A method for establishing HPLC fingerprint of Taibai tea

Abstract

The present invention provides a method for establishing an HPLC fingerprint of Taibai tea, comprising the following steps: S1, preparation of a test solution: taking Taibai tea sample powder, adding an extraction solvent, ultrasonically extracting, and filtering to obtain; S2, establishment of an HPLC fingerprint: performing HPLC detection on the test solution, recording a chromatogram, and obtaining an HPLC fingerprint of Taibai tea; S3, determination of a standard fingerprint: according to the methods of S1 to S2, establishing HPLC fingerprints for multiple batches of Taibai tea, and determining through analysis and comparison that the HPLC fingerprints of multiple batches of Taibai tea samples all have 8 common characteristic peaks; the 8 common characteristic peaks constitute the fingerprint characteristics of Taibai tea, which serve as the standard fingerprint of Taibai tea. The present invention establishes an HPLC fingerprint of Taibai tea in order to provide a feasible method for comprehensive quality evaluation of Taibai tea and lay a foundation for subsequent basic research on the efficacy of the medicinal material.

Description

Technical Field

[0001] The invention relates to the field of quality detection of Taibai tea, and in particular to a method for establishing a HPLC fingerprint of Taibai tea. Background Art

[0002] Taibai tea is the dried lichen of snow tea Thamnolia subuliformis (Ehrh.) W.Culb. or ground tea Tham noliavermicularis (Sw.) Ach.ex Schaer., which has the effects of clearing away heat and detoxifying, calming the mind and nourishing the heart, clearing the heart and improving eyesight. Taibai tea mainly grows in high-altitude cold areas. Modern research shows that Taibai tea is a unique complex biological organic symbiosis composed of symbiotic bacteria and symbiotic algae, and its metabolites are mainly divided into primary metabolites and secondary metabolites. Among them, primary metabolites mainly include polysaccharides, amino acids and other substances, and secondary metabolites are mainly lichen acids. Taibai tea has pharmacological activities such as antibacterial, immune regulation, and anti-aging, and is used to assist in the treatment of hypertension, Alzheimer's disease, neurasthenia and other diseases. It has high medicinal value. At present, research on the quality control of Taibai tea at home and abroad is relatively scarce. Therefore, it is necessary to study the quality control of Taibai tea to provide a reference for the establishment of the quality standard of Taibai tea.

[0003] At present, the quality control indicators of Taibai tea in local standards only include properties, inspection items, microscopic identification and thin-layer chromatography identification of reference medicinal materials; the 2015 edition of the Shaanxi Provincial Medicinal Materials Standard does not have a content determination item, and the 2010 edition of the Sichuan Provincial Traditional Chinese Medicine Standard uses glycine as a reference for total amino acid determination. There is currently no unified quality control standard, and it is impossible to conduct effective quality evaluation. Therefore, it is necessary to conduct quality standard research. In recent years, the chemical composition and pharmacological effects of Taibai tea have attracted the attention of many researchers, and a large number of studies have been conducted on it. A study used high-performance liquid chromatography to determine the amount of usnic acid in Taibai tea. The chromatographic conditions were: Sepax Sapphire C18 column (250mm×4.6mm, 5μm), methanol-(50mmol potassium dihydrogen phosphate-triethylamine buffer 500:1) (70:30) as the mobile phase, and the detection wavelength was 285nm. However, when we used this method to establish the quality standard of Taibai tea, we could not detect usnic acid well. Mass spectrometry showed that the medicinal material contained usnic acid, but the response value of usnic acid was particularly low. The chromatographic peak with high content detected by HPLC lacked corresponding reference substances on the market. Therefore, we intend to establish the HPLC fingerprint of Taibai tea, in order to provide a reference for the quality evaluation of Taibai tea and the development and utilization of new drugs, and at the same time provide a model for the development of innovative Chinese medicine resources from ethnic / folk traditional medicines. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for establishing a HPLC fingerprint of Taibai tea, by establishing the HPLC fingerprint of Taibai tea, it is used to achieve the authenticity identification of Taibai tea and the geographical protection of the product.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows.

