A method for simultaneously determining contents of sibiricine, puerarin and salvianolic acid B in health-care food by two-dimensional liquid chromatography

Abstract

The application discloses a method for simultaneously determining contents of shugan su, puerarin and salvianolic acid B in health-care food by two-dimensional liquid chromatography, which comprises the following steps: extracting a sample to be measured by a solvent to obtain an extract, detecting the extract by a two-dimensional high performance liquid chromatograph, on-line center cutting, and quantifying by an external standard method. The method separates shugan su, puerarin and salvianolic acid B in the sample based on the on-line center cutting two-dimensional liquid chromatography, solves the problem that impurity peaks of salvianolic acid B exist under different mobile phase systems for health-care food with complex components, improves the separation degree and accuracy of salvianolic acid B, and the two-dimensional detection is on-line synchronous, which greatly improves the detection efficiency.

Description

Technical Field

[0001] This invention belongs to the field of health food testing technology, specifically relating to a method for simultaneously determining the content of silymarin, puerarin and salvianolic acid B in health foods using two-dimensional liquid chromatography. Background Technology

[0002] The harmful effects of alcohol on the human body, especially on the liver, are increasingly attracting attention. Developing products that can effectively inhibit ethanol-induced liver damage is a research hotspot in the health food and pharmaceutical industries. Traditional Chinese medicinal herbs such as kudzu flower, kudzu root, milk thistle, schisandra fruit, galangal, and salvia miltiorrhiza are important raw materials for hangover remedies and liver-protecting products, and there are many such products on the market.

[0003] The content of active ingredients in traditional Chinese medicine (TCM) herbs is crucial to the efficacy and quality of hangover relief and liver protection products. Take milk thistle, kudzu root, and danshen tablets as an example. This health food product, developed using milk thistle, danshen, and kudzu root as raw materials, is designed to relieve hangovers and protect the liver. Silymarin, puerarin, and salvianolic acid B are the functional components of these three herbs, respectively. Silymarin, also known as a "natural liver protectant," has a significant protective and stabilizing effect on liver cells. Puerarin, an isoflavone derivative isolated from kudzu root, has a good therapeutic effect on acute liver injury. Salvianolic acid B, formed by the condensation of three molecules of salvianolic acid and one molecule of caffeic acid, is one of the most studied salvianolic acids and can effectively alleviate pathological damage to liver tissue. The content of these three components is key to the product's hangover relief and liver protection efficacy. Therefore, developing a method to simultaneously determine the content of silymarin, puerarin, and salvianolic acid B in health foods can provide a basis for the quality control of health foods and has certain research significance.

[0004] Currently, there are no literature reports on methods for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food products. Researchers have found that methods for detecting the content of silymarin, puerarin, and salvianolic acid B suffer from problems such as diverse determination conditions and inconsistent chromatographic conditions. In addition, due to the complex composition of traditional Chinese medicine health food products and the difficulty in sample preparation, impurity peaks may interfere with chromatographic detection, leading to difficulties in accurate quantification and low detection efficiency. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a method for simultaneously determining the content of silybin, puerarin, and salvianolic acid B in health food products using two-dimensional liquid chromatography. This invention uses a center-cut two-dimensional liquid chromatography method to separate silybin, puerarin, and salvianolic acid B in a sample, improving the separation degree and accuracy of salvianolic acid B, while also increasing detection efficiency.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for simultaneously determining the contents of silymarin, puerarin, and salvianolic acid B in health food products using two-dimensional liquid chromatography includes the following steps:

[0008] (1) The sample to be tested was extracted by ultrasonic extraction with solvent to obtain an extract;

[0009] (2) The retention times of puerarin, salvianolic acid B, and silymarin in the first dimension of the two-dimensional high performance liquid chromatography were determined by the first dimension of the two-dimensional high performance liquid chromatography.

[0010] (3) The extract was detected by two-dimensional high performance liquid chromatography. Based on the retention time of salvianolic acid B in the first-dimensional liquid chromatography, it was cut online at the center and quantified by external standard method.

[0011] The system suitability chromatographic conditions for the two-dimensional high-performance liquid chromatograph are as follows:

[0012] The first-dimensional liquid chromatography conditions were as follows: the first-dimensional column was packed with octadecylsilane-bonded silica gel; gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol as mobile phase C; the detection wavelengths were 247 nm for 0-15.60 min and 287 nm for 15.60-37 min.

