0077] Different tea standard samples and mixed tea standard samples were filtered through a 0.22 μm PTPE membrane for QC analysis, followed by UPLC-HRMS analysis to obtain UPLC-HRMS spectra. Raw UPLC-HRMS data were extracted from the spectra. MZmine software parameters were set (see Table 1), and MS information was extracted from the raw UPLC-HRMS data using MZmine software to obtain raw MS information, including precursor ion values, retention times, and MS values. 1 MS 2 Peak area. Among them, the precursor ion value is the precise mass of the parent ion obtained after mass spectrometry scanning.

[0078] Table 1 MZmine Software Parameters

[0079]

[0080]

[0081] 2.2.2 Obtain MS information from one filter:

[0082] The RSD and FC values ​​are calculated using the original MS information, and then the original MS information is filtered using the RSD and FC values ​​to obtain MS information after one filtering. The specific process is as follows:

[0083] RSD and difference factor FC are calculated according to the following equations (1) and (2).

[0084] The formula for calculating RSD is as follows:

[0085] RSD=IntSQC / IntMQC×100% (1);

[0086] Wherein, IntSQC represents the standard deviation of the peak intensity of a specific chromatographic peak in the mixed tea standard sample QC, and IntMQC represents the average value of the peak intensity of a specific chromatographic peak in the mixed tea standard sample QC.

[0087] The formula for calculating the difference factor FC is as follows:

[0088] Difference factor FC = IntMQC / IntMBK(2);

[0089] Wherein, IntMQC represents the average peak intensity of a specific chromatographic peak in the mixed tea standard sample QC, and IntMBK represents the average peak intensity of a specific chromatographic peak in the blank sample; the above IntSQC, IntMQC, IntMBK, and IntMQC were all obtained using MZmine software.

[0090] The original MS information with RSD≤20% and FC≥10% was retained to obtain the MS information of the first screening. The MS information of the first screening includes the precursor ion value and peak area.

[0091] Among them, specific chromatographic peaks refer to chromatographic peaks in known proanthocyanidins that can represent proanthocyanidins and chromatographic peaks in known polyester-type catechins that can represent proanthocyanidins.

[0092] 2.3 Obtaining potential mass spectrometry peaks

[0093] 2.3.1 Calculation and prediction of precursor ion values

[0094] The formulas for predicting the precursor ion value are shown in equations (3) and (4) below:

[0095] MW = M1 + M2 + ... + M n -(2n-2)×1.008 (3);

[0096] EW = MW - 1.008 (4);

[0097] Where MW is the predicted molecular mass; EW is the predicted molecular mass of the oligomer in negative ion mode; n is the number of catechin monomers participating in polymerization, i.e., the degree of polymerization; Mn represents the molecular mass of the nth catechin monomer; and 1.008 represents the monoisotopic atomic mass of element H.

[0098] 2.3.2 Constructing a precursor ion list

[0099] The predicted precursor ion values ​​are calculated using the formula for predicting precursor ion values ​​in section 2.3.1, and a precursor ion list is constructed using the calculated predicted precursor ion values.

[0100] For example, the predicted precursor ion value of a polymer compound with EGCG as the monomer is calculated as follows:

[0101] First, when the polymeric compound is a dimer, two EGCG molecules, with a precise molecular weight of 458.0849, can condense into a PAs-B dimer, EGCG-EGCG, by losing two hydrogen atoms. Therefore, the theoretical molecular weight (MW) of this dimer is calculated using the precursor ion value calculation formula as follows:

[0102] MW=M1+M2-(2n-2)×1.008=458.0849+458.0849-(2×2-2)×1.008=914.1538.

[0103] Therefore, the precursor ion peak [MH] of this dimer compound in negative ion mode –Theoretically, the m / z value should be EW = 913.1458. The predicted precursor ion values ​​for n-mer compounds (trimeric, tetrameric, pentamer, etc.) are calculated using the method described above. A precursor ion list is then constructed using the calculated predicted precursor ion values ​​for the n-mer compounds.

