Liquid chromatography method for determining contents of nine flavonoid compounds in natural perfume raw materials

Abstract

The invention discloses a liquid chromatography method for determining the content of nine flavonoid compounds in a natural perfume raw material. The method is based on an ultra-high performance liquid chromatography technology, chromatographic conditions are optimized, two HSS T3 chromatographic columns are connected in series, and efficient separation of various flavonoid compounds is successfully achieved. The method is particularly suitable for analysis of complex samples, can be used for simultaneously determining the content of various flavonoid compounds in single detection, and has relatively high sensitivity and separation degree. In addition, the method is also combined with multi-wavelength detection, the flavonoid compounds in the sample are quantitatively analyzed through an external standard method, and efficient separation and accurate determination of the flavonoid compounds can be realized. Compared with a traditional LC / MS method, the method has the advantages that analysis efficiency and accuracy are improved, sample treatment steps and analysis cost are reduced, and the method has wide industrial application potential.

Description

Technical Field

[0001] The invention belongs to the technical field of analytical chemistry, and in particular relates to a liquid chromatography method for determining the contents of nine flavonoid compounds in natural fragrance raw materials. Background Art

[0002] With the continuous deepening of scientific research, the various biological activities of flavonoids (such as antioxidant, anti-inflammatory, antiviral, anti-tumor, etc.) have gradually attracted widespread attention. As a class of polyphenolic compounds widely present in nature, flavonoids are of great significance in the quantitative analysis of food industry, pharmaceutical development and cosmetics. However, due to the complex chemical structure of flavonoids and the large differences in their content in different natural samples, how to accurately and effectively detect these components has become an important technical challenge. The mobile phase selection of existing HPLC-based determination methods is mainly phosphate, and dilute phosphoric acid as a mobile phase will cause irreversible damage to the chromatographic column and piping system. Although LC / MS performs well in the quantitative analysis of various flavonoids, its high analytical cost and interference caused by matrix effects make it challenging to simultaneously detect multiple flavonoids in complex samples (such as plant extracts). The structural similarity of flavonoids also further increases the difficulty of separation, which may lead to response suppression or errors in mass spectrometry detection. Therefore, accurate determination of its content is crucial for the quality control of these products. Summary of the invention

[0003] In order to solve these problems, the present invention provides a liquid chromatography method suitable for complex matrix analysis, which uses ultra-high performance liquid chromatography (UPLC / PDA) technology to determine the contents of nine flavonoid compounds in natural fragrance raw materials. The method can be widely used in food safety testing, drug development, natural fragrance quality control and other fields, and can effectively solve the deficiencies of existing analytical methods in sensitivity and separation, and provides a method for efficiently and accurately determining the contents of nine flavonoid compounds in natural fragrance raw materials.

[0004] The method of the present invention can not only effectively improve the separation degree of various flavonoid compounds by optimizing separation conditions and detection parameters, but also reduce the influence of matrix interference on the test results, thereby ensuring the accuracy and reproducibility of the measurement results. The present invention utilizes ultra-high performance liquid chromatography technology in combination with two HSS T3 chromatographic columns in series, and successfully achieves the simultaneous and efficient separation of various flavonoid compounds. Specifically, the present invention significantly improves the separation effect by accurately controlling chromatographic conditions, such as the combination of mobile phases, column temperature, flow rate and detection wavelength, while ensuring the high sensitivity of detection. In addition, the present invention adopts a multi-wavelength detection method (228nm, 254nm), which can cover the absorption characteristics of different flavonoid compounds, thereby achieving accurate quantification of each component in the sample.

[0005] The first object of the present invention is to provide a liquid chromatography method for determining the contents of nine flavonoid compounds in natural fragrant raw materials, comprising the following steps:

[0006] S1. Weigh the natural incense raw material sample to be tested, perform ultrasonic extraction with the extract, and the extract is centrifuged and filtered to obtain the sample solution to be tested;

[0007] S2. The sample solution to be tested is subjected to ultra-high performance liquid chromatography detection; the conditions for the ultra-high performance liquid chromatography detection are:

[0008] Chromatographic column: two HSS T3 chromatographic columns are used in series, the HSS T3 is a HSS T3 2.1×100mm, 1.7μm chromatographic column;