[0013] The conditions for the second-dimensional liquid chromatography were as follows: the second-dimensional column was packed with pentafluorobenzene; isocratic elution was performed using 0.1% phosphoric acid as mobile phase A1 and acetonitrile as mobile phase B1; and the detection wavelength was 286 nm.

[0014] Capture column: filled with octadecylsilane-bonded silica gel;

[0015] The elution procedure is shown in the table below:

[0016]

[0017]

[0018] Preferably, in step (1), the solvent is a methanol aqueous solution with a volume concentration of 60-80%; the ultrasonic extraction time is 20-40 min; and the mass-volume ratio of the sample to the solvent is 1:40-60 g / ml.

[0019] Preferably, in steps (2) and (3), the column temperature of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography is 30°C; the flow rate of the first-dimensional liquid chromatography is 0.8 mL / min, and the flow rate of the second-dimensional liquid chromatography is 0.5 mL / min.

[0020] Preferably, in step (3), the time for online center cutting is the retention time of salvianolic acid B in the first-dimensional liquid chromatography ± 0.1 min.

[0021] In a preferred embodiment of the present invention, the first-dimensional chromatographic column is an Agilent ZORBAX EclipsePlus C18, 4.6×250mm, 5μm.

[0022] In a preferred embodiment of the present invention, the second-dimensional chromatographic column is an Agilent Poroshell 120PFP, 4.6×100mm, 4μm.

[0023] In a preferred embodiment of the present invention, the capture column is an Agilent Poroshell 120EC C18, 4.6×50mm 2.7μm.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] This invention provides a method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food using two-dimensional liquid chromatography, which standardizes the sample preparation method and simplifies the pretreatment process.

[0026] This invention utilizes online center-cut two-dimensional liquid chromatography to separate silybin, puerarin, and salvianolic acid B from a sample. First, in the first dimension, a methanol-water system is used to separate silybin, puerarin, and salvianolic acid B. Then, salvianolic acid B from the first dimension is captured and further separated in the second dimension using an acetonitrile-water system. This method solves the problem of interference from impurity peaks in salvianolic acid B in complex health foods under different mobile phase systems, improving the separation accuracy and precision of salvianolic acid B. Furthermore, the two-dimensional detection is performed online simultaneously, significantly increasing detection efficiency.

[0027] This invention demonstrates the scientific effectiveness of the content determination method for silymarin, puerarin, and salvianolic acid B in health foods through linearity, precision, robustness (stability), specificity (blank), limit of detection, limit of quantitation, accuracy (recovery rate) tests, and actual sample measurements. The method can effectively control the content of silymarin, puerarin, and salvianolic acid B in health foods. Attached Figure Description

[0028] Figure 1 The diagram shows the chromatographic flow path connections: (1 is the injector; 2 is the first-dimensional pump; 3 is the first-dimensional column; 4 is the capture column; 5 is the second-dimensional column; 6 is the UV detector; 7 is the second-dimensional pump; 8 is the UV detector; V1 is valve position 1; V2 is valve position 2; V3 is valve position 3; V4 is valve position 4; V5 is valve position 5; V6 is valve position 6).

[0029] Figure 2 This is a blank chromatogram;

[0030] Figure 3 Chromatograms of standard solutions of silymarin, puerarin, and salvianolic acid B;

[0031] Figure 4 The standard working curve for salvianolic acid B;

[0032] Figure 5 The standard operating curve for silymarin;

[0033] Figure 6 The standard working curve for puerarin;

[0034] Figure 7 The chromatogram of a single-dimensional methanol-water system (V / V) (20-80:80-20);

[0035] Figure 8 The chromatogram is for a single-dimensional acetonitrile-water system (V / V) (20-80:80-20). Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. However, the embodiments of the present invention are not limited to the following examples.

[0037] Example 1:

[0038] 1 Instrument

[0039] Two-dimensional high performance liquid chromatograph: equipped with ultraviolet detector; electronic balance with a sensitivity of 0.1 mg; ultrasonic generator.

[0040] 2 Reagents / References

[0041] Unless otherwise specified, all reagents used were of analytical grade.

