High performance liquid chromatography detection method of anthocyanin

Abstract

The invention discloses a high performance liquid chromatography detection method of anthocyanin, and belongs to the field of food detection. According to the method for detecting the anthocyanin in the maqui berry or the berry fruit extract, seven anthocyanin components in the maqui berry can be effectively separated, and particularly the separation effect on anthocyanin isomers is enhanced. The detection method is good in stability and high in precision.

Description

Technical Field

[0001] The invention belongs to the field of food detection, and particularly relates to a high performance liquid chromatography detection method for anthocyanins. Background Art

[0002] Anthocyanins, also known as anthocyanins, belong to the flavonoids and are a class of compounds formed by the glycosidic bond between anthocyanidins and sugars. They are the main source of pigments that give plants their brilliant colors, such as red, purple, and blue, and are widely found in the cell sap of flowers, fruits, stems, leaves, and roots of plants.

[0003] Anthocyanins have certain potential in the prevention and treatment of cardiovascular diseases, diabetes, cancer, eye diseases, etc. It can lower cholesterol and blood lipid levels, improve vascular elasticity, prevent atherosclerosis; regulate blood sugar metabolism, enhance insulin sensitivity; inhibit the growth and metastasis of tumor cells; protect retinal cells, improve vision, etc.

[0004] Maqui berry (Aristotelia chilensis) is rich in anthocyanins, mainly glycoside derivatives of cyanidin and delphinidin.

[0005] At present, there are many methods for detecting anthocyanins, such as traditional methods such as colorimetry, which is based on the relationship between the absorbance and concentration of anthocyanins at a specific wavelength for quantitative analysis. Although the operation is relatively simple and the cost is low, the specificity is poor and it is easily interfered by other pigments and impurities. It can only measure the total anthocyanin content, and cannot separate and accurately identify different types of anthocyanins, and cannot meet the needs of detailed analysis of anthocyanins in maqui berry.

[0006] High performance liquid chromatography is currently a commonly used method for detecting anthocyanins. It can achieve the separation and quantitative analysis of different types of anthocyanins. However, there are still some problems with the existing HPLC detection methods for anthocyanins in maqui berry. On the one hand, the chemical composition of maqui berry fruit is complex. In addition to anthocyanins, it also contains polysaccharides, proteins, fats and other substances. These impurities will affect the separation and detection of anthocyanins, resulting in broadening and tailing of chromatographic peaks, poor separation, and thus affecting the accuracy of quantification. On the other hand, the existing detection methods also need to be improved in terms of sensitivity, analysis time and stability. Some methods have low sensitivity and are difficult to detect low-content anthocyanin components in maqui berry; some methods have too long analysis time and low efficiency, which are not suitable for rapid detection of large-scale samples; moreover, the stability and reproducibility of the test results are not ideal, and there may be large differences in the test results between different laboratories or different operators. Summary of the invention

[0007] The purpose of the present invention is to provide a high performance liquid chromatography detection method for anthocyanins in view of the deficiencies of the prior art.

[0008] In one aspect, the present invention provides a high performance liquid chromatography method for detecting anthocyanins, comprising the following steps:

[0009] (1) ultrasonically dissolving the sample to be tested, fixing the volume, and filtering to obtain a solution to be tested;

[0010] (2) preparing anthocyanin standard stock solution and single-point calibration working solution;

[0011] (3) The test solution is subjected to high performance liquid chromatography analysis to separate various anthocyanin components.

[0012] In some embodiments, the source of anthocyanins includes, but is not limited to, one or more combinations of blueberry, strawberry, blackberry, cranberry, raspberry, maqui berry, black currant, raspberry, lingonberry, mulberry, and aronia berry.

[0013] In some embodiments, aronia berries include, but are not limited to, one or a combination of Aronia Melanocarpa, Aronia Arbutifolia, and Aronia Prunifolia.

[0014] In some embodiments, the source of anthocyanins is a berry extract.

[0015] In some embodiments, the solvent used for ultrasound in step (1) is an acidified organic solvent.

[0016] In some embodiments, the mass ratio of the acidified organic solvent to the sample to be tested in step (1) is 1 mL: 1-24 mg, preferably 1 mL: 2.5-12 mg, and further preferably 1 mL: 4-6 mg.

