Method for detecting content of puerarin and phloretin in plant extract
Through the quantitative detection of triple quadrupole liquid-mass junction (LCMS/MS), the problem that the prior art cannot simultaneously detect the concentration of puerarin and rosocholin in plant extracts is solved, achieving high accuracy and high efficiency detection effects.
Patent Information
- Application Number
- CN202311176966.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-13
- Publication Date
- 2025-03-21
AI Technical Summary
There is no method in the prior art to detect the concentrations of puerarin and rosorin in plant extracts simultaneously.
Quantitative detection was performed using triple quadrupole liquid-mass junction (LCMS/MS). Samples and standard curve solutions were prepared, combined with liquid chromatography and mass spectrometry technology, simultaneous detection of puerarin and rosorin was achieved.
Accurate quantification detection of puerarin and rosalin in plant extracts is achieved, with high accuracy, high precision and short time consuming, suitable for the detection and research of active ingredients related to Rosaceae plants.
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Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting and analyzing the contents of puerarin and phloretin in a plant extract. Background Art
[0002] Liquid chromatography-mass spectrometry tandem detection technology has developed rapidly in recent years due to its high sensitivity, strong specificity, short analysis time and good reproducibility. It is not only widely used in the fields of medicine, chemical industry, food, agriculture, etc., but is also gradually used in clinical testing and auxiliary diagnosis, providing technical support for precise detection and analysis.
[0003] Puerarin Phloretin It is commonly found in plants of the Rosaceae family. Studies have shown that it not only has pharmacological effects such as anti-tumor and anti-inflammatory, but also has a variety of cosmetic effects such as delaying aging, whitening skin, anti-inflammatory, and removing acne. With the increase in the market share of functional cosmetics, puerarin and phloretin as cosmetic functional ingredients have received extensive attention from researchers in the beauty industry. The existing technology has a method for detecting the active ingredient of puerarin alone, but there is currently no method for simultaneously detecting the concentration of puerarin and phloretin. Summary of the invention
[0004] In view of this, the present invention aims to provide a method for quantitatively detecting the concentrations of puerarin and phloretin in plant extracts using triple quadrupole liquid chromatography-mass spectrometry (LCMS / MS). The present invention realizes a method for simultaneously testing the concentrations of puerarin and phloretin in plant extracts.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: A method for detecting the concentration of puerarin and phloretin in a plant extract comprises the following steps: S1: Sample solution preparation, Take the plant extract test sample, dilute it with a diluent, and obtain a sample solution to be tested; S2: Preparation of standard curve solution, Weigh puerarin and phloretin standard products respectively, dilute with diluent to obtain puerarin standard working solution and phloretin standard working solution; S3: LC-MS / MS determination: Take the sample solution and use liquid chromatography-tandem mass spectrometry for qualitative or quantitative detection; Wherein, the liquid chromatography mobile phase A is water-formic acid (100:0.1); the mobile phase B is methanol.
[0006] In some embodiments, the liquid chromatography is eluted using the following elution mode: Table 1 Gradient elution mode .
[0007] In some embodiments, the diluent is methanol and water in a volume ratio of 1:1.
[0008] In some embodiments, the chromatographic column of the liquid chromatography is Agilent ZORBAX RRHD Eclipse Plus C18: column length 100 mm, diameter 3.0 mm, particle size 1.8 µm, column temperature 35-45°C, the flow rate of the mobile phase of the liquid chromatography is 0.26 ml / min-0.34 ml / min, and the injection volume of the mobile phase of the liquid chromatography is 1-4 ul.
[0009] In some embodiments, the column temperature is 40° C., the flow rate of the mobile phase is 0.30 ml / min, and the feed volume is 2 ul.
[0010] In some embodiments, the ion source of the mass spectrometer is an electrospray ion source; the drying gas flow rate is 5 L / min, the sheath gas temperature is 180°C, the sheath gas flow rate is 11 L / min, the nebulizer pressure is 35 psi, the capillary voltage is 3500 V, and the scanning mode of the mass spectrometer is a negative ion scanning mode.
[0011] In some embodiments, the ion pairs in the mass spectrum are shown in the following table: Table 2 Mass spectrometry ion pairs .