[0009] Mobile phase: Phase A is methanol: formic acid-ammonium acetate solution with a volume ratio of 5:95, wherein the formic acid-ammonium acetate solution is an aqueous solution containing 2mmol / L formic acid and 10mmol / L ammonium acetate; Phase B is 0.1% formic acid aqueous solution: methanol with a volume ratio of 1:1; Phase C is 0.1% formic acid acetonitrile solution; Phase D is 0.1% formic acid methanol solution;

[0010] Gradient elution program: 0-9 min, 100% phase A; 9-12 min, 100%-70% phase A, 0-16% phase B, 0-14% phase C; 12-18 min, 70%-50% phase A, 16%-36% phase B, 14% phase C; 18-27 min, 50%-42% phase A, 36%-44% phase B, 14% phase C; 27-33 min, 42%-4% phase A, 44%-82% phase B, 14% phase C; 33-45 min, 4% phase A, 82% phase B, 14% Phase C; 45-48 min, 4%-0 phase A, 82%-86% phase B, 14% phase C; 48-51 min, 0-45% phase A, 86%-0 phase B, 14%-0 phase C, 0-55% phase D; 51-57 min, 45%-10% phase A, 55%-90% phase D; 57-66 min, 10% phase A, 90% phase D; 66-72 min, 10%-100% phase A, 90%-0 phase D; balance 15 min; flow rate: 0-27 min, 0.15 mL min -1 ; 27~72min, 0.2mL·min -1 ; 72~87min, 0.15mL·min -1 ;

[0011] Column temperature: 30°C;

[0012] Detection wavelength: 228nm, 254nm;

[0013] Injection volume: 10 μL;

[0014] S3. Use the external standard method to conduct qualitative and quantitative analysis on the nine flavonoid compounds in the samples.

[0015] Preferably, the nine flavonoid compounds are epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol, and isorhamnetin.

[0016] Preferably, the extract is an aqueous solution of ethanol with a volume fraction of 70%.

[0017] Preferably, the centrifugation is performed at 8000 r / min for 10 min.

[0018] Preferably, the filtration is performed using a 0.22 μm filter membrane.

[0019] Preferably, the qualitative analysis in step S3 is: comparing the ultra-high performance liquid chromatogram obtained in step S2 with the ultra-high performance liquid chromatogram obtained from nine flavonoid standard solutions under the same detection conditions to obtain a qualitative analysis result; the nine flavonoid standard solutions are solutions prepared by dissolving nine flavonoid standard products in an extract.

[0020] Preferably, the quantitative analysis in step S3 comprises the following steps:

[0021] S31. Accurately weigh nine flavonoid standard products, dissolve them with extracts by ultrasonication and make up to volume, so that the concentrations of epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol and isorhamnetin are 7.53 μg / mL, 6.82 μg / mL, 7.38 μg / mL, 82.50 μg / mL, 10.45 μg / mL, 5.76 μg / mL, 5.52 μg / mL, 18.56 μg / mL and 60.98 μg / mL, respectively, as standard mother solutions; then accurately measure 0.3 mL, 0.5 mL, 1 mL, 3 mL and 5 mL of the standard mother solution into a 10 mL volumetric flask, dilute and make up to volume with the extract to obtain nine flavonoid series standard solutions;

[0022] S32. The standard solution is tested using the conditions of step S2, and a standard curve is drawn based on the peak area measured for each standard solution and its corresponding concentration. After a linear regression equation is obtained, the concentrations of the nine flavonoid compounds in the sample solution to be tested are calculated using the linear regression equation based on the peak area of ​​the sample to be tested, and then the contents of the nine flavonoid compounds in the sample to be tested are calculated.

[0023] The second object of the present invention is to provide the application of the liquid chromatography method for determining the contents of nine flavonoid compounds in natural fragrant raw materials in the detection of flavonoid compounds.

[0024] Preferably, the flavonoid compound is epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol, and isorhamnetin.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] In the ultra-high performance liquid chromatography analysis, the samples were separated in two HSS T3 columns in sequence. Through the optimized chromatographic gradient and mobile phase combination, the contents of multiple flavonoids can be simultaneously determined in a single test, and nine flavonoids were successfully separated. During the detection process, multi-wavelength detection technology was used to further improve the accuracy of the analysis results by quantitatively analyzing the chromatographic peaks at different wavelengths. The present invention solves the problem of separation and detection of flavonoids in complex matrices by combining tandem chromatographic columns and multi-wavelength detection, and significantly improves the analytical efficiency and sensitivity of the method.