[0006] A method for establishing a HPLC fingerprint of Taibai tea comprises the following steps:

[0007] S1. Preparation of test solution

[0008] Take Taibai tea sample powder, add extraction solvent, perform ultrasonic extraction, and filter to obtain;

[0009] S2. Establishment of HPLC fingerprint

[0010] The test solution of S1 was tested by HPLC, and the chromatogram was recorded to obtain the HPLC fingerprint of Taibai tea;

[0011] S3. Determination of standard fingerprint

[0012] According to the methods of S1 to S2, HPLC fingerprints were established for multiple batches of Taibai tea. Through analysis and comparison, it was determined that the HPLC fingerprints of multiple batches of Taibai tea samples all had 8 common characteristic peaks; the 8 common characteristic peaks constituted the fingerprint characteristics of Taibai tea and served as the standard fingerprint of Taibai tea.

[0013] Furthermore, in S1, the extraction solvent is methanol.

[0014] Furthermore, in S1, the usage ratio of Taibai tea sample powder to methanol is 0.5 g: 25-50 mL.

[0015] Furthermore, in S1, the ultrasonic extraction time is 30 to 60 minutes.

[0016] Further, in S2, the chromatographic conditions of HPLC are as follows:

[0017] C18 chromatographic column, 250×4.6 mm, 5 μm; column temperature, 30 °C;

[0018] Mobile phase A was methanol, and mobile phase B was 0.1% phosphoric acid in water;

[0019] The gradient elution program is 0-10 min, 30%-45% mobile phase A; 10-80 min, 45%-80% mobile phase A; 80-90 min, 80%-95% mobile phase A;

[0020] Flow rate: 1.0 mL min -1; The detection wavelength was 254nm; the injection volume was 10μL.

[0021] Furthermore, in S3, the retention times of the eight common characteristic peaks are 11.3, 18.755, 37.402, 53.487, 60.492, 67.328, 75.132, and 84.588, respectively.

[0022] Beneficial effects of the present invention:

[0023] 1. The present invention establishes the HPLC fingerprint of Taibai tea and uses UHPLC-QE×activ e Focus-MS / MS technology to perform qualitative analysis on its common peak components, in order to provide a feasible method for the comprehensive quality evaluation of Taibai tea and lay a foundation for subsequent basic research on the medicinal material of the medicinal material.

[0024] 2. The present invention establishes HPLC fingerprints for multiple batches of Taibai tea, and through analysis and comparison, determines that the HPLC fingerprints of multiple batches of Taibai tea samples all have 8 common characteristic peaks; the 8 common characteristic peaks constitute the fingerprint characteristics of Taibai tea, which are used as the standard fingerprint of Taibai tea. The method is simple to operate, the results are stable and reliable, and the repeatability is good, and it can be used to comprehensively evaluate the overall quality of Taibai tea medicinal materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The fingerprints of Taibai tea detected by different detection chromatographic columns are shown in Figure 1. A represents Pheromone, B represents Diamonsil, and C represents Aglient.

[0026] Figure 2 It is the fingerprint spectrum of Taibai tea at different detection wavelengths.

[0027] Figure 3 It is the fingerprint spectrum of Taibai tea with different detection mobile phases.

[0028] Figure 4 It is the fingerprint spectrum of Taibai tea at different detection column temperatures.

[0029] Figure 5 It is the fingerprint spectrum of Taibai tea with different detection extraction solvents.

[0030] Figure 6 It is the fingerprint spectrum of Taibai tea with different detection extraction solvent ratios.

[0031] Figure 7 It is the fingerprint of Taibai tea with different detection and extraction methods.

[0032] Figure 8 It is the fingerprint spectrum of Taibai tea with different detection and extraction times.

[0033] Fig. 9It is the HPLC superposition spectrum (S1~S10) of 10 batches of samples and the reference spectrum R. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods described in the following examples are all conventional methods; the reagents and materials described are all commercially available unless otherwise specified.

[0037] Example 1

[0038] The method for establishing the HPLC fingerprint of Taibai tea is as follows:

[0039] 1. Test samples

[0040] The source information of the 10 batches of Taibai tea samples is shown in Table 1. They were identified as dried branches of snow tea Thamnolia subuliformis (Ehrh.) W.Culb. or ground tea Thamnolia vermicularis (Sw.) Ach.ex Schaer.

