High performance liquid chromatography detection method for simultaneously determining 14 polyphenols

Abstract

The invention relates to the technical field of detection, and particularly provides a high performance liquid chromatography detection method for simultaneously detecting 14 polyphenols. The 14 kinds of polyphenols are linden glycoside, gallic acid, isoquercitrin, 1, 2, 3, 4, 6-pentagalloylglucose, quercetin, ellagic acid, hyperoside, protocatechuic acid, procyanidine B2, rutin, luteolin, apigenin, kaempferol and procyanidine B1. According to the method, the chromatographic conditions of the high performance liquid chromatography are accurately limited, different types of polyphenol substances are detected in different retention times, and simultaneous determination of 14 types of polyphenols is realized. The data of the embodiment shows that the 14 polyphenols can be simultaneously determined, the operation is simple and fast, and a special detector is not needed.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a high performance liquid chromatography detection method for simultaneously determining 14 polyphenols. Background Art

[0002] Polyphenols are a general term for substances with a benzene ring and a chemical structure combined with multiple hydroxyl groups, including flavonoids, tannins, phenolic acids and anthocyanins. Polyphenols have the effects of antioxidant, prevention of cardiovascular and cerebrovascular diseases, anti-cancer, beauty, anti-aging, anti-fatigue, antibacterial, and enhancing immune function.

[0003] The main analytical methods for polyphenols include the Folin phenol method, high performance liquid chromatography, gas chromatography, liquid chromatography-mass spectrometry analysis, etc. Among them, high performance liquid chromatography is a commonly used method for determining the content of polyphenol compounds. High performance liquid chromatography is simple to operate, highly accurate and reproducible. However, due to the differences in the properties of various components such as flavonoids and phenolic acids, multiple methods are often required for detection, which increases working time and cost. Summary of the invention

[0004] The purpose of the present invention is to provide a high performance liquid chromatography detection method for simultaneously determining 14 polyphenols. The detection method provided by the present invention has the characteristics of good separation degree and simple and fast operation.

[0005] The technical solution of the present invention is achieved as follows: The present invention provides a high performance liquid chromatography detection method for simultaneously determining 14 polyphenols, wherein the 14 polyphenols are tiliaoside, gallic acid, isoquercetin, 1,2,3,4,6-pentagalloyl glucose, quercetin, ellagic acid, hyperoside, protocatechuic acid, proanthocyanidin B2, rutin, luteolin, apigenin, kaempferol and proanthocyanidin B1, and the detection method is a high performance liquid chromatography analysis method, and the chromatographic conditions of the high performance liquid chromatography analysis are:

[0006] Analytical column: C18 column;

[0007] Mobile phase: Aqueous phase A: 0.2%-0.4% phosphoric acid aqueous solution; Organic phase B: acetonitrile;

[0008] Flow rate: 0.5 mL / min;

[0009] Elution mode: gradient elution, 0 min, A:B = 97:3, 12 min, A:B = 97:3, 13 min, A:B = 90:10, 25 min, A:B = 90:10, 30 min, A:B = 85:15, 50 min, A:B = 80:20, 60 min, A:B = 75:25, 61 min, A:B = 70:30, 100 min, A:B = 70:30;

[0010] Detection wavelength: 250-270nm;

[0011] Detection temperature: 40℃;

[0012] Injection volume: 10 μL;

[0013] Detector: VWD detector.

[0014] In some embodiments, the C18 chromatographic column is Cosmosil 5C18-MS-II or an equivalent chromatographic column, and the specifications of the C18 chromatographic column are 4.6×250 mm, 2.5 μm.

[0015] In some embodiments, before the solution to be tested is subjected to high performance liquid chromatography analysis, the solution to be tested is filtered using a 0.22 μm organic filter membrane to obtain an injection solution.

[0016] In some embodiments, aqueous phase A is preferably a 0.2% phosphoric acid aqueous solution.