[0027] Compared with the traditional LC / MS method, the present invention improves the analysis efficiency and accuracy while reducing the sample processing steps and analysis costs, and has a wide range of industrial application potentials.

[0028] In addition, the method of the present invention is easy to operate, has strong applicability, and can be widely used in quality control and component analysis of food, medicine and cosmetics; it can not only accurately determine the content of flavonoid compounds in natural spices, but also provide reliable analytical data support for the development and research of related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is an ultra-high performance liquid chromatogram of nine flavonoid standard solutions⑤; wherein Epig represents epigallocatechin gallate, Cate represents catechin, Epic represents epicatechin, Nari represents naringin, Isoq represents isoquercitrin, Ruti represents rutin, Quer represents quercetin, Kaem represents kaempferol, and Isor represents isorhamnetin, the same below.

[0030] Figure 2 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 1 solution.

[0031] Figure 3 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 2 solution.

[0032] Figure 4 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 3 solution.

[0033] Figure 5 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 4 solution.

[0034] Figure 6 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 5 solution.

[0035] Figure 7 This is the ultra-high performance liquid chromatogram of natural fragrance raw material sample No. 6 solution.

[0036] Figure 8 It is the ultra-high performance liquid chromatogram of the nine flavonoid compound standard solution ⑤ of comparative example 1. DETAILED DESCRIPTION

[0037] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0038] The following instruments and reagents were used:

[0039] The ultra-high performance liquid chromatograph was Waters ACQUITY UPLC H-Class (Waters Company), the chromatographic column was HSST3 column (Waters Company), and the UV detector was (Waters Company).

[0040] Ethanol, methanol, ammonium acetate, and acetonitrile were of chromatographic grade (Merck), and formic acid was of chromatographic grade (Shanghai Anpu).

[0041] Epigallocatechin gallate standard (Shanghai Anpu, content: 98.1%), catechin standard (Shanghai Anpu, content: 92.7%), epicatechin standard (Shanghai Anpu, content: 99.6%), naringin standard (Shanghai Anpu, content: 92.7%), isoquercetin standard (Shanghai Anpu, content: 97.8%), rutin standard (Shanghai Anpu, content: 95.6%), quercetin standard (Shanghai Anpu, content: 91.7%), kaempferol standard (Shanghai Anpu, content: 97.2%), isorhamnetin standard (Shanghai Anpu, content: 98.1%).

[0042] The method of Example 1 of the present invention is the best method that can finally achieve a relatively ideal effect after a lot of optimization. Before this, the biggest difficulty was that "epigallocatechin gallate and catechin" and "isoquercetin and rutin" were difficult to separate.

[0043] Example 1

[0044] 1. Preparation of nine flavonoid compound series standard solutions

[0045] Nine flavonoid compound standards (epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol, isorhamnetin) were accurately weighed, and ultrasonically dissolved and fixed to volume with extraction solution (i.e., 70% ethanol aqueous solution, the same below) to make the concentrations of the standards 7.53 μg / mL, 6.82 μg / mL, 7.38 μg / mL, 82.50 μg / mL, respectively. , 10.45μg / mL, 5.76μg / mL, 5.52μg / mL, 18.56μg / mL, 60.98μg / mL as standard stock solutions; then accurately measure 0.3mL, 0.5mL, 1mL, 3mL, 5mL of the standard stock solutions into 10mL volumetric flasks and number them as ①, ②, ③, ④, ⑤ respectively, dilute with the extract and make up to volume to obtain nine flavonoid compound series standard solutions.

[0046] 2. Extraction and Purification

[0047] Accurately weigh 0.5000 g of natural fragrance raw material sample (all plant extract samples) into a 25 mL brown volumetric flask, dissolve it with the extract and ultrasonically extract it for 10 minutes, then cool it to room temperature and dilute it with the extract to a fixed volume; after centrifugation at 8000 r / min for 10 minutes, take the supernatant to obtain the natural fragrance raw material sample solution.

[0048] 3. Determination

[0049] 3.1 UPLC conditions

[0050] 3.1.1 Chromatographic columns: HSS T3 (2.1×100mm, 1.7μm) and HSS T3 (2.1×100mm, 1.7μm) were connected in series. 3.1.2 Mobile phase: Phase A was "methanol: formic acid-ammonium acetate solution (an aqueous solution containing 2mmol / L formic acid and 10mmol / L ammonium acetate) = 5:95 (v / v)"; Phase B was "0.1% formic acid aqueous solution: methanol = 1:1 (v / v)"; Phase C was "0.1% formic acid acetonitrile solution by volume"; Phase D was "0.1% formic acid methanol solution by volume". The gradient elution conditions are detailed in Table 1.

