CN114910578A - Method for determining procyanidine C1 in grape seed extract - Google Patents
Method for determining procyanidine C1 in grape seed extract Download PDFInfo
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Abstract
The invention discloses a method for determining procyanidine C1 in a grape seed extract, which comprises the following steps: (1) adding a to-be-detected sample of the grape seed extract into an organic solvent for extraction to obtain a test solution; (2) taking a test sample solution, detecting by using a liquid chromatograph-mass spectrometer, and quantitatively calculating the content of procyanidine C1 in the sample by using an external standard method; wherein the chromatographic conditions are as follows: performing gradient elution with pentafluorophenyl column, phenyl column or C30 column, formic acid as mobile phase A, and methanol as mobile phase B. The method provided by the invention adopts a liquid chromatograph-mass spectrometer analysis technology for analysis, and can eliminate matrix interference of other polyphenols except procyanidin C1 in the grape seed extract, particularly interference of procyanidin C2 which is an optical isomer with procyanidin C1, so that the content of procyanidin C1 in the grape seed extract can be accurately determined, and effective control of quality of the grape seed extract is facilitated.
Description
Technical Field
The invention relates to the field of analytical chemistry, and particularly relates to a method for determining procyanidine C1 in a grape seed extract.
Background
Procyanidins are a general term for a large group of polyphenols widely existing in plants, and have the effects of resisting oxidation and eliminating free radicals. The nutritional and health research institute of the academy of sciences in 2021 released that procyanidin C1 in grape seed extract has the effect of directionally eliminating senescent cells, so that the development of a detection method for determining procyanidin C1 in grape seed extract has certain research significance.
A UPLC method for measuring the contents of procyanidine B-2 and procyanidine C-1 in cassia and an HPLC method for simultaneously measuring 6 polyphenol components in golden buckwheat relates to the content detection of procyanidine C1. Both of the two documents use a liquid phase instrument to measure the content of procyanidine C1 under the wavelength of 280nm/220nm, the grape seed extract has a plurality of polyphenol components with similar structures, and the polyphenol substances have strong ultraviolet absorption at 280nm/220nm, so that the existing detection method cannot be well suitable for measuring procyanidine in the grape seed extract. The application uses a method of UPLC for measuring contents of procyanidine B-2 and procyanidine C-1 in cinnamon as a document: c18 chromatographic column (2.0 mm × 100 mm, 2 μm), gradient eluting with mobile phase acetonitrile-0.2% acetic acid water, detecting procyanidin C1 in grape seed extract at detector wavelength of 280nm, and determining the result as shown in FIG. 1-1. The result shows that the grape seed extract detected by the method has a plurality of interference peaks, procyanidin C1 and other polyphenols cannot be effectively separated, and procyanidin C1 retention time is 11.340min (wherein procyanidin C1 and procyanidin C2 are optical isomers of each other and are difficult to separate). The application uses a method of simultaneously determining 6 polyphenol components in the golden buckwheat flakes by using a document 'HPLC method': the procyanidin C1 in grape seed extract is measured by using C18 chromatographic column (250 mm × 4.6mm, 5 μm) and acetonitrile and phosphoric acid water solution as mobile phase, and detecting wavelength of 220nm, and the result is shown in figure 1-2. The results show that the grape seed extract tested by the method can not effectively separate procyanidin C1 and other polyphenols (the retention time of procyanidin C1 is 9.819 min). In addition, as can be seen from fig. 1-1 and fig. 1-2, the problems of matrix interference, baseline elevation and insufficient specificity of procyanidin C1 in the determination of procyanidin C1 in the grape seed extract by using the literature methods have resulted in the failure to accurately quantify procyanidin C1, and therefore, a determination method with strong specificity and capable of accurately quantifying procyanidin C1 content in the grape seed extract is required to be developed.
Disclosure of Invention
In order to solve the above problems, the present invention aims to provide a method for determining procyanidin C1 in a grape seed extract, which can eliminate matrix interference of other polyphenols in the grape seed extract, has strong specificity, and can accurately quantify the content of procyanidin C1 in the grape seed extract.
The invention is realized by the following technical scheme:
a method for determining procyanidine C1 in grape seed extract comprises the following steps:
(1) adding an organic solvent into a to-be-detected sample of the grape seed extract for extraction to obtain a test solution;
(2) taking a test sample solution, detecting by using a liquid chromatograph-mass spectrometer, and quantitatively calculating the content of procyanidine C1 in the sample by using an external standard method; wherein the chromatographic conditions are as follows: performing gradient elution by using a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column and taking formic acid with the mass concentration of 0.05-0.5% as a mobile phase A and methanol as a mobile phase B, wherein the gradient elution procedure is as follows:
the initial ratio of the mobile phase A is 80-95%, and the mobile phase A is kept for 1 min; then reducing the proportion of the mobile phase A to 70-85% in 3 min; then further reducing the proportion of the mobile phase A to 50-65% in 8min, and then keeping for 5 min; then the ratio of the mobile phase A is rapidly increased to 80-95% in 13.01min and kept for 3 min.