[0017] In some embodiments, the detection wavelength is preferably 270 nm.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a high performance liquid chromatography detection method for simultaneously determining 14 types of polyphenols. By accurately defining the chromatographic conditions of the high performance liquid chromatography, different types of polyphenol substances are detected at different retention times, and the simultaneous determination of 14 types of polyphenols can be achieved. The data of the embodiment show that the present invention achieves the simultaneous determination of 14 types of polyphenols, and the operation is simple and fast, and no special detector is required. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the HPLC detection spectrum of the mixed standard solution of Example 1 of the present invention;

[0022] Figure 2 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 1 of the present invention;

[0023] Figure 3 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 2 of the present invention;

[0024] Figure 4 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 3 of the present invention;

[0025] Figure 5 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 4 of the present invention;

[0026] Figure 6 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 5 of the present invention;

[0027] Figure 7 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 6 of the present invention;

[0028] Figure 8 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 7 of the present invention;

[0029] Fig. 9 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 8 of the present invention;

[0030] Fig.10 This is the HPLC detection spectrum of the mixed standard solution of Comparative Example 9 of the present invention. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0033] Unless otherwise specified, the methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents and instruments used are all conventional materials, reagents and instruments in the art, and can be obtained by those skilled in the art through commercial channels.

[0034] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including the range "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range. In the present specification and claims, range definitions can be combined and / or interchanged, and if not otherwise stated, these ranges include all subranges contained therein.

[0035] Example 1

[0036] This example proposes a high performance liquid chromatography detection method for simultaneously determining 14 polyphenols, wherein the 14 polyphenols are tiliaoside, gallic acid, isoquercetin, 1,2,3,4,6-pentagalloyl glucose, quercetin, ellagic acid, hyperoside, protocatechuic acid, proanthocyanidin B2, rutin, luteolin, apigenin, kaempferol and proanthocyanidin B1, and specifically comprises the following steps:

[0037] (1) Weigh HPLC-pure tiliaoside, gallic acid, isoquercetin, 1,2,3,4,6-pentagalloylglucose, quercetin, ellagic acid, hyperoside, protocatechuic acid, proanthocyanidin B2, rutin, luteolin, apigenin, kaempferol and proanthocyanidin B1, respectively, and prepare 1 mg / mL standard solutions of individual samples with methanol or dimethyl sulfoxide (except apigenin, the solvent used for preparing the test solution is methanol solution; the solvent used for preparing the apigenin solution is dimethyl sulfoxide). Take appropriate volumes of the standard solutions of the individual samples and mix them to obtain a mixed standard solution, which is filtered with a 0.22 μm organic filter membrane and analyzed by high performance liquid chromatography.

[0038] (2) The mixed standard solution is subjected to high performance liquid chromatography analysis, and the chromatographic conditions of the high performance liquid chromatography analysis are:

[0039] Analytical column: Cosmosil 5C18-MS-II, 4.6×250 mm, 2.5 μm;

[0040] Mobile phase: Aqueous phase A: 0.2% phosphoric acid aqueous solution; Organic phase B: acetonitrile;

[0041] Flow rate: 0.5 mL / min;

[0042] Elution mode: gradient elution, 0 min, A:B = 97:3, 12 min, A:B = 97:3, 13 min, A:B = 90:10, 25 min, A:B = 90:10, 30 min, A:B = 85:15, 50 min, A:B = 80:20, 60 min, A:B = 75:25, 61 min, A:B = 70:30, 100 min, A:B = 70:30;

[0043] Detection wavelength: 270m;

[0044] Detection temperature: 40℃;

[0045] Injection volume: 10 μL;

[0046] Detector: VWD detector.

[0047] (3) Comparing the chromatogram of the sample to be tested with a predetermined standard spectrum to obtain the type of polyphenols in the sample to be tested.

[0048] The predetermined standard spectra are a series of spectra obtained by detecting the standard solution of each individual sample using the detection method in the above step (2).

[0049] The HPLC detection spectrum of the standard solution is as follows Figure 1 As shown by Figure 1 It can be seen that the detection method provided by the present invention realizes the simultaneous determination of 14 polyphenols. Figure 1 Among them, 1 is gallic acid, 2 is protocatechuic acid, 3 is proanthocyanidin B1, 4 is proanthocyanidin B2, 5 is ellagic acid, 6 is rutin, 7 is hyperoside, 8 is isoquercetin, 9 is 1,2,3,4,6-pentagalloylglucose, 10 is tiliacoside, 11 is quercetin, 12 is luteolin, 13 is apigenin, and 14 is kaempferol.