[0041] Table 1 Source information of Taibai tea samples

[0042] Serial number batch number Origin Serial number batch number Origin S1 20211203 Taibai County, Shaanxi S6 20211104 Taibai County, Shaanxi S2 20210801 Zhouzhi County, Shaanxi S7 20210720 Taibai County, Shaanxi S3 20211126 Shangri-La, Yunnan S8 20210628 Taibai County, Shaanxi S4 20211216 Lijiang, Yunnan S9 20210610 Taibai County, Shaanxi S5 20211119 Aba, Sichuan S10 20220520 Mei County, Shaanxi

[0043] 2. Experimental methods

[0044] 2.1 Chromatographic conditions

[0045] 2.1.1. Selection of chromatographic column

[0046] Three different manufacturers' chromatographic columns were tested: Agilent C18 (250 × 4.6 mm, 5 μm), Phimlo C18 (250 × 4.6 mm, 5 μm), Diamonsil C18 (250 × 4.6 mm, 5 μm), see Figure 1 .

[0047] Through experimental analysis, the peak shape and separation of each chromatographic peak of Agilent C18 (250×4.6mm, 5μm) are relatively good. Therefore, Agilent C18 (250×4.6mm, 5μm) was selected as the chromatographic column. Figure 1 .

[0048] 2.1.2. Selection of detection wavelength

[0049] The separation spectra at three detection wavelengths of 254nm, 300nm and 365nm were examined respectively. Figure 2 .Depend on Figure 2 The results showed that the apparent abundance of the chromatographic peak at 254 nm was high and the baseline was relatively stable, so 254 nm was selected as the detection wavelength.

[0050] 2.1.3. Selection of mobile phase

[0051] Agilent C18 (250×4.6mm, 5μm) was used as the chromatographic column, the detection wavelength was 254nm, and the gradient elution was performed with different mobile phase systems. The elution at the bottom of different mobile phase systems was compared. The results are shown in Table 2 and Figure 3 .

[0052] Table 2 Comparison results of gradient elution with different mobile phase systems

[0053] serial number Mobile phase Gradient elution ① Methanol-water system The response value of the chromatographic peak is low and the baseline is unstable ② Acetonitrile-water system The response value of the chromatographic peak is low and the baseline is unstable ③ Methanol-0.1% phosphoric acid system The baseline is stable, the chromatographic peak shape is good, and the separation is good ④ Acetonitrile-0.1% phosphoric acid system The chromatographic peak response value is relatively large, the peak emerges quickly, but the separation is poor

[0054] According to Table 2 and Figure 3 As a result, the mobile phase was finally selected as methanol-0.1% phosphoric acid system.

[0055] Since Taibai tea contains a large amount of lichenic acid components with similar polarity, when the mobile phase ratio starts from 10%, the distribution of the chromatographic peaks is unreasonable and the separation is poor, so the initial mobile phase ratio is 30%. After a lot of experimental exploration and ratio adjustment, the best elution ratio is 0-10min, 30%-45% methanol; 10-80min, 45%-80% methanol; 80-90min, 80%-95% methanol.

[0056] 2.1.4. Selection of column temperature

[0057] The effects of different column temperatures (20°C, 25°C, 30°C, 35°C, 40°C) on the separation effect were compared. The results are shown in Figure 4 .

[0058] according to Figure 4 The results showed that when the column temperature was between 20℃ and 30℃, the peak shape of the chromatographic peak remained unchanged and the peak elution time moved forward as the temperature increased; however, when the column temperature was at 35℃ and 40℃, the separation of the chromatographic peaks was poor; therefore, the column temperature could be either 25℃ or 30℃.

[0059] The final chromatographic conditions were determined as follows: Agilent C18 (250×4.6 mm, 5 μm), the mobile phase was methanol (A)-0.1% phosphoric acid aqueous solution (B), and the flow rate was 1.0 mL·min -1; Column temperature was 30°C; Detection wavelength was 254nm; Injection volume was 10μL, Gradient elution ratio was 0-10min, 30%-45% methanol; 10-80min, 45%-80% methanol; 80-90min, 80%-95% methanol.

[0060] 2.2 Preparation of test samples

[0061] 2.2.1. Selection of extraction solvent

[0062] (1) Type of extraction solvent: Use water, methanol, chloroform, and ethanol as extraction solvents, weigh about 0.5g of Taibai tea sample powder, place it in a 100mL stoppered conical flask, add 25mL of extraction solvent, weigh it, ultrasonically extract for 30min, let it cool to room temperature, make up the weight, filter it through a 0.22μm microporous filter membrane, and the test solution is obtained. Take 10uL of each, inject it into HPLC, and detect it. Figure 5 .Depend on Figure 5 The results showed that, in comparison, the spectrum obtained with methanol as the extraction solvent had better main peak number, peak position and peak value, so methanol was selected as the extraction solvent.