[0104] 2.3.3 Obtaining potential mass spectrometry peaks

[0105] Match the precursor ion values ​​in the MS information of the first screening in 2.2.2 with the predicted precursor ion values ​​in the precursor ion list in 2.3.2, and retain the precursor ion values ​​in 2.2.2 that are the same as the predicted precursor ion values ​​to obtain the precursor ion values ​​of the second screening; the mass spectrometry peaks corresponding to the precursor ion values ​​of the second screening are the potential mass spectrometry peaks.

[0106] 2.4 Obtaining mass spectrometric characteristics of proanthocyanidins and polyester-type catechins in potential mass spectrometric peaks:

[0107] MS of known proanthocyanidins and known polyester-type catechins in the potential mass spectrometry peaks of 2.3.3 2 The fragmentation pathways were summarized, and the mass spectrometry characteristics of known proanthocyanidins and polyester catechins were obtained, namely, the EIC chromatograms of proanthocyanidins and polyester catechins in multiple tea standard samples, such as... Figure 1 and Figure 2 As shown. Figure 1 The response intensity of b is 0 to 5*10. 8 The response intensity of c is 0 to 4*10 7 ; Figure 2 The response intensity of b is 0 to 3*10. 6 The response intensity of c is 0 to 3*10 5 Mass spectrometry feature information includes feature fragmentation information and neutral loss information.

[0108] MS of known proanthocyanidins and known polyester catechins 2 The specific process of summarizing the pyrolysis pathway is as follows:

[0109] MS analysis of 17 catechin oligomers was performed comprehensively. 2Fragmentation pathways. This included 5 standards: procyanidin B1, procyanidin B2, procyanidin C1, cinnamtannin A2, and proanthocyanidin B5-3-3′-gallate, as well as 12 oligomers from the literature, including 8 proanthocyanidins: EC pentamer, EC hexamer, EC-EGC, EC-EGCG, EC-ECG, EC-EC-EGC, EC-EGC-EGC, and EGC-EGC, and 4 polyester-type catechins: Theasinensin A, Theasinensin D, Theasinensin C, and Theasinensin B. MS analysis of 17 known catechin oligomers was conducted. 2 In-depth analysis of the information summarized the fragmentation pathways of polyester-type catechins and proanthocyanidins, further revealing characteristic fragment information and neutral loss information. The specific analysis process is as follows:

[0110] 1) For polyester-type catechins:

[0111] TheasinensinB, a compound formed by the polymerization of two pyrogallol-type catechins, is used to explain and summarize the characteristic neutral loss process. Figure 5 a shows the EIC and MS of TheasinensinB. 2 The spectra show that this compound produced two main fragment ions, m / z 609.13 and m / z 591.12. It is inferred that m / z 609.13 was generated by the loss of a portion of the galloyl group (152.01 Da) from the precursor ion, while m / z 591.12 was generated by the loss of an H₂O molecule (18 Da) from m / z 609.13. Subsequently, a characteristic fragment ion ([609.13-138.03 Da]) was generated by cleaving the C ring of the catechin monomer unit via RDA. – With an m / z value of 471.10, the fragment continues to lose a water molecule, producing a distinct fragment ion [M - 156.04 Da - H]. – The m / z value is 453.08. Furthermore, at m / z 471.09, a further loss of 138.03 Da yields the characteristic fragment ion with m / z 333.06, which has been found in other similar compounds. When the proton ion structure consists of a catechol-type catechin and a pyrogallol-type catechin, the difference between these two structures is observed in MS. 2 The difference in the spectrum is reflected by the 16 Da phase difference between the corresponding characteristic ions.

[0112] Based on the above analysis, the characteristic fragments and neutral loss of polyester-type catechins can be summarized as follows:

[0113] Polyester-type catechin TSs exhibit a set of characteristic neutral loss values ​​of 156.04 Da and 138.03 Da. When the parent nucleus structure of TSs consists of two pyrogallol-type catechins, characteristic fragment ions with m / z values ​​of 609.13, 591.12, 453.08, 471.10, and 333.06 are readily generated. When the precursor ion structure consists of one catechol-type catechin and one pyrogallol-type catechin, [593-H2O] with m / z value of 593.13 is readily generated. – The value is m / z 575.12, 9[593-H2O-138Da]. – Characteristic fragment ions with m / z 437.0, m / z 455.10, and m / z 281.05.