[0051] 3.1.3 Column temperature: 30℃.

[0052] 3.1.4 Detection wavelength: 228nm, 254nm.

[0053] 3.1.5 Injection volume: 10 μL.

[0054] Table 1 Chromatographic gradient elution conditions

[0055]

[0056]

[0057] 3.2 Chromatographic determination

[0058] 3.2.1 Determination method

[0059] Blank test: Use the extract as a blank sample and filter it through a 0.22 μm filter membrane. The filtrate is then measured according to the UPLC conditions in 3.1.

[0060] The nine flavonoid compound series standard solutions and the natural aroma raw material sample solutions were filtered through a 0.22 μm filter membrane, and the filtrate was tested according to the ultra-high performance liquid chromatography conditions of 3.1, and qualitative and quantitative analysis was performed based on retention time and peak area.

[0061] 3.2.2 Sample determination

[0062] The results of the ultra-high performance liquid chromatography of the nine flavonoid compound standard solutions ⑤ are shown in Figure 1 .

[0063] A total of 6 batches of natural incense raw material samples (6 batches numbered 1-6) were tested, and the results of their ultra-high performance liquid chromatography are shown in Figure 2-7 .

[0064] A standard curve was drawn based on the peak area measured for each standard solution and its corresponding concentration, and a linear regression equation was obtained (see Table 2).

[0065] Table 2 Linear regression equation table

[0066]

[0067] 4. Calculation of results

[0068] The standard curve was drawn based on the peak area and its corresponding concentration measured for each standard solution. After obtaining the linear regression equation in Table 2, the concentration C (μg / mL) of the nine flavonoid compounds in the sample solution was calculated by the linear regression equation based on the peak area of ​​the sample. The content of the nine flavonoid compounds was then calculated according to formula ①. The content of the nine flavonoid compounds in the sample is represented by W and is calculated according to formula ①:

[0069]

[0070] Wherein, W is the content of nine flavonoid compounds (mg / g); C is the actual concentration of the sample (μg / mL); V is the volume of the sample solution (mL); and M is the weight of the sample (g).

[0071] After calculation, the contents of nine flavonoid compounds in the six batches of natural incense raw material samples tested are shown in Table 3.

[0072] Table 3 Test results of six batches of incense raw material samples

[0073]

[0074] Note: - means not detected.

[0075] according to Figure 1 , Figure 2-7 As shown in the results in Table 3, this detection method can completely separate nine flavonoids (epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol, isorhamnetin) at the same time. The retention times of "naringin" measured in samples 3 to 6 were 26.853min, 26.855min, 26.921min, and 26.946min, respectively, with a maximum and minimum difference of 0.093min, indicating that the method has good stability and reproducibility; and the separation degree has reached baseline separation, and the separation degree between the nine flavonoid components is ≥1.5. The above results show that this detection method is suitable for samples with complex matrices, and the detection has high sensitivity; according to the drawn standard curve, the content results of flavonoids calculated have high accuracy and reproducibility.

[0076] Comparative Example 1

[0077] In this comparative example, the method of "3. Determination" in Example 1 is the same as that in Example 1 except for the following conditions; specifically, the different conditions in this comparative example are:

[0078] Mobile phase: M phase is "methanol: formic acid-ammonium acetate solution (an aqueous solution containing 2 mmol / L formic acid and 10 mmol / L ammonium acetate) = 5:95 (v / v)"; N phase is "0.1% formic acid methanol solution by volume". The gradient elution conditions are detailed in Table 4.

[0079] Table 4 Chromatographic gradient elution conditions of Comparative Example 1

[0080]

[0081]

[0082] The ultra-high performance liquid chromatograms of the nine flavonoid compound standard solutions ⑤ measured under the conditions of this comparative example are shown in Figure 8 .Depend on Figure 8 It can be clearly seen that the separation of “epigallocatechin gallate and catechin” is insufficient, and “isoquercetin and rutin” completely overlap.