[0063] (2) Concentration of extraction solvent: 40%, 60%, 80%, 100% methanol was used as extraction solvent, and about 0.5g of Taibai tea sample powder was weighed and placed in a 100mL stoppered conical flask. 25mL of extraction solvent was added, weighed, and ultrasonically extracted for 30min. The mixture was cooled to room temperature, reweighted, and filtered through a 0.22μm microporous filter membrane to obtain the test solution. 10uL was taken and injected into HPLC for detection. Figure 6 .Depend on Figure 6 The results showed that when 100% pure methanol was used as the extraction solvent, the number of chromatographic peaks was large and the absorption intensity was good. In addition, the effects of different solid-liquid ratios (1:50, 1:100, 1:200 times) on sample extraction were investigated. The results showed that when the solid-liquid ratio was 1:50, the peak height and peak area of ​​each chromatographic peak were ideal.

[0064] 2.2.2. Selection of extraction method

[0065] Weigh about 0.5g of Taibai tea sample powder, place it in a 100mL stoppered conical flask, add 25mL of methanol, weigh it, soak it overnight, extract it by ultrasonic wave for 30min, and extract it by reflux for 30min, let it cool to room temperature, add methanol to make up the weight, filter it through a 0.22μm microporous filter membrane, and the test solution is obtained. Take 10uL of it, inject it into HPLC, and test it. Figure 7 .Depend on Figure 7 The results showed that the chromatograms obtained by ultrasonic extraction and reflux extraction were similar in terms of peak shape and peak number. Both extraction methods were acceptable, and ultrasonic extraction was chosen after comprehensive consideration.

[0066] 2.2.3. Selection of extraction time

[0067] Weigh about 0.5g of Taibai tea sample powder, place it in a 100mL stoppered conical flask, add 25mL of methanol, weigh it, and ultrasonically extract for 30min, 45min, and 60min respectively, let it cool to room temperature, make up the weight with methanol, and filter it through a 0.22μm microporous filter membrane to obtain the test solution. Take 10uL of it respectively, inject it into HPLC, and test it. Figure 8 .Depend on Figure 8 The results showed that with the increase of extraction time, the number of chromatographic peaks and their response values ​​did not change significantly, so ultrasonic extraction was performed for 30 minutes.

[0068] The final preparation method of the test solution was determined as follows: weigh about 0.5 g of Taibai tea sample powder, place it in a 100 mL stoppered conical flask, add 25 mL of methanol, weigh it, perform ultrasonic extraction for 30 minutes, cool to room temperature, make up the weight with methanol, and filter through a 0.22 μm microporous filter membrane to obtain the solution.

[0069] 2.3 Methodological Investigation

[0070] 2.3.1 Precision test

[0071] Take the same sample solution and continuously inject and measure 6 times according to the chromatographic conditions finally determined in 2.1. Take the retention time and chromatographic peak area of ​​Peak 4 as reference to calculate the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of each common peak is less than 0.13% (n=6), and the RSD of the relative peak area is less than 1.82% (n=6), indicating that the precision of the instrument is good.

[0072] 2.3.2 Stability test

[0073] Take the same sample solution and place it at room temperature for 0, 2, 4, 8, 10, and 12 hours. Determine the chromatographic conditions for injection according to 2.1. Take the retention time and chromatographic peak area of ​​peak 4 as reference to calculate the relative retention time and relative peak area of ​​each common peak. The results show that the RSD of the relative retention time of the 8 common peaks is less than 0.08%, and the RSD of the relative peak area is less than 2.99%, indicating that the sample solution has good stability within 12 hours at room temperature.

[0074] 2.3.3 Repeatability test

[0075] Take 6 portions of medicinal powder from the same batch, 0.5 g each, prepare the test solution according to method 2.2, and then measure it according to the chromatographic conditions of 2.1. Take the retention time and chromatographic peak area of ​​peak 4 as reference, calculate the relative retention time and relative peak area of ​​each common peak, and the results show that the RSD of the relative retention time of the 8 common peaks is less than 0.11% (n=6), and the RSD of the relative peak area is less than 6.06% (n=6), indicating that this method has good repeatability.