[0114] 2) Regarding proanthocyanidins:

[0115] Procyanidin C1 and cinnamtannin A2 are oligomers formed by the polymerization of three or four EC monomers via C4→C8 linkage. For example... Figure 5 As shown in b, the cleavage pathway of procyanidin C1 includes fragment ions at m / z 713.15, m / z 575.12, m / z 407.08, m / z 287.06, and a characteristic neutral loss of 152.05 Da. The fragment ion at m / z 713.15 is generated by the loss of the B ring from a precursor ion with a 2-phenylbenzopyran ring structure via RDA cleavage [M-152.05Da-H]. – The m / z value of 575.1234 represents a fragment ion [M - 290.08 Da - H] resulting from the loss of one EC molecule from the precursor ion. – m / z 407.08 is the product ion resulting from the RDA cleavage of m / z 575.12. m / z 287.06 is the fragment ion [M-578.15Da-H] produced by the loss of one molecule of proanthocyanidin B1 from the precursor ion. – CinnamtanninA2's EIC and MS 2 See diagram Figure 5 c. The major fragment ions of Cinnamtannin A2 are m / z 1001.21, m / z 983.20, m / z 865.20, m / z 575.12, m / z 407.08, m / z 287.06, and m / z 125.02, with an intrinsic neutral loss of 152.05 Da. The fragment ions of Cinnamtannin A2 are similar to those of procyanidin C1. m[M-152.05Da-H] – For / z 1001.21 and [M-152.05Da-18.01Da-H] –The m / z value of 983.20 is due to the neutral loss of 152.05 Da from the precursor ion and further loss of H₂O. The m / z value of 863.19 is due to the loss of one EC from the precursor ion, [M - 290 Da - H]. – The two standards mentioned above are polymers of pyrogallol-type catechins, such as EC and ECG. The same characteristic fragmentation pathway was also found in other compounds containing at least one pyrogallol-type catechin, with m / z 407.08 and a neutral loss of 152.05 Da, such as proanthocyanidins B1 / B2. When the structure of a type B proanthocyanidin compound contains at least one pyrogallol-type catechin, the characteristic fragment ions of this type of compound differ from those of pyrogallol-type catechins by 16 Da: m / z 423.07, m / z 407.08 and neutral losses of 168.04 Da and 152.05 Da, respectively.

[0116] Based on the above analysis, the characteristic fragments and neutral loss of proanthocyanidins can be summarized as follows:

[0117] It has a pair of characteristic neutral loss values ​​of 152.05 Da and 168.04 Da, and a pair of characteristic fragment ions with m / z values ​​of 407.08 and 423.07.

[0118] Among them, when the overall structure contains at least one catechol-type catechin, it is easy to produce an m / z of 407.08 and a neutral loss of 152.05 Da;

[0119] When the overall structure contains at least one pyrogallol-type catechin, it is prone to m / z 423.07 and neutral loss of 168.04 Da.

[0120] In addition, the precursor ions of proanthocyanidins are easily cleaved in succession to produce fragment ions of the corresponding monomer flavan-3-ol.

[0121] In summary, the characteristic fragments and neutral loss of proanthocyanidins and polyester-type catechins are as follows:

[0122] The characteristic fragments of proanthocyanidins are m / z 407.08 and m / z 423.07, with neutral loss values ​​of 152.05 Da and 168.04 Da, respectively.

[0123] The characteristic fragment ions of polyester-type catechins are m / z 609.13 and m / z 593.13, m / z 591.12 and m / z 575.12, m / z 453.08 and m / z 437.09, m / z 471.10 and m / z 455.10, and m / z 333.06 and m / z 317.07; the neutral loss is 156.04 Da and 138.03 Da.

[0124] 2.5. The structures corresponding to each peak in the EIC diagrams of proanthocyanidins and polyester catechins in the multiple tea standard samples obtained in 2.4 were annotated, and the annotation results are shown in Table 2.