[0042] 2.1 Reagents:

[0043] Grade I water; methanol: chromatographic grade; acetonitrile: chromatographic grade; phosphoric acid.

[0044] 2.2 Reference Standard:

[0045] Silymarin reference standard (CAS: 22888-70-6, purity: 99.7%, source: Shanghai Yuanye Biotechnology, batch number: C27N11Y131954);

[0046] Puerarin reference standard (CAS: 3681-99-0, purity: 96.8%, source: China National Institutes for Food and Drug Control, batch number: 110752-202217);

[0047] Tanshinone B reference standard (CAS: 115939-25-8, purity: 97.5%, source: China National Institutes for Food and Drug Control, batch number: 111562-202318);

[0048] 2.3 Reagent Preparation:

[0049] 75% (V / V) methanol: Measure 75 mL of methanol, dissolve it in water and dilute to 100 mL, then mix well;

[0050] 0.1% phosphoric acid solution: Measure 1.0 ml of phosphoric acid, add water to make up to 1000 mL, and mix well;

[0051] Nylon filter head (pore size 0.45μm);

[0052] The test sample was milk thistle, kudzu root and salvia miltiorrhiza tablets (source: By-Health Co., Ltd.).

[0053] 3. Analytical Methods

[0054] 3.1 Chromatographic conditions for system suitability test:

[0055] Chromatographic columns: First-dimensional column: Agilent ZORBAX Eclipse Plus C18, 4.6×250mm, 5μm. Second-dimensional column: Agilent Poroshell 120PFP, 4.6×100mm, 4μm.

[0056] Capture column: Agilent Poroshell 120EC C18, 2.7μm.

[0057] Column temperature: 30℃.

[0058] Flow rate: First dimension: 0.8 mL / min; Second dimension: 0.5 mL / min.

[0059] Detection wavelengths: First dimension: 0-15.60 min: 247 nm, 15.60-37 min: 287 nm; Second dimension: 286 nm.

[0060] Valve position:

[0061] Figure 1 As shown in Figure a, it is the initial flow path connection diagram of two-dimensional liquid chromatography. The sample to be detected enters the first-dimensional chromatographic column through the injector along with the first-dimensional mobile phase under the pressure of the first-dimensional pump. Then it passes through valves V2-V3 to the ultraviolet detector and then to the waste liquid collector. The signal of puerarin can be obtained on the first-dimensional ultraviolet detector.

[0062] Figure 1As shown in Figure b, this is the flow path connection diagram of two-dimensional liquid chromatography after the central cutting switching valve. At 22.18 min (specifically 0.1 min before the peak start time of salvianolic acid in the first-dimensional liquid chromatography), the valve is adjusted. After passing through the first-dimensional liquid chromatography, the sample to be detected passes through valves V2-V1, flows through the capture column, and then passes through valves V4-V3 to the waste liquid collector. This valve cutting operation can enrich salvianolic acid B in the capture column.

[0063] At 23.31 min (specifically 0.1 min after the peak of salvianolic acid in the first-dimensional liquid chromatography ended), adjust the valve to switch the two-dimensional liquid chromatography flow path back to the initial state (i.e., Figure 1 (As shown in a). After passing through the first-dimensional chromatographic column, silymarin enters through valves V2-V3 for separation by first-dimensional liquid chromatography, and salvianolic acid B is separated by second-dimensional liquid chromatography.

[0064] Mobile phase: Gradient elution conditions are shown in Table 1 below.

[0065] Table 1

[0066]

[0067] 3.2 Solution Preparation

[0068] 3.2.1 Preparation of standard solutions:

[0069] Preparation of standard stock solutions of silybin, puerarin, and salvianolic acid B (silybin: 3.8524 mg / mL, puerarin: 1.5052 mg / mL, salvianolic acid B: 1.3738 mg / mL): Weigh 38.64 mg, 15.55 mg, and 14.09 mg of silybin, puerarin, and salvianolic acid B reference standards respectively into 10 mL volumetric flasks, add an appropriate amount of 75% methanol and sonicate to dissolve (silybin is dissolved by heating with pure methanol appropriately), dilute to the mark with methanol, and shake well.