[0076] 2.4. Sample determination

[0077] Prepare each batch of sample solution according to the preparation method of the sample solution selected in 2.2, and measure according to the chromatographic conditions determined in 2.1.

[0078] 3. Results

[0079] 3.1、Establishment of fingerprint

[0080] Take 10 batches of medicinal material samples, prepare the test solution according to the method selected in 2.2, and then inject and measure according to the chromatographic conditions selected in 2.1, and record the chromatogram. Using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", the S6 sample chromatogram is used as the reference chromatogram, the time window width is 0.1, and the reference spectrum is generated by the median method. The HPLC superimposed spectrum of the 10 batches of Taibai Tea medicinal materials has 8 common peaks, see Fig. 9 The retention times of the eight common characteristic peaks were 11.3, 18.755, 37.402, 53.487, 60.492, 67.328, 75.132, and 84.588, respectively. Taking peak 4 as the reference peak, the relative retention time and relative peak area of ​​the common peaks of the 10 batches of medicinal materials were calculated. The results are shown in Tables 3 and 4.

[0081] Table 3 Relative retention time of common peaks in 10 batches of samples

[0082] Peak S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 RSD% 1 0.2114 0.2113 0.2113 0.2112 0.2116 0.2117 0.2112 0.2115 0.2116 0.2113 0.1708 2 0.3507 0.3507 0.3506 0.3504 0.3511 0.3512 0.3507 0.3509 0.3510 0.3507 0.1111 3 0.6998 0.6999 0.6993 0.6987 0.7002 0.70055 0.7004 0.7011 0.7013 0.70099 0.0739 4 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 0.0000 5 1.1313 1.1313 1.1310 1.1317 1.1313 1.1308 1.1299 1.13044 1.1305 1.1306 0.0280 6 1.2588 1.2589 1.2588 1.25999 1.2589 11.2582 1.2572 1.2570 1.2566 1.2573 0.0555 7 1.4047 1.4047 1.4047 1.4046 1.1044 1.4034 1.4013 1.4014 1.4014 1.4017 0.1108 8 1.5816 1.5810 1.5815 1.5836 1.5808 1.5799 1.5775 1.5770 1.5772 1.5773 0.1217

[0083] Table 4 Relative peak areas of common peaks of 10 batches of samples

[0084] Peak S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 1 0.0459 0.0174 0.0168 0.0174 0.0259 0.0168 0.0178 0.0179 0.0181 0.0204 2 0.1519 0.0078 0.0050 0.0063 0.0064 0.0367 0.0150 0.0374 0.0503 0.0562 3 0.1250 0.0617 0.0261 0.0333 0.0216 0.0855 0.0932 0.0546 0.0597 0.0854 4 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 5 0.0406 0.0177 0.0201 0.0214 0.0164 0.0302 0.0272 0.0372 0.0292 0.0385 6 0.5181 0.7942 0.6416 0.8106 0.6230 0.7380 0.4740 0.7394 0.7320 0.7999 7 0.0399 0.0131 0.0026 0.0028 0.0047 0.0079 0.0148 0.0013 0.0117 0.0176 8 0.0230 0.0128 0.0100 0.0197 0.0116 0.0153 0.0134 0.0164 0.0142 0.0217

[0085] As shown in Table 4, there are 8 common peaks in the chromatographic peaks of 10 batches of Taibai tea. Peak No. 4 with the largest peak area is taken as the reference peak, and the peak area is set to 1. The other common peaks in each Taibai tea spectrum are divided by it to obtain the relative peak area. Similarly, Table 3 provides the relative retention time of the 8 common peaks. From the data in Table 3, it can be seen that the standard deviation (SD) of the retention time of the common peaks in each Taibai tea chromatogram is <1, indicating that the selection of these peaks meets the characteristics of the common peaks.

[0086] 3.2 Similarity Evaluation

[0087] The similarity evaluation system of Chinese medicine chromatographic fingerprint was used (2012 edition) to evaluate the similarity of 10 batches of samples, and the results are shown in Table 5. The software similarity calculation uses the vector angle cosine method, and the similarity result is between 0.9 and 1.0 to meet the requirements.