[0125] As shown in Table 2, a total of 7 monomeric catechins and 52 oligomers were discovered and annotated. The 52 oligomers were 2-6 polymers, and proanthocyanidins with a degree of polymerization of 5-6 were detected in tea for the first time.

[0126] Table 2. Identification results of proanthocyanidins and polyester-type catechins.

[0127]

[0128]

[0129]

[0130]

[0131]

[0132]

[0133]

[0134] Note: m / z is the mass-to-charge ratio, t R For retention time, NAME is the name, Formula is the molecular formula, error is the error value, and DP is the degree of polymerization.

[0135] Where error = [(detected precursor ion value - predicted precursor ion value) / predicted precursor ion value] × 10 6 .

[0136] 2.6 Qualitative Analysis of Proanthocyanidins and Polyester-type Catechins in Tea Leaves to be Tested

[0137] The potential mass spectrometry peaks of the tea leaves to be tested were obtained according to methods 2.1–2.3. The MS values ​​of the potential mass spectrometry peaks were then analyzed. 2 The characteristic fragment information and neutral loss information in the fragmentation information are compared with the characteristic fragment information and neutral loss information in the mass spectrometry characteristic information in 2.4 to determine the categories of proanthocyanidins and polyester catechins in the tea to be detected. Then, based on the annotation results obtained in 2.5, the qualitative results of proanthocyanidins and polyester catechins in the tea to be detected are obtained.

[0138] To further demonstrate that the method of the present invention can more accurately identify proanthocyanidins and polyester-type catechins, we conducted the following experiments.

[0139] Extracts were extracted from four types of tea: black tea, green tea, dark tea, and white tea, to prepare different tea samples for testing. The specific methods are as follows:

[0140] Four types of tea samples were ground into powder using a grinder and passed through a 60-mesh sieve to obtain tea powder. Then, 0.1g of each tea powder was weighed into a 5mL centrifuge tube, and 4mL of 70% methanol was added to each tube for ultrasonic extraction at 50℃ (200W, 35kHz); centrifugation was performed at 10000rpm for 10min. The supernatant was collected to obtain extracts of the four types of tea. 1mL of each extract was then used to obtain four tea samples for testing. Six replicates were performed for each group. The four tea samples were filtered through a 0.22μm PTPE membrane and then analyzed by UPLC-HRMS to obtain the TIC chromatograms of the four tea samples, as shown below. Figure 1 As shown in a, and Figure 2 As shown in Figure a.

[0141] The TIC plots of the four types of tea to be tested are compared with the EIC plots of proanthocyanidins and polyester catechins identified and annotated using method 2.5, as shown below. Figure 1 As shown in b and c in the middle and Figure 2 Compare the values ​​shown in b and c. Figure 1 In the figure, 'a' represents the TIC (Total Indices) of four types of tea to be tested, m / z 120-1200; 'b' and 'c' represent the EIC (Enhanced Organic Indices) of proanthocyanidins and polyester catechins after annotation according to the method standard in section 2.5. Figure 2 In the figure, 'a' represents the TIC (Total Indices) of four types of tea to be tested, m / z 1200-2600; 'b' and 'c' represent the EIC (Enhanced Organic Indices) of proanthocyanidins and polyester catechins after annotation according to method standard 2.5.

[0142] from Figure 1 and Figure 2 The TIC spectra of the four tea samples clearly show that many proanthocyanidins and polyester catechins are masked by MS peaks with high response intensity. However, after the first filtration in section 2.2 and the second filtration in section 2.3, the masked MS peaks can be extracted quickly and efficiently. Figure 1 and Figure 2 The specific results of the mass spectrometry information corresponding to the numbers marked on each peak are shown in Table 2.

[0143] The number of mass spectrometry peaks in the original UPLC dataset for four types of tea was compared with the number of mass spectrometry peaks identified and annotated using method 2.5. The results are as follows: Figure 3 As shown. Figure 3 In the figure, a represents the number of mass spectrometry peaks in the original UPLC dataset of the four types of tea, and b represents the number of mass spectrometry peaks after being filtered by the method of this invention.

[0144] from Figure 3It can be seen that the method of the present invention can efficiently extract and annotate the chromatographic-mass spectrometry information of proanthocyanidins and polyester catechins.