[0012] In some embodiments, the detection limit of puerarin and phloretin is 0.03 ng / ml, the quantification limit is 0.1 ng / ml, puerarin and phloretin have a good linear relationship in the range of 0.1 ng / ml to 5 ng / ml, and the recovery rate is 80% to 120%.
[0013] In some embodiments, the plant extract is a Rosaceae plant extract.
[0014] Beneficial effects: In the detection method of the present invention, by selecting a suitable diluent, chromatographic column, mobile phase, elution gradient, and mass spectrometry conditions, simultaneous quantitative detection of phloretin and puerarin in plant extracts can be achieved, and the detection results are highly accurate, highly precise, and time-saving, and can be effectively applied to the detection research of active ingredients related to phloretin and puerarin in Rosaceae plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 : Negative ion scanning mode (a) TIC graph of reference substance (1ug / ml) and blank solution (50% methanol) (b) Negative ion mass spectrum of puerarin (c) Negative ion mass spectrum of phloretin Figure 2 :EIC diagram of test product Figure 3 : Puerarin linearity and range test results Figure 4 :Results of linearity and range test of phloretin Figure 5 :EIC diagram of comparative example 1 Figure 6 :Comparative Example 2 EIC diagram Figure 7 :Comparative Example 3 (a) TIC graph of positive ion scanning mode reference substance (1ug / ml) and blank solution (50% methanol) (b) Positive ion mass spectrum of puerarin (c) Positive ion mass spectrum of phloretin Figure 8 : Comparative Example 4 EIC Diagram Fig. 9 :Comparative Example 5 EIC Diagram DETAILED DESCRIPTION
[0016] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings, but the embodiments do not limit the present invention in any form.
[0017] In the examples and comparative examples of the present application, an Agilent G6470B liquid phase mass spectrometer was used, and the chromatographic column in the example was an Agilent ZORBAX RRHD Eclipse Plus C18: column length 100 mm, diameter 3.0 mm, particle size 1.8 µm. Phloretin and puerarin standards were from Shanghai Anpu Experimental Technology Co., Ltd., the sample Rosa serrata plant extract to be tested was taken from a commercial Rosa serrata extract, and methanol and formic acid were both commercially available HPLC or HPLC grade reagents.
[0018] The chromatographic and mass spectrometric conditions of this example are shown in Table 3 Table 3 Chromatographic and mass spectrometry conditions
[0019] 1.1 Solution preparation Test solution: Take about 100 mg of the Rosa microstemmata extract test sample, accurately weigh it, place it in a 10 ml volumetric flask, add diluent to make up to volume, and use it as the test sample stock solution.
[0020] Take 1 ml of the test sample stock solution and place it in a 10 ml volumetric flask, add diluent to make up to volume, and the concentration is 1 mg / ml. Transfer the solution as the test sample solution.
[0021] Preparation of reference substance solution (1 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); take 1 ml of reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance solution (1 ng / ml).
[0022] Preparation of spiked test solution: Take 1 ml of the test sample stock solution (1) and 1 ml of the reference sample stock solution (2) respectively and place them in a 10 ml volumetric flask. Dilute to the mark with diluent to prepare the spiked test solution.
[0023] 1.2 Mass spectrometry negative ion mode The reference substance (1ug / ml) and blank solution (50% methanol) were scanned in negative ion mode. Figure 1 , RT = 6.42min, m / z = 415.1, deduced as M 葛根素 -H+, identified as puerarin, was in good abundance.
[0024] Full scan comparison of the reference substance (1ug / ml) and blank solution (50% methanol). RT = 11.15min, m / z = 273.0, deduced as M 根皮素 -H+ , identified as phloretin, was found in good abundance.
[0025] 1.3 Experimental Results Accurately measure 2 ml of blank solution, reference solution, test solution and spiked test solution, inject into LC-MS instrument and record the spectrum. The test results of the test solution are shown in Figure 2 ,After testing, the concentration of puerarin in the test sample was 1.05ng / ml, and the concentration of phloretin was 0.98ng / ml.
[0026] Results and analysis: The impurity peaks in the blank solution and the test sample had no interference with the detection of puerarin and phloretin, and the method had good specificity.