[0070] Preparation of mixed standard working solution of silybin, puerarin, and salvianolic acid B (silybin: 0.3082 mg / mL, puerarin: 0.1204 mg / mL, salvianolic acid B: 0.0824 mg / mL): Accurately measure 0.80 mL, 0.80 mL, and 0.60 mL of silybin, puerarin, and salvianolic acid B standard stock solutions into 10 mL volumetric flasks, respectively, add methanol to the mark, and shake well.

[0071] 3.2.2 Preparation of standard curve solution:

[0072] Standard working solutions were injected in increments of 1.0 μL, 2.0 μL, 3.0 μL, 4.0 μL, and 5.0 μL respectively under conditions 5.1, and the results were analyzed to plot the external standard method standard working curve.

[0073] 3.2.3 Preparation of test solution:

[0074] Take an appropriate amount of sample, grind it into a homogenized powder, accurately weigh 0.2 g of the sample, place it in a 10 mL volumetric flask, add an appropriate amount of 75% methanol solution, sonicate at 45℃ for 30 min, cool, and dilute to volume with 75% methanol, then mix well. Centrifuge for 1 min, and filter through a 0.45 μm nylon membrane for analysis. Perform a blank test simultaneously.

[0075] 3.3 Measurement

[0076] 3.3.1 Flow path connection (e.g.) Figure 1 (As shown)

[0077] 3.3.2 Determine the cutting time of salvianolic acid B and establish a complete acquisition method.

[0078] Following the chromatographic conditions in 3.1, a single injection of a mixed standard working solution of silymarin, puerarin, and salvianolic acid B was performed to obtain the retention time of salvianolic acid B on the first-dimensional column. This retention time ±0.1 min is the salvianolic acid B cutoff time for the complete method. The cutoff start time is the salvianolic acid B peak initiation time minus 0.1 min, and the switchover end time is the salvianolic acid B peak return to baseline time plus 0.1 min.

[0079] 3.3.3 Inject the mixed standard working solution and 1.0 μL of the test solution into the two-dimensional liquid chromatograph. The chromatogram of the test sample should show a chromatographic peak with the same retention time as the reference standard peak. Calculate its concentration using the external standard method through the above standard curve.

[0080] 3.4 Result Calculation

[0081]

[0082] Where: X—content of silymarin, puerarin, and salvianolic acid B in the sample, g / 100g;

[0083] C—Concentration of silymarin, puerarin, and salvianolic acid B in the sample solution, mg / mL;

[0084] M—mass of the sample, in g;

[0085] V—Volume of sample dilution, mL;

[0086] K—Unit conversion factor, K = 0.1.

[0087] 4. Methodological Validation

[0088] 4.1 Specificity test (blank)

[0089] 4.1.1 Test Methods

[0090] Without adding samples, the blank solution was prepared according to the sample preparation method in 3.2.3, and the blank solution was determined according to the chromatographic conditions in 3.1. The peak times of the blank solution were compared with those of the standard working solutions of silymarin, puerarin, and salvianolic acid B.

[0091] 3.1.2 Test Results (see...) Figure 2-3 )

[0092] 3.1.3 Experimental Conclusion:

[0093] The blank solution showed no peaks at the elution times of silymarin, puerarin, and salvianolic acid B, indicating that the blank solution had virtually no interference with the determination results of silymarin, puerarin, and salvianolic acid B, demonstrating the good specificity of the method.

[0094] 3.2 Linear Range Confirmation

[0095] 3.2.1 The experimental data are shown in Table 2-4:

[0096] Table 2

[0097]

[0098] Table 3

[0099]

[0100] Table 4

[0101]

[0102] 3.2.2 Standard Working Curve Diagram

[0103] A standard working curve was plotted with silymarin, puerarin, and salvianolic acid B (mg / mL) on the x-axis and peak area (mAU*s) on the y-axis, as shown below. Figure 4-6 As shown.

[0104] 3.2.3 Conclusions of the Linearity Test

[0105] Linearity evaluation: The correlation coefficients (R) for silymarin, puerarin, and salvianolic acid B were 0.99999, 0.99999, and 0.99999, respectively. Therefore, this method shows good linearity for the determination of silymarin, puerarin, and salvianolic acid B in concentrations of 0.308193 mg / mL to 1.540963 mg / mL, 0.120419 mg / mL to 0.602096 mg / mL, and 0.082426 mg / mL to 0.412132 mg / mL, respectively, which meets the requirements of GB / T 27404-2008 Laboratory Quality Control Standard for Physicochemical Testing of Food [GB / T27404-2008 requires a correlation coefficient R ≥ 0.99].