[0017] In some embodiments, in the acidified organic solvent in step (1), the acid includes, but is not limited to, one or more combinations of formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, and trifluoroacetic acid.

[0018] In some embodiments, the organic solvent in step (1) includes, but is not limited to, one or more combinations of methanol, ethanol, acetonitrile, tetrahydrofuran, diethyl ether, dichloromethane, and isopropanol.

[0019] In some embodiments, the volume fraction of the acid in the acidified organic solvent in step (1) is 1-50%, preferably 1-25%, more preferably 1-10%, specifically 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%.

[0020] In some embodiments, in step (1), the ultrasonic time is 1-60 min, preferably 3-30 min, and more preferably 5-15 min.

[0021] In some embodiments, the solution used for volume adjustment in step (1) is an acidic aqueous solution.

[0022] In some embodiments, the acidic aqueous solution includes, but is not limited to, one or more combinations of an aqueous phosphoric acid solution, an aqueous acetic acid solution, an aqueous hydrochloric acid solution, an aqueous formic acid solution, and an aqueous sulfuric acid solution.

[0023] In some embodiments, the volume fraction of the acid in the acidic aqueous solution is 1-50%, preferably 5-30%, specifically 5%, 8%, 10%, 12%, 15%, 20%, 25%, 30%.

[0024] In some embodiments, in step (3), the HPLC column is a reverse phase HPLC column.

[0025] In some embodiments, the length of the reversed phase HPLC column is 100-300 mm, preferably 200-300 mm.

[0026] In some embodiments, the inner diameter of the reversed phase HPLC column is 1-10 nm, preferably 3-7 nm.

[0027] In some embodiments, the particle size of the filler of the reversed phase HPLC column is 1-10 μm, preferably 1-7 μm, more preferably 2-6 μm, specifically 2 μm, 3 μm, 4 μm, 5 μm, 6 μm.

[0028] In some embodiments, the flow rate in the HPLC analysis in step (3) is 0.1-10 mL / min, preferably 0.1-5 mL / min, and more preferably 0.1-1.1 mL / min.

[0029] In some embodiments, the detection wavelength in the high performance liquid chromatography analysis in step (3) is 200-700 nm, preferably 280-600 nm, and more preferably 280-520 nm.

[0030] In some embodiments, the injection volume in the HPLC analysis in step (3) is 1-20 μL, preferably 1-10 μL.

[0031] In some embodiments, the mobile phases A and B of the HPLC in step (3) are acidified organic aqueous solutions.

[0032] In some embodiments, in the acidified organic aqueous solution, the organic solvent includes, but is not limited to, one or a combination of methanol, ethanol, acetonitrile, tetrahydrofuran, diethyl ether, dichloromethane and isopropanol.

[0033] In some embodiments, in the acidified organic aqueous solution, the acid includes, but is not limited to, one or a combination of phosphoric acid, formic acid, acetic acid, and trifluoroacetic acid.

[0034] In some embodiments, the volume fraction of the acid in the acidified organic aqueous solution is ≥ 1%.

[0035] In some embodiments, the volume fraction of the acid in the acidified organic aqueous solution is 1-50%, preferably 1-25%, specifically 1%, 5%, 10%, 15%, 20%, 25%.

[0036] In some embodiments, the volume fraction of the organic solvent in the acidified organic aqueous solution is ≥5%.

[0037] In some embodiments, in the acidified organic aqueous solution, the volume fraction of the organic solvent in mobile phase A is 5-90%, preferably 15-90%, further preferably 30-90%, specifically 30%, 40%, 50%, 60%, 70%, 80%, 90%.

[0038] In some embodiments, in the acidified organic aqueous solution, the volume fraction of the organic solvent in the mobile phase B is 5-90%, preferably 5-70%, further preferably 5-50%, specifically 5%, 10%, 13%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%.