The method comprises the steps of extracting a sample to be detected of the grape seed extract by adopting an organic solvent, analyzing a sample solution after extraction by using a liquid chromatograph-mass spectrometer, carrying out gradient elution by using 0.05-0.5% formic acid and methanol as mobile phases, separating procyanidine isomers by using a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column, and quantifying by using an external standard method to obtain the content of procyanidine C1 in the grape seed extract.
By optimizing the chromatographic column, the mobile phase and the elution procedure, the method not only can separate other polyphenols in the grape seed extract, but also can separate the procyanidin C1 and the procyanidin C2 which are optical isomers of each other, and eliminates matrix interference of other polyphenols except the procyanidin C1, thereby realizing accurate quantification of the procyanidin C1.
Preferably, in step (2), the gradient elution procedure is as follows:
| time min | Mobile phase A% | Mobile phase B% |
| 0 | 95 | 5 |
| 1 | 95 | 5 |
| 3 | 85 | 15 |
| 8 | 65 | 35 |
| 13 | 65 | 35 |
| 13.01 | 95 | 5 |
| 15 | 95 | 5 |
Preferably, in step (1), the organic solvent is an ethanol solution, a methanol solution or an acetonitrile solution, and more preferably a 50-80% ethanol solution.
Preferably, in the step (1), the extraction mode is ultrasonic extraction or heating reflux extraction, preferably ultrasonic extraction, the temperature of the ultrasonic extraction is 25-35 ℃, and the extraction time is 5-15 min.
Preferably, in the step (2), the column has a column length of 15cm, an inner diameter of 4.6mm and a particle size of 2.6. mu.m.
Preferably, in the step (2), the formic acid concentration is preferably 0.1%.
Preferably, in the step (2), the temperature of the chromatographic column is 25-40 ℃, and the flow rate is 0.2-0.6 mL/min.
Compared with the prior art, the invention has the following advantages:
the method provided by the invention adopts a liquid chromatograph-mass spectrometer analysis technology for analysis, and can eliminate matrix interference of other polyphenols except procyanidin C1 in the grape seed extract, particularly interference of procyanidin C2 which is an optical isomer with procyanidin C1, so that the content of procyanidin C1 in the grape seed extract can be accurately determined, and effective control of quality of the grape seed extract is facilitated.
The detection method disclosed by the invention is strong in specificity, and the tests of linearity, precision, durability (stability), specificity (blank), detection limit, quantification limit and accuracy (recovery rate) all meet the requirements of GB/T27404-2008 laboratory quality control Specification, and the method is scientific and effective and can achieve the purpose of quality control on the content of procyanidine C1 in the glucose seed extract.
Drawings
FIG. 1-1 is a chromatogram of a sample from grape seed extract analyzed according to the method of UPLC for determining the contents of procyanidin B-2 and procyanidin C-1 in cinnamon.
FIGS. 1-2 are chromatograms of samples of grape seed extract in liquid phase analyzed according to the literature "HPLC method for simultaneous determination of 6 polyphenol components in Fagopyrum cymosum tablets".
Fig. 2 is a standard working curve diagram of procyanidin C1.
FIG. 3 is a chromatogram of C18 chromatographic column analysis of mixed control of procyanidin C1 and procyanidin C2.
FIG. 4 is a control chromatogram of a mixture of C1 and C2 procyanidins analyzed by pentafluorophenyl column.
FIG. 5 is a chromatogram of mixed control of procyanidin C1 and procyanidin C2 analyzed by phenyl column.
FIG. 6 is a chromatogram of a mixed control sample of C1 procyanidin and C2 procyanidin analyzed by C30 column.
FIG. 7 is a chromatogram of an acetonitrile-acetic acid water gradient elution procedure-analysis of a grape seed extract sample.
FIG. 8 is a chromatogram of a sample from the analysis of grape seed extract from methanol-formic acid water gradient elution procedure.
Figure 9-1 is a gradient elution procedure-analysis procyanidin C1 control chromatogram.
Fig. 9-2 is a gradient elution procedure-analysis procyanidin C2 control chromatogram.
FIG. 10-1 is a gradient elution procedure chromatogram of a control sample of procyanidin C1.
Fig. 10-2 is a gradient elution procedure tri-analysis procyanidin C2 control chromatogram.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the embodiments of the present invention, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions and alterations are intended to be included in the scope of the present invention.
Example 1:
instrument and reagent
1 apparatus
An electronic balance; an ultrasonic oscillator; liquid phase mass spectrometer: LC-30A liquid chromatograph (Shimadzu, Japan) was connected in series to a TRIPLE QUAD 4500 mass spectrometer (AB SCIEX, USA).
Reagent
All reagents used were analytical grade unless otherwise indicated.
Reagent: formic acid (chromatographically pure), primary water, methanol (chromatographically pure), ethanol.
Comparison products: procyanidin C1 (source: Dorpus sp., lot: PS011257, purity: 95.11%), procyanidin C2 (source: ZZ STANADS, lot: 22B200-A1, purity: 99.0%).