Claims

1. A liquid chromatography method for determining the contents of nine flavonoid compounds in natural incense raw materials, characterized in that: The following steps are involved: S1. Weigh the natural incense raw material sample to be tested, perform ultrasonic extraction with the extract, and the extract is centrifuged and filtered to obtain the sample solution to be tested; S2. The sample solution to be tested is subjected to ultra-high performance liquid chromatography detection; the conditions for the ultra-high performance liquid chromatography detection are: Chromatographic column: two HSS T3 chromatographic columns are used in series, the HSS T3 is a HSS T3 2.1×100mm, 1.7μm chromatographic column; Mobile phase: Phase A is methanol: formic acid-ammonium acetate solution with a volume ratio of 5:95, wherein the formic acid-ammonium acetate solution is an aqueous solution containing 2mmol / L formic acid and 10mmol / L ammonium acetate; Phase B is 0.1% formic acid aqueous solution: methanol with a volume ratio of 1:1; Phase C is 0.1% formic acid acetonitrile solution; Phase D is 0.1% formic acid methanol solution; Gradient elution program: 0-9 min, 100% phase A; 9-12 min, 100%-70% phase A, 0-16% phase B, 0-14% phase C; 12-18 min, 70%-50% phase A, 16%-36% phase B, 14% phase C; 18-27 min, 50%-42% phase A, 36%-44% phase B, 14% phase C; 27-33min, 42%-4% phase A, 44%-82% phase B, 14% phase C; 33-45min, 4% phase A, 82% phase B, 14% phase C; 45-48min, 4%-0 phase A, 82%-86% phase B, 14% phase C; 48-51min, 0-45% phase A, 86%-0 phase B, 14%-0 phase C, 0-55% phase D; 51-57min, 45%-10% phase A, 55%-90% phase D; 57-66 min, 10% phase A, 90% phase D; 66-72 min, 10%-100% phase A, 90%-0 phase D; equilibrium 15 min; flow rate: 0-27 min, 0.15 mL min -1 ; 27~72min, 0.2mL·min -1 ; 72~87min, 0.15mL·min -1 ; Column temperature: 30°C; Detection wavelength: 228nm, 254nm; Injection volume: 10 μL; S3. Use the external standard method to conduct qualitative and quantitative analysis on the nine flavonoid compounds in the samples.

2. The method according to claim 1, characterized in that The nine flavonoid compounds are epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol and isorhamnetin.

3. The method according to claim 1, characterized in that The extract is a 70% ethanol aqueous solution.

4. The method according to claim 1, characterized in that The centrifugation is performed at 8000 r / min for 10 min.

5. The method according to claim 1, characterized in that The filtration is carried out using a 0.22 μm filter membrane.

6. The method according to claim 1, characterized in that The qualitative analysis in step S3 is as follows: the ultra-high performance liquid chromatogram obtained in step S2 is compared with the ultra-high performance liquid chromatogram obtained from the nine flavonoid compound standard solutions under the same detection conditions to obtain the qualitative analysis results; the nine flavonoid compound standard solutions are solutions prepared by dissolving the nine flavonoid compound standards in the extract.

7. The method according to claim 1, characterized in that The quantitative analysis in step S3 comprises the following steps: S31. Accurately weigh nine flavonoid standard products, dissolve them with extracts by ultrasonication and make up to volume, so that the concentrations of epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol and isorhamnetin are 7.53 μg / mL, 6.82 μg / mL, 7.38 μg / mL, 82.50 μg / mL, 10.45 μg / mL, 5.76 μg / mL, 5.52 μg / mL, 18.56 μg / mL and 60.98 μg / mL, respectively, as standard mother solutions; then accurately measure 0.3 mL, 0.5 mL, 1 mL, 3 mL and 5 mL of the standard mother solution into a 10 mL volumetric flask, dilute and make up to volume with the extract to obtain nine flavonoid series standard solutions; S32. The standard solution is tested using the conditions of step S2, and a standard curve is drawn based on the peak area measured for each standard solution and its corresponding concentration. After a linear regression equation is obtained, the concentrations of the nine flavonoid compounds in the sample solution to be tested are calculated using the linear regression equation based on the peak area of ​​the sample to be tested, and then the contents of the nine flavonoid compounds in the sample to be tested are calculated.

8. Use of the method according to any one of claims 1 to 7 in the detection of flavonoid compounds.

9. The use according to claim 8, characterized in that: The flavonoid compounds are epigallocatechin gallate, catechin, epicatechin, naringin, isoquercetin, rutin, quercetin, kaempferol and isorhamnetin.

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