[0027] 1.4 Limit of detection and limit of quantification test (1) Take appropriate amounts of puerarin and phloretin reference substances to prepare a series of solutions. Take 2 μl of each solution for analysis. The detection limit is when S / N ≥ 3. (2) Take appropriate amounts of puerarin and phloretin reference substances to prepare a series of solutions. Take 2 μl of each solution for analysis. The limit of quantification is when S / N ≥ 10. (3) Take appropriate amounts of puerarin and phloretin reference substances to prepare 6 solutions with quantitative limit concentrations. Take 2 μl of each solution for sample analysis and calculate the RSD of the peak area and retention time of the 6 samples.
[0028] 1.41 Experimental Results Accurately measure 2 μL of the above solution, inject it into the LC-MS instrument, and record the chromatogram. The experimental results are shown in Tables 4 and 5.
[0029] Table 4 Quantitation limit results
[0030] Table 5 Detection limit results
[0031] Results and analysis: The RSD of the quantitative limit peak area was less than 15.0%, and the RSD of the retention time was less than 2.0%. The detection limit and quantitative limit concentrations were much lower than their limit concentrations, and the method had good sensitivity.
[0032] 1.5 Linearity and range test results Preparation of calibration solution and standard working solution, Take puerarin and phloretin, add diluent to dissolve and dilute to make solutions containing 200 μg of each in 1 ml, as reference stock solutions; take 50 μl of reference stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference stock solution (1); take 100 μl of reference stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference stock solution (2) (10 ng / ml); take 1 ml of reference stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference solution (1 ng / ml); take stock solution (2) and operate as follows: 500 μl and place it in 1 ml, as linear solution A; then take 400 μl of linear solution A and place it in 1 ml, as linear solution B; then take 500 μl of linear solution B and place it in 1 ml, as linear solution C; then take 500 μl of linear solution C and place it in 1 ml, as linear solution D; then take linear solution D 200 μl was transferred to 1 ml as linear solution E to prepare a series of linear test solutions.
[0033] Accurately measure 10 μl of each linear test solution for sample analysis, and use the quantitative limit as the starting point to calculate the linear relationship between peak area and concentration. The results are shown in Table 6-7. Figure 3-4 .
[0034] Table 6 Puerarin linearity and range test results
[0035] Table 7 Results of linearity and range test of phloretin
[0036] Results and analysis: In the concentration range of 0.1ng / ml~5ng / ml, the peak area and concentration of puerarin and phloretin were linearly fitted, and the linear relationship was good. The correlation coefficients were all greater than 0.99.
[0037] 1.6 Solution stability 1.6.1 Solution preparation Test solution: Take about 100 mg of the Rosa serrata test sample, weigh it accurately, place it in a 10 ml volumetric flask, add diluent to make up the volume, and use it as the test sample stock solution. Take another 1 ml of the test sample stock solution, place it in a 10 ml volumetric flask, add diluent to make up the volume, and the concentration is 1 mg / ml. Transfer the solution as the test sample solution.
[0038] Preparation of reference substance solution (1 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); take 1 ml of reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance solution (1 ng / ml).
[0039] Preparation of spiked test solution: Take 1 ml of the test sample stock solution (1) and 1 ml of the reference sample stock solution (2) respectively and place them in a 10 ml volumetric flask. Dilute to the mark with diluent to prepare the spiked test solution.
[0040] 1.6.2 Experimental Results The reference solution and the test solution were placed at room temperature, and the samples were injected and analyzed at 0, 2, 4, 6, 8, 10, and 12 hours to investigate the changes of puerarin and phloretin and their stability. The results are shown in Tables 8 and 9.
[0041] Table 8 Results of stability test of reference solution
[0042] Table 9 Stability test results of spiked test solution
[0043] Results and analysis: (1) The reference solution was placed at room temperature for 12 hours, and the RSD of the peak areas of puerarin and phloretin was no more than 10%. The reference solution was stable at room temperature for 12 hours. (2) The spiked test solution was placed at room temperature for 12 hours, and the RSD of the peak areas of puerarin and phloretin was no more than 10%. The reference solution was stable at room temperature for 12 hours.
[0044] 1.7. Injection precision 1.7.1 Solution preparation Preparation of reference substance solution (1 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); take 1 ml of reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance solution (1 ng / ml).
[0045] 1.7.2 Experimental Results Take the reference solution (1 ng / ml) and inject 6 times continuously, record the mass spectrum, and calculate the RSD of the peak area and retention time. The results are shown in Table 10-11.