[0050] Comparative Example 1

[0051] The other steps of this comparative example are the same as those of Example 1, except that the detection wavelength is changed to 350 nm.

[0052] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 2 Compared with Example 1, the detection wavelength was changed, gallic acid, protocatechuic acid, proanthocyanidin B1, and proanthocyanidin B2 were not detected, and the method of Comparative Example 1 could not simultaneously determine 14 polyphenols.

[0053] Comparative Example 2

[0054] The other steps of this comparative example are the same as those of Example 1, except that the elution method is changed to: gradient elution, 0 min, A:B=97:3, 12 min, A:B=97:3, 13 min, A:B=90:10, 25 min, A:B=90:10, 30 min, A:B=85:15, 50 min, A:B=80:20, 100 min, A:B=65:35.

[0055] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 3 Compared with Example 1, the elution mode was changed, and the peaks of ellagic acid and rutin overlapped, and the peaks of tiliaoside and quercetin overlapped.

[0056] Comparative Example 3

[0057] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0058] Flow rate: 0.9 mL / min;

[0059] Elution mode: gradient elution, 0 min, A:B = 97:3, 10 min, A:B = 92:8, 2 min, A:B = 88:12, 50 min, A:B = 80:20, 52 min, A:B = 97:3, 60 min, A:B = 97:3;

[0060] Detection wavelength: 255m;

[0061] Detection temperature: 30℃;

[0062] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 4 Compared with Example 1, the flow rate, elution mode, detection wavelength and detection temperature were changed, and proanthocyanidin B1, proanthocyanidin B2, linalool, quercetin, luteolin, celery and kaempferol were not detected.

[0063] Comparative Example 4

[0064] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0065] Flow rate: 1.0 mL / min;

[0066] Elution mode: gradient elution, 0 min, A:B=97:3, 10 min, A:B=97:3, 11 min, A:B=90:10, 31 min, A:B=90:10, 32 min, A:B=84:16, 42 min, A:B=84:16, 43 min, A:B=81:19, 63 min, A:B=81:19, 64 min, A:B=70:30, 80 min, A:B=70:30, 81 min, A:B=97:3;

[0067] Detection temperature: 35℃;

[0068] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 5 Compared with Example 1, the flow rate, elution mode and detection temperature were changed, 1,2,3,4,6-pentagalloylglucose was not detected, and one of basilicin, quercetin and luteolin was not detected.

[0069] Comparative Example 5

[0070] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0071] Mobile phase: Aqueous phase A: 0.1% formic acid + 2.5% acetonitrile in water; Organic phase B: 0.1% formic acid in acetonitrile;

[0072] Elution mode: gradient elution, 0 min, A:B = 100:0, 4 min, A:B = 90:10, 30 min, A:B = 85:15, 100 min, A:B = 65:35;

[0073] Detection wavelength: 350m;

[0074] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 6 Compared with Example 1, the mobile phase, elution mode and detection wavelength were changed, and gallic acid, protocatechuic acid, proanthocyanidin B1 and proanthocyanidin B2 were not detected.

[0075] Comparative Example 6

[0076] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0077] Analytical column: Eclipse XDB-phenyl, 4.6×250 mm, 2.5 μm;

[0078] Flow rate: 0.9 mL / min;

[0079] Elution mode: gradient elution, 0 min, A:B = 97:3, 10 min, A:B = 92:8, 2 min, A:B = 88:12, 50 min, A:B = 80:20, 52 min, A:B = 97:3, 60 min, A:B = 97:3;

[0080] Detection wavelength: 255m;

[0081] Detection temperature: 30℃;

[0082] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 7 Compared with Example 1, the chromatographic column, flow rate, elution mode, detection wavelength and detection temperature were changed. Compared with Comparative Example 3, the chromatographic column was changed, that is, the Hubei Provincial Traditional Chinese Medicine Formula Granule Standard-Rose Formula Granule Method was used. The responses of proanthocyanidin B1 and proanthocyanidin B2 were relatively low, and 1,2,3,4,6-pentagalloylglucose was not detected.