[0088] Table 5 Similarity analysis of Taibai tea fingerprints

[0089] serial number S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 R S1 1.000 0.971 0.985 0.970 0.989 0.981 0.991 0.979 0.981 0.976 0.987 S2 0.971 1.000 0.994 0.999 0.992 0.999 0.973 0.999 0.999 0.999 0.997 S3 0.985 0.994 1.000 0.994 1.000 0.997 0.990 0.997 0.998 0.993 0.999 S4 0.970 0.9999 0.994 1.000 0.992 0.998 0.9700 0.999 0.998 0.999 0.996 S5 0.989 0.992 1.000 0.992 1.000 0.995 0.990 0.996 0.997 0.992 0.998 S6 0.981 0.999 0.997 0.998 0.995 1.000 0.981 1.000 1.000 0.999 0.999 S7 0.991 0.973 0.990 0.970 0.990 0.981 1.0000 0.980 0.981 0.973 0.987 S8 0.979 0.999 0.997 0.999 0.996 1.0000 0.980 1.0000 1.000 0.999 0.999 S9 0.981 0.999 0.998 0.998 0.997 1.000 0.981 1.000 1.000 0.999 0.999 S10 0.976 0.999 0.993 0.999 0.992 0.999 0.973 0.999 0.999 1.000 0.997 R 0.987 0.997 0.999 0.996 0.998 0.999 0.987 0.999 0.999 0.997 1.000

[0090] From the data in Table 5, it can be seen that the similarity between each batch of samples and the control fingerprint is greater than 0.987, which meets the technical requirements of the fingerprint, indicating that there is good consistency among the 10 batches of medicinal materials. This method can be used to comprehensively evaluate the overall quality of Taibai tea medicinal materials.

[0091] Example 2

[0092] The characteristic peaks of Taibai tea determined in Example 1 were qualitatively analyzed using ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry (UHPLC-Q-Exactive Focus-MS / MS).

[0093] 1. Materials

[0094] The reference substances, mosnic acid (CHB210121) and usnic acid (CHB201214), with a purity of ≥98%, were purchased from Chengdu Kroma Biotechnology Co., Ltd.; dark tea stain (OK201912B), with a purity of ≥98%, was purchased from Beijing Wokai Biotechnology Co., Ltd.

[0095] 2. Chromatographic and mass spectrometry conditions

[0096] Chromatographic column: Waters Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm).

[0097] Mobile phase: acetonitrile (A)-0.1% formic acid water (B).

[0098] Gradient elution: 0-8 min, 10%-45% A; 8-15 min, 45%-60% A; 15-20 min, 60%-95% A; 20-22 min, 95% A; flow rate 0.3 mL min -1 .

[0099] The column temperature was 30°C and the injection volume was 1 μL.

[0100] A heated electrospray ion source (ESI) was used in positive and negative ion monitoring modes. The spray voltage in both positive and negative ion modes was 3.0 kV, the capillary temperature was 350 °C, and the atomization temperature was 320 °C. The scanning mode was FullMS, the FullMS resolution was 70000, the dd-MS resolution was 17500, the scanning range was m / z 100-1500, and the collision energy (CE) was set to 15 eV.

[0101] Data analysis was performed using Xcalibur 4.0 software.

[0102] 3. Identification of common peaks

[0103] Take the Taibai tea sample solution and measure it according to the chromatography-mass spectrometry conditions under item 2 to obtain the total ion current of the test mass spectrum. Combined with the reference substance and relevant literature reports, the structural attribution of some common peaks in the fingerprint spectrum was judged. Detailed information is shown in Table 6.

[0104] The main components of Taibai tea are lichenic acid components, mainly including dibenzofurans, desepoxides and their derivatives and single-ring derivatives. By comparing with existing reference substances, the single-benzene ring substituted lichenic acid, desepoxides black tea stains and dibenzofuran usnic acid in Taibai tea medicinal materials were identified. However, the response values ​​of the three in the mass spectrometry were low, and the chromatographic peaks in HPLC were also very small and almost undetectable, so the common characteristic peaks in the fingerprint were not added. The contents of the common peaks 4, 6, 7 and 8 in the article are relatively high, so they were enriched by preparative liquid phase, and qualitatively analyzed by UHPLC-Q-Exactive Focus-MS / MS, and they were identified as desepoxides compounds, namely, scaly lichenic acid, horn lichenic acid, Thamnoliadepsides B, and babaric acid.