[0039] In some embodiments, the elution condition of the high performance liquid chromatography in step (3) is gradient elution, and the elution gradient is: 0-8 min, the volume ratio of mobile phase A to mobile phase B is 0:100; 8-25 min, the volume ratio of mobile phase A to mobile phase B is (25-35):(65-75); 25-30 min, the volume ratio of mobile phase A to mobile phase B is (31-41):(59-69); 30-35 min, the volume ratio of mobile phase A to mobile phase B is 100:0; 35-40 min, the volume ratio of mobile phase A to mobile phase B is 100:0; 40-41 min, the volume ratio of mobile phase A to mobile phase B is 0:100; 41-50 min, the volume ratio of mobile phase A to mobile phase B is 0:100; further preferably, as shown in Table 1, Table 2 or Table 3.

[0040] Table 1

[0041] Running time / min A(％) B(％) 0 0 100 8 0 100 25 30 70 30 36 64 35 100 0 40 100 0 41 0 100 50 0 100

[0042] Table 2

[0043] Running time / min A(％) B(％) 0 0 100 8 0 100 25 25 75 30 31 69 35 100 0 40 100 0 41 0 100 50 0 100

[0044] Table 3

[0045] Running time / min A(％) B(％) 0 0 100 8 0 100 25 35 65 30 36 64 35 100 0 40 100 0 41 0 100 50 0 100

[0046] Beneficial effects: The present invention provides a method for detecting anthocyanins in maqui berry, which can effectively separate seven anthocyanin components in maqui berry. On the one hand, an acidic aqueous solution is used to make the volume constant in the pretreatment, and the pH value is adjusted to a suitable range so that the anthocyanins exist in a stable form; on the other hand, the polarity of the isomer cyanidin-3-sambubioside-5-glucoside is greater than that of cyanidin diglucoside, and the parameter settings of the chromatographic column and the elution gradient are optimized to further enhance the separation effect of the anthocyanin isomers. The detection method has good stability and high precision.

[0047] Terminology

[0048] Berry extract is defined as one or more of blueberry extract, strawberry extract, blackberry extract, cranberry extract, raspberry extract, maqui berry extract, black currant extract, raspberry extract, bilberry extract, mulberry extract, aronia extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 This is the HPLC graph of Maqui berry sample testing. DETAILED DESCRIPTION

[0050] In order to facilitate the understanding of those skilled in the art, the present invention is further described below in conjunction with embodiments, and the contents mentioned in the implementation modes are not intended to limit the present invention.

[0051] Example 1

[0052] 1. Sample processing:

[0053] Weigh 125 mg of sample (accurate to 0.1 mg) into a 100 mL volumetric flask, add 25 mL of solvent (methanol: hydrochloric acid = 49:1) and ultrasonically dissolve for 10 min. After cooling, dilute to the mark with diluent (10% phosphoric acid water (85% phosphoric acid: water = 9:1)), shake well, filter through a 0.45 um filter membrane, dilute five times and inject (use 10% phosphoric acid aqueous solution to dilute or adjust the injection volume, berry extract does not need to be diluted and injected directly) for liquid chromatography determination.

[0054] 2. Prepare anthocyanin standard stock solution and single-point calibration working solution:

[0055] Stock solution: Weigh 5.00 mg of delphinidin-3-sambubioside-5-glucoside or 4.26 mg of delphinidin-3,5-diglucoside or 4.85 mg of delphinidin-3-glucoside, dissolve in methanol to 25 mL, and prepare the stock solutions of the three.

[0056] Single-point calibration working solution: respectively pipette 100 μL of delphinidin 3-sambubioside-5-glucoside stock solution, 100 μL of delphinidin-3,5-diglucoside stock solution, and 100 μL of delphinidin-3-glucoside stock solution and use 10% phosphoric acid water to prepare 1 mL series of working solutions.

[0057] 3. The solution to be tested is subjected to high performance liquid chromatography analysis under the following chromatographic conditions:

[0058] (1) Chromatographic column: Poroshell 120SB-C18 4.6*250mm, particle size 4μm;

[0059] (2) Mobile phase: A: water + methanol + formic acid = 30 + 60 + 10, mobile phase B: water + methanol + formic acid

[0060] =77+13+10 gradient elution.

[0061] (3) Flow rate: 0.6 mL / min.

[0062] (4) Detection wavelength: 520nm.

[0063] (5) Injection volume: 5 μL.