[0106] 3.3 Limit of Detection and Limit of Quantification

[0107] The limits of detection (DL) and quantitation (QL) of the analytical method are calculated using the signal-to-noise ratio (S / N). DL is defined as the concentration of the analyte when S / N = 3, and QL is defined as the concentration of the analyte when S / N = 10.

[0108] 3.3.1 Limit of Detection

[0109] When the signal-to-noise ratio (S / N) is 3, the detection limits for silybin, puerarin, and salvianolic acid B are 0.00058608 mg / mL, 0.000063630 mg / mL, and 0.000087832 mg / mL, respectively. When the sample volume is 0.2 g and the volume is adjusted to 10 mL, the detection limits for silybin, puerarin, and salvianolic acid B are 0.00058608×10 / 0.2×1000=29.304 μg / g, 0.000063630×10 / 0.2×1000=3.1815 μg / g, and 0.000087832×10 / 0.2×1000=4.3916 μg / g.

[0110] 3.3.2 Limit of Quantification

[0111] When the signal-to-noise ratio (S / N) is 10, the detection limits for silybin, puerarin, and salvianolic acid B are 0.0019536 mg / mL, 0.0002121 mg / mL, and 0.000292773 mg / mL, respectively. When the sample volume is 0.2 g and the volume is adjusted to 10 mL, the quantification limits for silybin, puerarin, and salvianolic acid B are 0.0019536×10 / 0.2×1000=97.68 μg / g, 0.0002121×10 / 0.2×1000=10.605 μg / g, and 0.000292773×10 / 0.2×1000=14.63865 μg / g.

[0112] 3.4 Precision Test

[0113] 3.4.1 Test Methods

[0114] Weigh 6 samples and process them according to the test solution preparation method in 3.2.3. Detect the contents of silymarin, puerarin, and salvianolic acid B in the samples and calculate their RSD (%).

[0115] 3.4.2 The experimental data are shown in Table 5-7:

[0116] Table 5

[0117]

[0118]

[0119] Table 6 Table 7

[0120]

[0121] 3.4.3 Experimental Conclusions

[0122] The RSDs of silymarin, puerarin, and salvianolic acid B in the six samples were 0.6%, 0.7%, and 1.1%, respectively, indicating that the method has good precision and meets the requirements of GB / T 27404-2008 Laboratory Quality Control Standard for Physicochemical Testing of Food [GB / T 27404-2008 requires RSD ≤ 2.0%].

[0123] 3.5 Durability test (stability)

[0124] 3.5.1 Test Method:

[0125] After the mixed standard working solution of silymarin, puerarin, and salvianolic acid B and the test sample solution were placed at room temperature for 0 h, 2.5 h, 5.0 h, 10.0 h, 15.0 h, and 20.0 h, respectively, the peak area (mAU*s) of the mixed standard working solution and the test sample solution were measured according to the conditions in 3.1, and their RSD (%) were calculated.

[0126] 3.5.2 The experimental data are shown in Table 8-9:

[0127] Table 8 Table 9

[0128]

[0129] 3.5.3 Experimental Conclusions

[0130] After the mixed standard working solution of silymarin, puerarin, and salvianolic acid B and the test sample solution were placed at room temperature for 0 h, 2.5 h, 5.0 h, 10.0 h, 15.0 h, and 20.0 h, respectively, the peak area RSDs of the mixed standard working solution of silymarin, puerarin, and salvianolic acid B were 1.1%, 2.0%, and 1.7%, respectively, and the peak area RSDs of the test sample solution of silymarin, puerarin, and salvianolic acid B were 0.6%, 0.5%, and 0.7%, respectively. This indicates that the mixed standard working solution of silymarin, puerarin, and salvianolic acid B and the test sample solution have good stability at room temperature for 20 hours.