[0105] 4. Mass spectrometry fragmentation rules of main chemical components

[0106] In the negative ion mode, the single benzene ring substituted compounds are mainly [MH] - The quasi-molecular ion peak exists. When cleavage occurs, the mother nucleus is prone to continuous loss of small molecules such as CO2 and H2O. When the retention time is 4.19min, it is identified as orsenoic acid based on the structure and the secondary fragment ion of the reference substance. The quasi-molecular ion peak of the primary mass spectrum is m / z 167.0357 [MH] - According to the database, the molecular formula is C8H8O4. One molecule of water is lost in the secondary mass spectrum, and the fragment ion 149.0245 is [MH-H2O] - , or directly lose 1 CO2, obtaining the fragment ion 123.0451 [MH-CO2] - , and then lose 1 molecule of water to obtain the fragment ion 105.0351 [MH-CO2-H2O]- .

[0107] When desuccinic acid compounds are cleaved, on the one hand, the mother nucleus is prone to continuous or combined loss of neutral molecules such as CO, CO2, and CH3. On the other hand, by breaking the ester bond of the eight-membered ring transition state hydrogen rearrangement, it competes with the salicylic acid part A and the phenol part B, causing them to break into two parts AB, and then the A or B part loses CH3, CO, CO2 or a combination of losses. Taking the reference substance black tea stain as an example, when its retention time is 16.59min, the primary mass spectrum shows a quasi-molecular ion peak m / z 373.0929 [MH] - The calculated molecular formula is C 19 H 18 O8, m / z 195.0670 is the part B of the corresponding ester bond rupture, m / z 195.0670, m / z 163.0395 and m / z 119.0505 are the ion fragments formed by the loss of CH3, H2O and CO2 from the part B, respectively; and m / z 177.0198 and m / z 105.0349 are the ion fragments formed by the continuous loss of CO2 and CHO from the part A of the corresponding ester bond rupture.

[0108] When dibenzofurans are cracked, on the one hand, the parent nucleus continuously removes neutral small molecules such as CH3 and CO, and on the other hand, these compounds are C 1-12 and C 2-3 It is easy to undergo reverse Diels-Alder (RDA) rearrangement, and the characteristic fragments of m / z84Da (C4H4O2) and m / z 112Da (C5H4O3) are mostly formed. When the retention time is 16.05min, it is identified as usnic acid based on the structure and the secondary fragment ion of the reference substance, and the primary mass spectrum shows a quasi-molecular ion peak of m / z 343.0837 [MH] - , the calculated molecular formula is C 18 H 16 O7, on the one hand, lost one methyl group in the secondary mass spectrum to obtain m / z 328.0579 [MH-CH3] - , and then lose two CH3 and CH2 successively to obtain m / z 313.0345 [MH-2CH3] - and m / z 299.0908 [MH-2CH3-CH2] - On the other hand, RDA rearrangement occurred to give m / z 259.0596 [MH-C4H4O2] - Fragment and m / z 231.0648 [MH-C5H4O3] - .

[0109] Table 6 High-resolution HPLC-MS analysis results of the main chemical components in Taibai tea

[0110]

[0111] 5. Discussion

[0112] 5.1. Selection of extraction solvent and method

[0113] This experiment investigated different extraction solvents (chloroform, methanol, ethanol, water), and found that when methanol was used as the extraction solvent, the peak number was optimal; at the same time, different proportions (50%, 60%, 70%, 80%, 100%) of methanol-water extracts were investigated, and the results showed that when pure methanol was used as the extraction solvent, the instrument response was higher, the spectrum lines were relatively stable, and the number of peaks was larger, so pure methanol was selected as the extraction solvent. The two extraction methods, ultrasonic method and reflux method, were investigated separately, and it was found that the extraction efficiency of most components under the two conditions was equivalent, but the heating reflux method would increase the response value of the chromatographic peak.