[0145] The neutral loss, characteristic fragments, and cleavage pathways of proanthocyanidins and polyester-type catechins obtained by the methods of the embodiments of the present invention are summarized, and the results are as follows: Figure 4 As shown. Figure 4 In the diagram, a is the inferred mass spectrometry fragmentation pathway of proanthocyanidins, b is the inferred mass spectrometry fragmentation pathway of polyester catechins, where TS-PP is a polyester catechin with a parent structure of two pyrogallol-type catechins, and TS-CP is a polyester catechin with a parent structure of one pyrogallol-type catechin and one catechol-type catechin; c is a summary diagram of the inferred fragmentation pathway structures of proanthocyanidins and polyester catechins, where CFLO refers to proanthocyanidins and polyester catechins, Int refers to the response intensity, FI refers to the fragment ion value, NL refers to the neutral loss value, PAs-4-8 refers to proanthocyanidins linked via C4→C8, and PAs-4-6 refers to proanthocyanidins linked via C4→C6.

[0146] The above experimental comparisons demonstrate that the method of this invention can be used for rapid identification and annotation of proanthocyanidins and polyester-type catechins in complex UPLC-HRMS data. Based on the monomer composition of the polymers and their polymerization mechanisms, a corresponding list of precursor ions is calculated using formulas and applied to the identification of potential proanthocyanidins and polyester-type catechins. Based on the characteristic fragmentation and neutral loss of known proanthocyanidins and polyester-type catechins, the extracted potential proanthocyanidins and polyester-type catechins are structurally annotated. Ultimately, a total of 7 monomeric catechins and 52 oligomers were comprehensively discovered and annotated from four types of tea, and proanthocyanidins and polyester-type catechins with a degree of polymerization of 5-6 were detected in tea for the first time.

[0147] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A qualitative method for identifying proanthocyanidins and polyester-type catechins in tea leaves, characterized in that, Includes the following steps: Multiple teas were extracted separately to obtain multiple tea standard samples; multiple tea standard samples were mixed in equal volumes to obtain tea mixed standard samples; multiple tea standard samples and tea mixed standard samples were analyzed by UPLC-HRMS, and raw MS information was extracted using MZmine software; RSD and FC values ​​were calculated using raw MS information, and raw MS information was screened using RSD and FC values ​​to obtain first-screened MS information; Calculate the predicted precursor ion values ​​for the n-mer compound and construct a list of precursor ions; The formula for predicting precursor ion values ​​is as follows: MW=M1+M2+…+M n -(2n-2)×1.008, EW=MW-1.008; Where MW is the predicted molecular weight, EW is the predicted molecular weight of the oligomer in negative ion mode, n is the number of catechin monomers participating in polymerization, Mn represents the molecular weight of the nth catechin monomer, and 1.008 represents the monoisotope atomic mass of element H. The precursor ion values ​​in the MS information of the first screening are compared with the predicted precursor ion values. The precursor ion values ​​that are the same as the predicted precursor ion values ​​are retained, and the mass spectrum peaks corresponding to the precursor ion values ​​are the potential mass spectrum peaks. MS of potential mass spectrometry peaks 2 The lysis pathway was analyzed to obtain mass spectrometry characteristics of multiple tea standard samples and mixed tea standard samples; The tea leaves to be tested were analyzed using the method described above to obtain the potential mass spectrometry peaks. The MS values ​​of the potential mass spectrometry peaks of the tea leaves were then analyzed. 2 The fragmentation information was compared with the mass spectrometry characteristic information to identify the types of proanthocyanidins and polyester catechins in the tea sample to be tested. MS of the potential mass spectrometry peak 2 The specific process for analyzing the pyrolysis pathway is as follows: MS based on the known proanthocyanidins in the potential mass spectrometry peaks 2 Fragmentation information, MS of known proanthocyanidins 2 The fragmentation pathways were analyzed and summarized to obtain the mass spectrometry characteristics of known proanthocyanidins; MS based on the known polyester-type catechins in the potential mass spectrometry peaks 2 Pyrolysis information, MS of known polyester catechins 2 The fragmentation pathways were analyzed and summarized to obtain the mass spectrometry characteristics of known polyester catechins; MS of the known proanthocyanidins 2 The specific process of fragmentation pathway analysis is as follows: When the proanthocyanidin structure is known to contain at least one catechol-type catechin, it is known that the proanthocyanidin can produce characteristic fragments. m / z 407.08 and neutral loss of 152.05 Da; When the proanthocyanidin structure is known to contain at least one pyrogallol-type catechin, it is known that the proanthocyanidin can produce characteristic fragments. m / z 423.07 and neutral loss of 168.04 Da; When the precursor ions in the known proanthocyanidin structure are continuously cleaved, it is known that proanthocyanidins can produce fragment ions of the corresponding monomer flavan-3-ol. m / z 289.07 m / z 305.07 m / z 441.09、 m / z 457.08; At this point, when the compounds linked by C4→C8 bonds in proanthocyanidins are known, the fragment ions of proanthocyanidins are also known. m / z 423.07 or m / z The relative response intensity of 407.08 is lower than that of the fragment ions of the monomer flavan-3-ol; when proanthocyanidins contain compounds linked by C4→C6 bonds, the fragment ions of proanthocyanidins are known. m / z 423.07 or m / z The relative response intensity of 407.08 is higher than that of the fragment ions of the monomer flavan-3-ol; MS of the known polyester-type catechins 2 The specific process of fragmentation pathway analysis is as follows: When the parent structure of polyester-type catechins is known to be two pyrogallol-type catechins, it is known that polyester-type catechins can produce... m / z 609.13, m / z 591.12, m / z 453.08, m / z 471.10, m / z 333.06 characteristic fragment ions; When the core structure of polyester-type catechins is known to consist of one pyrogallol-type catechin and one catechol-type catechin, it is known that polyester-type catechins can produce... m / z 593.13, m / z 575.12, m / z 437.09, m / z 455.10, m / z 317.07 Feature fragments.