[0064] (6) Gradient elution conditions are shown in Table 4:

[0065] Table 4

[0066]

[0067]

[0068] The measured spectrum is as follows Figure 1 As shown in the figure, this detection method can effectively separate seven anthocyanin components in maqui berry, including a pair of isomers. Figure 1 The anthocyanin components A1-A7 are shown in Table 5.

[0069] Table 5

[0070]

[0071]

[0072] Example 2

[0073] The stability data of the test results are shown in Table 6. The RSDs of the six tests all meet the requirements of Appendix F.3 of GB / T 27404, indicating that the test method has good stability and high precision.

[0074] Table 6

[0075]

[0076] Example 3

[0077] 1. Sample processing:

[0078] Weigh 100 mg of sample (accurate to 0.1 mg) into a 100 mL volumetric flask, add 25 mL of solvent (ethanol: sulfuric acid = 49.5:0.5) and ultrasonically dissolve for 15 minutes. After cooling, dilute to the mark with diluent (5% hydrochloric acid water (hydrochloric acid: water = 19:1)), shake well, filter through a 0.45 um filter membrane, dilute five times and inject (use 10% phosphoric acid aqueous solution to dilute or adjust the injection volume, berry extract does not need to be diluted and injected directly) for liquid chromatography determination.

[0079] 2. Prepare anthocyanin standard stock solution and single-point calibration working solution:

[0080] Stock solution: Weigh 5.00 mg of delphinidin-3-sambubioside-5-glucoside or 4.26 mg of delphinidin-3,5-diglucoside or 4.85 mg of delphinidin-3-glucoside, dissolve in methanol to 25 mL, and prepare the stock solutions of the three.

[0081] Single-point calibration working solution: respectively pipette 100 μL of delphinidin 3-sambubioside-5-glucoside stock solution, 100 μL of delphinidin-3,5-diglucoside stock solution, and 100 μL of delphinidin-3-glucoside stock solution and use 10% phosphoric acid water to prepare 1 mL series of working solutions.

[0082] 3. The solution to be tested is subjected to high performance liquid chromatography analysis under the following chromatographic conditions:

[0083] (1) Chromatographic column: Poroshell 120SB-C18 4.6*150mm, particle size 2.7μm;

[0084] (2) Mobile phase: A: water + ethanol + acetic acid = 50 + 45 + 5, mobile phase B: water + ethanol + acetic acid

[0085] =80+15+5 gradient elution.

[0086] (3) Flow rate: 1.1 mL / min.

[0087] (4) Detection wavelength: 520nm.

[0088] (5) Injection volume: 2 μL.

[0089] (6) Gradient elution conditions are shown in Table 2:

[0090] Table 2

[0091] Running time / min A(％) B(％) 0 0 100 8 0 100 25 25 75 30 31 69 35 100 0 40 100 0 41 0 100 50 0 100

[0092] Example 4

[0093] 1. Sample processing:

[0094] Weigh 150 mg of sample (accurate to 0.1 mg) into a 100 mL volumetric flask, add 25 mL of solvent (tetrahydrofuran: phosphoric acid = 40:10) and ultrasonically dissolve for 5 min (temperature not exceeding 30°C), cool and dilute to scale with diluent (15% acetic acid water (acetic acid: water = 17:3)), shake well, filter through a 0.45 um filter membrane, dilute five times and inject (use 10% phosphoric acid aqueous solution to dilute or adjust the injection volume, berry extract does not need to be diluted and injected directly) for liquid chromatography determination.

[0095] 4. Prepare anthocyanin standard stock solution and single-point calibration working solution:

[0096] Stock solution: Weigh 5.00 mg of delphinidin-3-sambubioside-5-glucoside or 4.26 mg of delphinidin-3,5-diglucoside or 4.85 mg of delphinidin-3-glucoside, dissolve in methanol to 25 mL, and prepare the stock solutions of the three.

[0097] Single-point calibration working solution: respectively pipette 100 μL of delphinidin 3-sambubioside-5-glucoside stock solution, 100 μL of delphinidin-3,5-diglucoside stock solution, and 100 μL of delphinidin-3-glucoside stock solution and use 10% phosphoric acid water to prepare 1 mL series of working solutions.