[0046] Table 10. Puerarin injection precision test results
[0047] Table 11 Phloretin injection precision test results
[0048] Results and analysis: The reference solution was injected 6 times continuously, the RSD of the peak area was 1.2%, and the RSD of the retention time was 0.1%. The instrument injection precision was good.
[0049] 1.8. Repeatability 1.8.1 Solution preparation Preparation of spiked test solution: Take 1 ml of the test sample stock solution (1) and 1 ml of the reference sample stock solution (2) respectively and place them in a 10 ml volumetric flask. Dilute to the mark with diluent to prepare the spiked test solution.
[0050] 1.8.2 Experimental Results Accurately measure 2 μl of blank solution and spiked test solution, inject 6 times in succession, record the mass spectrum, and calculate the RSD of the peak area. The results are shown in Table 12.
[0051] Table 12. Repeatability test results
[0052] Results and analysis: The RSD of the contents in the 6 spiked test solutions was less than 10.0%, and the repeatability of this method was good.
[0053] 1.9. Intermediate precision 1.9.1 Solution preparation Preparation of reference substance solution (1 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); take 1 ml of reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance solution (1 ng / ml).
[0054] Preparation of spiked test solution: Measure 1 ml of the test sample stock solution (1) and 1 ml of the reference sample stock solution (2) into a 10 ml volumetric flask, dilute to the mark with diluent, and use this as the spiked test solution. Repeatability tests were performed by different analysts on different days.
[0055] 1.9.2 Experimental Results The content and RSD were calculated by linear regression equation. The results are shown in Table 13.
[0056] Table 13. Intermediate precision test results
[0057] Results and analysis: The results of 12 tests by different instruments and different personnel showed that the RSD of puerarin and phloretin content was no more than 10.0%, and the intermediate precision of this method was good.
[0058] 1.10. Accuracy test (recovery test) 1.10.1 Solution preparation Preparation of linear solution: Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference stock solution; take 50 μl of reference stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference stock solution (1); take 100 μl of reference stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference stock solution (2) (10 ng / ml); take 1 ml of reference stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference solution (1 ng / ml); take stock solution (2) and operate as follows: 500 μl and place it in 1 ml, as linear solution A; then take 400 μl of linear solution A and place it in 1 ml, as linear solution B; then take 500 μl of linear solution B and place it in 1 ml, as linear solution C; then take linear solution C 500μl was placed in 1ml as linear solution D; then 200μl of linear solution D was placed in 1ml as linear solution E to prepare a series of linear test solutions.
[0059] Preparation of reference substance stock solution (10 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, dissolve and dilute with diluent to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); prepare 3 copies in parallel.
[0060] 50% reference solution: Accurately measure 0.5 ml of the reference stock solution (10 ng / ml) and place it in a 10 ml volumetric flask. Dilute to the mark with diluent and shake well. This is used as the reference solution (0.5 ng / ml). Prepare 3 copies in parallel.
[0061] 100% reference substance solution: Accurately measure 1 ml of the reference substance stock solution (10 ng / ml) and place it in a 10 ml volumetric flask. Dilute to the mark with diluent and shake well. This is used as the reference substance solution (1 ng / ml). Prepare 3 copies in parallel.
[0062] 150% reference solution: Accurately measure 1.5 ml of the reference stock solution (10 ng / ml) and place it in a 10 ml volumetric flask. Dilute to the mark with diluent and shake well. This is used as the reference solution (1.5 ng / ml). Prepare 3 copies in parallel.
[0063] Test solution: Take about 100 mg of the Rosa gracilis extract test sample, weigh it accurately, place it in a 10 ml volumetric flask, add diluent to make up to volume, and use it as the test sample stock solution (10 mg / ml). Take 1 ml of the test sample stock solution, place it in a 10 ml volumetric flask, add diluent to make up to volume, and its concentration is 1 mg / ml, which is used as the test solution.