[0083] Comparative Example 7

[0084] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0085] Analytical column: Agilent Zorbax SB-C18, 4.6 × 250 mm, 2.5 μm;

[0086] Mobile phase: aqueous phase A: 0.2% glacial acetic acid solution; organic phase B: acetonitrile;

[0087] Flow rate: 0.8 mL / min;

[0088] Elution mode: isocratic elution, A:B=90:10;

[0089] Detection wavelength: 310m;

[0090] Detection temperature: 25℃;

[0091] The HPLC detection spectrum of the mixed standard solution is as follows: Figure 8 Compared with Example 1, only two substances were detected by changing the chromatographic column, mobile phase, flow rate, elution method, detection wavelength and detection temperature.

[0092] Comparative Example 8

[0093] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0094] Analytical column: Agilent Zorbax SB-C18, 4.6 × 250 mm, 2.5 μm;

[0095] Mobile phase: aqueous phase A: 0.4% phosphoric acid solution; organic phase B: methanol;

[0096] Flow rate: 1.0 mL / min;

[0097] Elution mode: isocratic elution, A:B=55:45;

[0098] Detection wavelength: 360m;

[0099] Detection temperature: 30℃;

[0100] The HPLC detection spectrum of the mixed standard solution is as follows: Fig. 9 Compared with Example 1, by changing the chromatographic column, mobile phase, flow rate, elution method, detection wavelength and detection temperature, only 7 substances were detected, and the peak width was wider and the peak was not sharp.

[0101] Comparative Example 9

[0102] The other steps of this comparative example are the same as those of Example 1, except that the following conditions are replaced:

[0103] Analytical column: Agilent Zorbax SB-C18, 4.6 × 250 mm, 2.5 μm;

[0104] Mobile phase: aqueous phase A: 0.5% glacial acetic acid solution; organic phase B: methanol;

[0105] Flow rate: 1.0 mL / min;

[0106] Elution mode: gradient elution, 0 min, A:B = 80:20, 10 min, A:B = 50:50, 18 min, A:B = 20:80, 25 min, A:B = 20:80, 28 min, A:B = 80:20;

[0107] Detection wavelength: 358m;

[0108] The HPLC detection spectrum of the mixed standard solution is as follows: Fig.10 Compared with Example 1, only 7 substances were detected by changing the chromatographic column, mobile phase, flow rate, elution method and detection wavelength, and the separation degree was poor.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A high performance liquid chromatography detection method for simultaneously determining 14 polyphenols, wherein the 14 polyphenols are: tilia glycosides, gallic acid, isoquercetin, 1,2,3,4,6-pentagalloyl glucose, quercetin, ellagic acid, hyperoside, protocatechuic acid, proanthocyanidin B2, rutin, luteolin, apigenin, kaempferol and proanthocyanidin B1, characterized in that: The detection method is a high performance liquid chromatography analysis method, and the chromatographic conditions of the high performance liquid chromatography analysis are: Analytical column: C18 column; Mobile phase: Aqueous phase A: 0.2%-0.4% phosphoric acid aqueous solution; Organic phase B: acetonitrile; Flow rate: 0.5 mL / min; Elution mode: gradient elution, 0 min, A:B = 97:3, 12 min, A:B = 97:3, 13 min, A:B = 90:10, 25 min, A:B = 90:10, 30 min, A:B = 85:15, 50 min, A:B = 80:20, 60 min, A:B = 75:25, 61 min, A:B = 70:30, 100 min, A:B = 70:30; Detection wavelength: 250-270nm; Detection temperature: 40℃; Injection volume: 10 μL; Detector: VWD detector.

2. The high performance liquid chromatography detection method for simultaneously determining 14 polyphenols as claimed in claim 1, characterized in that: The C18 chromatographic column is Cosmosil 5C18-MS-II or an equivalent chromatographic column, and the specification of the C18 chromatographic column is 4.6×250 mm, 2.5 μm.

3. The high performance liquid chromatography detection method for simultaneously determining 14 polyphenols as claimed in claim 1, characterized in that: The detection wavelength is 270 nm.

4. The high performance liquid chromatography detection method for simultaneously determining 14 polyphenols as claimed in claim 1, characterized in that: The aqueous phase A is a 0.2% phosphoric acid aqueous solution.

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