[0114] 5.2. Selection of detection wavelength and mobile phase

[0115] When establishing the fingerprint, the sample solution was first scanned at a full wavelength of 190-400nm. It was found that the chromatographic peaks at 254nm, 300nm, and 365nm were clearly expressed. Through experimental comparison, 254nm was selected as the detection wavelength of Taibai tea. In the early stage, different mobile phase systems such as methanol-water, methanol-0.1% phosphoric acid aqueous solution, methanol-0.1% formic acid aqueous solution, acetonitrile-water, acetonitrile-0.1% phosphoric acid aqueous solution, and acetonitrile-0.1% formic acid aqueous solution were also compared. The results showed that when methanol-0.1% phosphoric acid aqueous solution was used as the mobile phase for gradient elution, the chromatographic peak separation effect of each component was the best, while when acetonitrile-0.1% formic acid aqueous solution was used as the mobile phase for gradient elution in UHPLC-Q-Exactive Focus-MS analysis, the chromatographic peak shape of each component was better. Therefore, these two mobile phases were selected for elution during fingerprint establishment and UHPLC-Q-Exactive Focus-MS analysis.

[0116] 5.3 Results Analysis

[0117] The Taibai tea fingerprint established in this experiment identified 8 common peaks, and evaluated the similarity of the fingerprints of 10 batches of samples. The results showed that there was good similarity with the control fingerprint. According to the separation of the chromatographic peaks, peak 4 has a good separation, a relatively large peak area, and a central position, so this peak was selected as the reference peak. When using UHPLC-Q-ExactiveFocus-MS to analyze and identify common peaks, each component was detected in positive and negative ion modes, respectively. It was found that each compound had a higher response in the negative mode, which may be related to the fact that the main chemical components in Taibai tea are lichenic acids, so the negative ion mode was finally used for sample determination.

[0118] The ion fragment information of the characteristic components in Taibai tea was collected and analyzed, and four components in its fingerprint were identified in combination with literature and data analysis. Since the reference materials of the main components in Taibai tea are difficult to obtain, the four common peaks were speculated by the fragment ion peaks of the compounds obtained by preparative liquid chromatography and mass spectrometry technology. There are still four common peaks that have not been identified. Subsequent research can further determine their structures after separation and purification by preparative chromatography.

[0119] In summary, this study constructed the HPLC fingerprint of Taibai tea medicinal materials, and used UHPLC-Q-Exactive Focus-MS technology to identify and analyze the four common characteristic peaks in the fingerprint, which provided a basis for the quality evaluation of Taibai tea medicinal materials and laid the foundation for subsequent research on the material basis of Taibai tea's efficacy.

[0120] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for establishing a HPLC fingerprint of Taibai tea, characterized in that: The following steps are involved: S1. Preparation of test solution Take Taibai tea sample powder, add extraction solvent, perform ultrasonic extraction, and filter to obtain; S2. Establishment of HPLC fingerprint The test solution of S1 was tested by HPLC, and the chromatogram was recorded to obtain the HPLC fingerprint of Taibai tea; S3. Determination of standard fingerprint According to the methods of S1 to S2, HPLC fingerprints of multiple batches of Taibai tea were established. Through analysis and comparison, it was determined that the HPLC fingerprints of multiple batches of Taibai tea samples all had 8 common characteristic peaks; the 8 common characteristic peaks constituted the fingerprint characteristics of Taibai tea, which served as the standard fingerprint of Taibai tea; In S1, the extraction solvent is methanol; In S2, the HPLC chromatographic conditions are as follows: C18 column, 250 × 4.6 mm, 5 μm; column temperature, 30 °C; Mobile phase A was methanol, and mobile phase B was 0.1% phosphoric acid in water; The gradient elution program was 0-10 min, 30%-45% mobile phase A; 10-80 min, 45%-80% mobile phase A; 80-90 min, 80%-95% mobile phase A; Flow rate: 1.0 mL min -1 ; The detection wavelength was 254nm; the injection volume was 10μL.

2. The method for establishing the HPLC fingerprint of Taibai Tea according to claim 1, characterized in that: In S1, the usage ratio of Taibai tea sample powder to methanol is 0.5 g: 25-50 mL.

3. The method for establishing the HPLC fingerprint of Taibai Tea according to claim 1, characterized in that: In S1, the ultrasonic extraction time is 30 to 60 min.

4. The method for establishing the HPLC fingerprint of Taibai Tea according to claim 1, characterized in that: In S3, the retention times of the eight common characteristic peaks are 11.3, 18.755, 37.402, 53.487, 60.492, 67.328, 75.132, and 84.588, respectively.

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