2. The qualitative method for identifying proanthocyanidins and polyester-type catechins in tea leaves according to claim 1, characterized in that, The specific process of the first screening is as follows: Calculate the relative standard deviation (RSD) and the factor of difference (FC), retain MS information with RSD ≤ 20% and FC ≥ 10%, and obtain the MS information of the first filtering. RSD and difference factor FC are calculated according to the following equations (1) and (2); The formula for calculating RSD is as follows: RSD = IntSQC / IntMQC×100% (1) Where IntSQC represents the standard deviation of the peak intensity of a specific chromatographic peak in the mixed standard sample QC, and IntMQC represents the average value of the peak intensity of a specific chromatographic peak in the mixed standard sample QC; The formula for calculating the difference factor FC is as follows: Difference factor FC = IntMQC / IntMBK (2) Wherein, IntMQC represents the average peak intensity of a specific chromatographic peak in the mixed standard sample QC, and IntMBK represents the average peak intensity of a specific chromatographic peak in the blank sample.

3. The qualitative method for identifying proanthocyanidins and polyester catechins in tea leaves according to claim 1, characterized in that, The formula for calculating the predicted precursor ion value is derived based on the known chemical structure rules of proanthocyanidins and known polyester catechins.

4. The qualitative method for identifying proanthocyanidins and polyester catechins in tea leaves according to claim 3, characterized in that, The specific rules governing the chemical structure are as follows: The known proanthocyanidins and the known polyester catechins are formed by linking monomeric catechins through C–C bonds. When two monomeric catechins are linked through C–C bonds, hydrogen is removed. The amount of hydrogen lost is twice the amount of the monomeric catechins minus 2.

5. The qualitative method for identifying proanthocyanidins and polyester-type catechins in tea leaves according to claim 1, characterized in that, The mass spectrometry feature information includes feature fragmentation information and neutral loss information; MS of the potential mass spectrometry peaks of the tea leaves to be detected 2 Fragmentation information includes characteristic fragment information and neutral loss information.

6. The qualitative method for identifying proanthocyanidins and polyester-type catechins in tea leaves according to claim 1, characterized in that, The original MS information includes precursor ion value, retention time, MS information, and MS... 2 Information, peak area.

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