[0064] 50% recovery test solution: Take 0.5ml of reference solution (10ng / ml) and 1ml of test solution (10mg / ml) respectively and place them in the same 10ml volumetric flask, dilute to the mark with diluent. Prepare 3 portions in parallel as 50% recovery test solution; 100% recovery test solution: Take 1 ml of reference solution (10 ng / ml) and 1 ml of test solution (10 mg / ml) respectively and place them in the same 10 ml volumetric flask, dilute to the mark with diluent. Prepare 3 portions in parallel as 100% recovery test solution; 150% recovery test solution: Take 1.5ml of reference solution (10ng / ml) and 1ml of test solution (10mg / ml) respectively and place them in the same 10ml volumetric flask, dilute to the mark with diluent. Prepare 3 portions in parallel as 150% recovery test solution; 1.10.2 Experimental Results Accurately measure 2 μl of blank solution, linear test solution, reference solution, test solution and recovery test solution, respectively, and inject them into the mass spectrometer to record the mass spectrum. Calculate the recovery rate according to the linear regression equation method. The experimental results are shown in Table 14.
[0065] Table 14. Puerarin recovery test results
[0066] Table 15. Phloretin recovery test results
[0067] Results and Analysis: The average recoveries of puerarin and phloretin at different concentrations were between 80.0% and 120.0%, and the RSD was less than 10.0%. The accuracy of this method was good.
[0068] 1.11 Durability test 1.11.1 Solution preparation Preparation of reference substance solution (1 ng / ml): Take appropriate amount of puerarin and phloretin, weigh accurately, add diluent to dissolve and dilute to make a solution containing about 200 μg of each in 1 ml, as reference substance stock solution; measure 50 μl of reference substance stock solution and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (1); take 100 μl of reference substance stock solution (1) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance stock solution (2) (10 ng / ml); take 1 ml of reference substance stock solution (2) and place it in a 10 ml volumetric flask, dilute to the mark with diluent, as reference substance solution (1 ng / ml).
[0069] Preparation of spiked test solution: Take 1 ml of the test sample stock solution (1) and 1 ml of the reference sample stock solution (2) respectively and place them in a 10 ml volumetric flask. Dilute to the mark with diluent to prepare the spiked test solution.
[0070] 1.11.2 Experimental Results Precisely measure the blank solution, reference solution, and spiked test solution, and inject them under standard conditions and column temperature ±5°C, flow rate ±0.04ml / min, and organic phase ratio ±2%. In each condition, inject two injections of reference solution and spiked test solution, and record the mass spectrum. Calculate the contents of puerarin and root bark under standard chromatographic conditions and column temperature ±5°C, flow rate ±0.04ml / min, and organic phase ratio ±2%, and calculate the percentage ratio with the standard conditions to examine the durability. The experimental results are shown in Table 17.
[0071] Table 16 Durability Chromatographic Conditions
[0072] Table 17 Puerarin durability test results
[0073] Results and analysis: When the chromatographic conditions were changed by single factor: column temperature ±5℃, and the proportion of organic phase in the elution gradient ±2%, the blank solution had no interference with the detection of puerarin, and the ratio of puerarin content to the sample content under standard conditions was in the range of 80.0%~120.0%. This shows that this method has good durability under changing conditions such as column temperature and organic phase proportion.
[0074] Table 18 Phloretin durability test results
[0075] Results and analysis: When the chromatographic conditions were changed by single factor: column temperature ±5℃, and the proportion of organic phase in the elution gradient ±2%, the blank solution had no interference with the detection of puerarin, and the ratio of phloretin content to the sample content under standard conditions was in the range of 80.0%~120.0%. This shows that this method has good durability under changing conditions such as column temperature and organic phase proportion.
[0076] Comparative Example 1 Gradient elution mode change Other chromatographic and mass spectrometric conditions are the same as those in Table 3. The test reference solution 1ug / ml is prepared as described in the example. Only the gradient elution mode uses the gradient elution mode 2 in Table 19. The test results are shown in Table 3. Figure 5 .
[0077] Table 19 Gradient elution mode 2
[0078] It can be seen that the gradient elution mode 2 method of comparative example 1 can scan puerarin and detect the puerarin component, but cannot scan phloretin and cannot test the concentration of phloretin.
[0079] Table 20 Gradient elution mode 3 time Mobile phase A Mobile phase B 0 95 5 2 95 5 15 82 18 17 78 22 18 82 95 20 95 95 It can be seen that the gradient elution mode 3 method of comparative example 1 cannot scan phloretin and cannot test the concentration of phloretin.