[0131] 3.6 Accuracy Test (Recovery Rate)

[0132] 3.6.1 Test Methods

[0133] Spiking method: Accurately weigh 9 samples of 0.2g each (the contents of silybin, puerarin, and salvianolic acid B in the samples are known to be 2.78g / 100g, 1.06g / 100g, and 0.91g / 100g, respectively), divide them into 3 groups, 3 samples in each group, and accurately add silybin, puerarin, and salvianolic acid B standard spiking solutions (concentrations of silybin: 3.8524mg / mL, puerarin: 1.5052mg / mL, salvianolic acid B: 1.3738mg / mL) to each group respectively. Process the samples according to the test solution preparation method in 3.2.3, and calculate the recovery rates of silybin, puerarin, and salvianolic acid B in the samples respectively.

[0134] 3.6.2 The experimental data are shown in Table 10 below (where V = 10 mL):

[0135] Table 10 Table 11

[0136]

[0137]

[0138] Table 12

[0139]

[0140] The measured amount of spiked sample = C × V;

[0141] Measured amount of sample = M × X × 10;

[0142] Measured spiking amount = Measured amount of spiked sample - Measured amount of sample;

[0143] Recovery rate (%) = Measured spiking amount / Theoretical spiking amount × 100%.

[0144] 3.6.3 Experimental Conclusions

[0145] The average recoveries of silymarin, puerarin, and salvianolic acid B in the samples were 99.3%, 99.5%, and 97.8%, respectively, which meet the requirements of GB / T27404-2008 Laboratory Quality Control Standard for Physicochemical Testing of Food [GB / T27404-2008 requires a recovery rate of 95-105%].

[0146] 4. Conclusion

[0147] The linearity, precision, robustness (stability), specificity (blank), limit of detection, limit of quantitation, and accuracy (recovery) tests of the content determination methods for silymarin, puerarin, and salvianolic acid B all met the requirements of GB / T27404-2008 Laboratory Quality Control Standard for Physicochemical Testing of Food, proving that the content determination methods are scientific and effective and can achieve the purpose of quality control of the content of silymarin, puerarin, and salvianolic acid B in milk thistle, kudzu root, and danshen tablets.

[0148] Comparative Example 1: Comparative Experiment of Different Extraction Methods

[0149] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol to each, and heat under reflux for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase membrane to obtain the sample solution.

[0150] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol to each, and sonicate for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase membrane to obtain the sample solution.

[0151] The results of the comparative experiment are shown in Table 13:

[0152] Table 13

[0153]

[0154] As shown in the table above, through experimental comparison of different extraction methods in this invention, the method of heating and refluxing for 30 min has a larger RSD and the extraction of salvianolic acid B is incomplete. This indicates that the extraction method of ultrasonic extraction in the pretreatment of this invention has higher extraction efficiency and more accurate quantitative results.

[0155] Comparative Example 2: Comparative Experiment on the Resolution and Precision of Tanshinone B in Different Mobile Phase Systems

[0156] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol, and sonicate at 45℃ for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase filter membrane to obtain the sample solution. Mobile phase: single-dimensional methanol-water system (V / V) (20-80:80-20), chromatogram shown in [reference needed]. Figure 7 Puerarin and silymarin can be separated relatively well, but salvianolic acid B still has interference from impurity peaks.

[0157] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol, and sonicate at 45℃ for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase filter membrane to obtain the sample solution. Mobile phase: acetonitrile-water system (V / V) (20-80:80-20), chromatogram shown in [reference needed]. Figure 8 Puerarin can be separated relatively well, but the introduction of acetonitrile will reduce the separation of the silymarin bimodal peaks, and salvianolic acid B has impurity peak interference.

[0158] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol, and sonicate at 45℃ for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase membrane to obtain the sample solution. Mobile phase: First dimension methanol-water system (V / V) (20-80:80-20), second dimension acetonitrile-water system (V / V) (20-80:80-20).

[0159] The results of the comparative experiment are shown in Table 14:

[0160] Table 14

[0161]

[0162] As shown in the table above, through comparative experiments on different mobile phase systems in this invention, the separation degree of salvianolic acid B in milk thistle, kudzu root, and danshen tablets was relatively low in the single-dimensional methanol-water or acetonitrile-water systems, and the RSD value of the detection results of salvianolic acid B with continuous injection was relatively high, indicating a certain measurement deviation. The two-dimensional methanol-water + acetonitrile-water system significantly improved the separation degree of salvianolic acid B in milk thistle, kudzu root, and danshen tablets, and the RSD value of the detection results of salvianolic acid B with continuous injection was low, indicating stable measurement results.