[0098] 5. The solution to be tested is subjected to high performance liquid chromatography analysis under the following chromatographic conditions:

[0099] (1) Chromatographic column: Poroshell 120SB-C18 4.6*100 mm, particle size 2.7 μm;

[0100] (2) Mobile phase: A: water + isopropanol + sulfuric acid = 10 + 75 + 15, mobile phase B: water + isopropanol +

[0101] Sulfuric acid = 80+10+10 gradient elution.

[0102] (3) Flow rate: 0.1 mL / min.

[0103] (4) Detection wavelength: 520nm.

[0104] (5) Injection volume: 8 μL.

[0105] (6) Gradient elution conditions are shown in Table 3:

[0106] Table 3

[0107] Running time / min A(％) B(％) 0 0 100 8 0 100 25 35 65 30 36 64 35 100 0 40 100 0 41 0 100 50 0 100

[0108] It should be understood that the detailed description of the technical solutions of the present invention by means of the preferred embodiments is illustrative rather than restrictive. A person skilled in the art may modify the technical solutions described in the embodiments, or replace some of the technical features by equivalents, based on reading the specification of the present invention; and these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A high performance liquid chromatography detection method for anthocyanins, characterized in that: The following steps are involved: (1) ultrasonically dissolving the sample to be tested, fixing the volume, and filtering to obtain a solution to be tested; (2) preparing anthocyanin standard stock solution and single-point calibration working solution; (3) The test solution is subjected to high performance liquid chromatography analysis to separate various anthocyanin components.

2. The method according to claim 1, characterized in that The anthocyanin source is one or more of blueberry, strawberry, blackberry, cranberry, raspberry, maqui berry, black currant, raspberry, lingonberry, mulberry and aronia berry.

3. The method according to claim 1, characterized in that The solvent used for ultrasound in step (1) is an acidified organic solvent.

4. The method according to claim 3, characterized in that The organic solvent is one or more of methanol, ethanol, acetonitrile, tetrahydrofuran, ether, dichloromethane and isopropanol.

5. The method according to claim 3, characterized in that: The acid is one or more of formic acid, acetic acid, hydrochloric acid, sulfuric acid, phosphoric acid, and trifluoroacetic acid.

6. The method according to claim 1, characterized in that The solution used for volume determination in step (1) is an acidic aqueous solution.

7. The method according to claim 6, characterized in that The acidic aqueous solution is one or more of a phosphoric acid aqueous solution, an acetic acid aqueous solution, a hydrochloric acid aqueous solution, a formic acid aqueous solution, and a sulfuric acid aqueous solution.

8. The method according to claim 1, characterized in that In the step (3), the chromatographic column of the high performance liquid chromatography is a reverse phase high performance liquid chromatography column.

9. The method according to claim 8, characterized in that The particle size of the chromatographic column filler is 1-10 μm.

10. The method according to claim 1, characterized in that In step (3), the mobile phases A and B of the HPLC are acidified organic aqueous solutions.

11. The method according to claim 10, characterized in that In the step (3) of acidifying the organic aqueous solution, the organic solvent is one or more of methanol, ethanol, acetonitrile, tetrahydrofuran, ether, dichloromethane and isopropanol.

12. The method according to claim 10, characterized in that In the step (3) of acidifying the organic aqueous solution, the acid is one or more of phosphoric acid, formic acid, acetic acid and trifluoroacetic acid.

13. The method according to claim 10, characterized in that In the acidified organic aqueous solution, the volume fraction of the acid is ≥ 1%.

14. The method according to claim 10, characterized in that In the acidified organic aqueous solution, the volume fraction of the organic solvent is ≥5%.

15. The method according to claim 10, characterized in that In step (3), the elution condition of the high performance liquid chromatography is gradient elution, and the elution gradient is: 0-8min, the volume ratio of mobile phase A to mobile phase B was 0:100; 8-25min, the volume ratio of mobile phase A to mobile phase B is (25-35):(65-75); 25-30min, the volume ratio of mobile phase A to mobile phase B is (31-41):(59-69); 30-35min, the volume ratio of mobile phase A to mobile phase B was 100:0; 35-40min, the volume ratio of mobile phase A to mobile phase B was 100:0; 40-41min, the volume ratio of mobile phase A to mobile phase B was 0:100; 41-50min, the volume ratio of mobile phase A to mobile phase B was 0:100.

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