[0080] Comparative Example 2: Changes in mobile phase composition Other chromatographic column and mass spectrometry conditions, gradient elution mode is the same as Table 3, only the mobile phase is changed to A: 0.1% formic acid-water, B: acetonitrile, the test reference substance 1ug / ml solution preparation method is described in the embodiment, the test results are shown in Figure 6 .
[0081] Depend on Figure 6 It can be seen that this method can scan puerarin, while the method in comparative example 2 cannot scan phloretin, so phloretin cannot be detected.
[0082] Comparative Example 3 Change of mass spectrometry scanning mode A full scan was performed using a positive ion pair reference substance (1ug / ml) and a blank solution (50% methanol). The scan results are shown in Figure 7 , RT = 6.36min, m / z = 417.1, deduced to be M 葛根素 +H + , m / z=417.1, derived as M 葛根素 +Na + , identified as puerarin, with a low abundance.
[0083] It can be seen that phloretin was not detected using the positive ion mode scan. Therefore, the positive ion scanning mode of Comparative Example 3 cannot test phloretin.
[0084] Comparative Example 4 Screening of feed amount and feed concentration Puerarin + phloretin (1ug / ml) was used, the detection method in Table 3 was adopted, the diluent was methanol, the feed volume was 10ul, and the detection results were shown in Figure 8 .have Figure 8 It can be seen that the peak shape of the samples in the test results is poor, the peak front of puerarin is large, and the peak tail of phloretin is large. This may be due to the solvent effect, which makes it impossible to accurately detect the content of puerarin and phloretin.
[0085] Comparative Example 5 Feed Amount and Solution Preparation Diluent Screening Puerarin + phloretin (100 ng / ml) was used, the detection method in Table 3 was adopted, the diluent was methanol, the feed volume was 5 ul, and the detection results were shown in Fig. 9 .
[0086] In the test results, the sample peak shape is slightly improved compared with comparative example 4, but the peak shape of puerarin still has a relatively obvious front, and the peak shape of phloretin has a tail, and the content of puerarin or phloretin cannot be accurately detected.
Claims
1. A method for detecting the concentration of puerarin and phloretin in a plant extract, characterized in that: The following steps are involved: S1: Sample solution preparation, Take the plant extract test sample, dilute it with a diluent, and obtain a sample solution to be tested; S2: Preparation of standard curve solution, Weigh puerarin and phloretin standard products respectively, dilute with diluent to obtain puerarin standard working solution and phloretin standard working solution; S3: LC-MS / MS determination: Take the sample solution and use liquid chromatography-tandem mass spectrometry for qualitative or quantitative detection; Wherein, the liquid chromatography mobile phase A is water-formic acid (100:0.1); the mobile phase B is methanol.
2. The method according to claim 1, characterized in that The liquid chromatography was eluted using the following elution mode: 。 3. The method according to claim 1, characterized in that The diluent is methanol and water in a volume ratio of 1:
1.
4. The method according to claim 1, characterized in that The chromatographic column of the liquid chromatography is Agilent ZORBAXRRHD Eclipse Plus C18: column length 100mm, diameter 3.0mm, particle size 1.8µm, column temperature 35-45°C, flow rate of the mobile phase of the liquid chromatography is 0.26ml / min-0.34ml / min, and the injection volume of the mobile phase of the liquid chromatography is 1-4ul.
5. The method according to claim 4, characterized in that The column temperature was 40° C., the flow rate of the mobile phase was 0.30 ml / min, and the feed volume was 2 ul.
6. The method according to claim 1, characterized in that The ion source of the mass spectrometer is an electrospray ion source; the drying gas flow rate is 5 L / min, the sheath gas temperature is 180°C, the sheath gas flow rate is 11 L / min, the nebulizer pressure is 35 psi, the capillary voltage is 3500 V, and the scanning mode of the mass spectrometer is a negative ion scanning mode.
7. The method according to claim 6, characterized in that The ion pairs in the mass spectrum are shown in the following table: 。 8. The method according to claim 6, characterized in that The detection limit of puerarin and phloretin is 0.03 ng / ml, the quantification limit is 0.1 ng / ml, puerarin and phloretin have a good linear relationship in the range of 0.1 ng / ml to 5 ng / ml, and the recovery rate is 80% to 120%.
9. The method according to claim 1, characterized in that The plant extract is a Rosaceae plant extract.