[0163] Comparative Example 3: Comparison of the resolution of salvianolic acid B in samples using second-dimensional chromatographic columns with different packing materials.

[0164] Simultaneously, weigh 1.0 g of each of the homogenized milk thistle, kudzu root, and salvia miltiorrhiza tablets, add 30 mL of 75% methanol, and sonicate at 45℃ for 30 min. After cooling, transfer the test solution to a 50 mL volumetric flask, dilute to volume with 75% methanol, centrifuge, and filter through an organic phase membrane to obtain the sample solution. The first dimension uses a column packed with octadecylsilane-bonded silica gel, and the second dimension uses columns packed with EC C18, HC C18, and PFP for selection.

[0165] The results of the comparative experiment are shown in Table 15:

[0166] Table 15

[0167]

[0168] As shown in the table above, through the comparison test of the resolution of salvianolic acid B in the sample by second-dimensional chromatographic columns with different packing materials, the chromatographic column with PFP (pentafluorobenzene) packing material has a better resolution of salvianolic acid in the sample.

Claims

1. A two-dimensional liquid chromatography method for the simultaneous determination of silymarin, puerarin, and salvianolic acid B in health food products. The method is characterized by, Includes the following steps: (1) The sample to be tested is extracted by ultrasonic extraction with solvent to obtain the extract; (2) The retention times of puerarin, salvianolic acid B, and silymarin in the first dimension of the two-dimensional high performance liquid chromatography were determined by the first dimension of the two-dimensional high performance liquid chromatography. (3) The extract was detected by two-dimensional high performance liquid chromatography. Based on the retention time of salvianolic acid B in the first-dimensional liquid chromatography, it was cut online at the center and quantified by external standard method. The chromatographic conditions of the two-dimensional high-performance liquid chromatograph are as follows: The first-dimensional liquid chromatography conditions were as follows: the first-dimensional column was packed with octadecylsilane-bonded silica gel; gradient elution was performed using 0.1% phosphoric acid as mobile phase A and methanol as mobile phase C; the detection wavelengths were 247 nm for 0-15.60 min and 287 nm for 15.60-37 min. The conditions for the second-dimensional liquid chromatography were as follows: the second-dimensional column was packed with pentafluorophenyl; isocratic elution was performed using 0.1% phosphoric acid as mobile phase A1 and acetonitrile as mobile phase B1; and the detection wavelength was 286 nm. Capture column: filled with octadecylsilane-bonded silica gel; The elution procedure is shown in the table below: 。 2. The two-dimensional liquid chromatography method according to claim 1 for the simultaneous determination of silymarin and puerarin in health food products. The method for determining the content of salvianolic acid B is characterized in that, In step (1), the solvent is a methanol aqueous solution with a volume concentration of 60-80%; the ultrasonic extraction time is 20-40 min; and the mass-volume ratio of the sample to the solvent is 1:40-60 g / ml.

3. The method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food by two-dimensional liquid chromatography according to claim 1, characterized in that, In steps (2) and (3), the column temperature of the first-dimensional liquid chromatography and the second-dimensional liquid chromatography is 30℃; the flow rate of the first-dimensional liquid chromatography is 0.8mL / min, and the flow rate of the second-dimensional liquid chromatography is 0.5mL / min.

4. The method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food by two-dimensional liquid chromatography according to claim 1, characterized in that, In step (3), the online center cut time is the retention time of salvianolic acid B in the first-dimensional liquid chromatography ± 0.1 min.

5. The method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food by two-dimensional liquid chromatography according to claim 1, characterized in that, The first-dimensional chromatographic column was an Agilent ZORBAX Eclipse Plus C18, 4.6×250mm, 5μm.

6. The method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food by two-dimensional liquid chromatography according to claim 1, characterized in that, The second-dimensional chromatographic column was an Agilent Poroshell 120 PFP, 4.6 × 100 mm, 4 μm.

7. The method for simultaneously determining the content of silymarin, puerarin, and salvianolic acid B in health food by two-dimensional liquid chromatography according to claim 1, characterized in that, The capture column was an Agilent Poroshell 120 EC C18, 4.6 × 50 mm, 2.7 μm.

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