Second, analysis method
2.1 Instrument conditions and parameters:
2.1.1 chromatographic conditions
A chromatographic column: filler Phlomis F5 (column length 15cm, inner diameter 4.6mm, particle diameter 2.6 μm);
column temperature: 40 ℃;
flow rate: 0.4 mL/min;
mobile phase: a is 0.1% formic acid solution, B is methanol, gradient elution according to Table 1 below;
table 1:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95-80 | 5-20 |
| 1 | 95-80 | 5-20 |
| 3 | 85-70 | 15-35 |
| 8 | 65-50 | 35-50 |
| 13 | 65-50 | 35-50 |
| 13.01 | 95-80 | 5-20 |
| 15 | 95-80 | 5-20 |
2.1.2 Mass Spectrometry conditions are shown in tables 2 and 3 below:
MRM mode:
table 2:
| air curtain air CUR | Collision gas CAD | Ion voltage IS | Atomization temperature TEM | Atomizer GS1 | Auxiliary |
| 15 | 8 | 5500 | 400 | 50 | 50 |
Table 3:
| name (R) | Ionization mode | Q1 | Q3 | Time（ms） | DP | CE |
| Procyanidin C1 | ESI+ | 868.0 | 716.0* | 200 | 100 | 30 |
| Procyanidin C1 | ESI+ | 868.0 | 698.3 | 200 | 100 | 32.9 |
Quantitative ion pairs
2.2 preparation of control solutions
Accurately weighing 12.98mg of procyanidine C1 reference substance, placing in a 50mL volumetric flask, adding 70% ethanol solution, ultrasonically dissolving to constant volume to scale, and shaking to obtain 0.247mg/mL reference stock solution.
Precisely transferring 1.00mL of the control stock solution, putting the control stock solution into a 50mL volumetric flask, adding 70% ethanol solution to a constant volume to a scale, and shaking up to obtain a control intermediate solution of 4.94 mu g/mL.
Precisely transferring 1.00mL of the control intermediate solution, placing the control intermediate solution in a 10mL volumetric flask, adding 70% ethanol solution to a constant volume to a scale, and shaking up to obtain 0.494 mug/mL of control working solution.
2.3 Standard working Curve plotting
Precisely absorbing the sample volume of the working standard solution: 0.5. mu.L, 1. mu.L, 2. mu.L, 4. mu.L, 8. mu.L and the sample amount of the sample test solution: 2 mu L of the solution is injected into a liquid chromatogram-mass spectrometer to establish a standard curve equation.
2.4 sample preparation of test solutions
Precisely weighing about 0.2g of uniform sample, placing the uniform sample in a 50mL volumetric flask, adding 70% ethanol solution, carrying out ultrasonic treatment at room temperature until the uniform sample is fully dissolved for 5-15min, taking out the uniform sample, carrying out constant volume with the 70% ethanol solution until the volume is scaled by the volumetric flask, and shaking up to obtain a sample intermediate solution 1. Precisely transferring 1mL of the sample intermediate solution 1, placing the sample intermediate solution in a 50mL volumetric flask, adding 70% ethanol solution to the volumetric flask to a constant volume, and shaking up to obtain a sample intermediate solution 2. Precisely transferring 1mL of sample intermediate solution 2, placing in a 10mL volumetric flask, adding 70% ethanol solution to constant volume to the scale of the volumetric flask, shaking up, and filtering with a filter membrane to obtain sample test solution.
2.5 results calculation:
in the formula: x represents the content of procyanidine C1 in the sample, and g/100 g;
c is the concentration of procyanidin C1 in the sample solution, mu g/mL;
m-mass of sample, g;
v-volume of sample dilution, mL;
k is the coefficient of conversion per unit,
。
third, methodology verification
3.1 specificity test (blank)
The blank solution was processed according to the 2.4 sample preparation method and the peak time of the blank solution was measured according to the 2.1 chromatographic conditions and compared with the procyanidin C1 working standard. The results show that the blank solution has no peak at the peak-out time of the procyanidin C1, which indicates that the blank has no substantial interference with the determination results.
Linear range validation
3.2.1 test data for the working standard solutions are shown in Table 4 below:
table 4:
3.2.2 Standard working Curve
The concentration is used as the abscissa, and the peak area is used as the ordinate, and a standard working curve is drawn as shown in fig. 2.
3.2.3 conclusion of Linear test
And (3) linear evaluation: the correlation coefficients R of the procyanidine C1 are respectively 0.9999, so the procyanidine C1 determined by the method has good linearity between the concentration of 0.124 mu g/mL and 1.976 mu g/mL, and meets the requirements of GB/T27404-2008 laboratory quality control Specification [ GB/T27404-2008 requires that the correlation coefficient R is more than or equal to 0.99 ].
Detection limit and quantification limit
The detection limit DL and the quantification limit QL of the analytical method are calculated from the signal-to-noise ratio (S/N). DL is defined as the concentration to be analyzed corresponding to S/N =3, and QL is defined as the concentration to be analyzed corresponding to S/N = 10.
3.3.1 detection Limit
When the signal to noise ratio (S/N) is 3, the detection limit of procyanidin C1 is 0.01 mu g/mL; when the sample sampling amount is 0.2g and the constant volume is 25000mL, the detection limit of the procyanidin C1 in the method is 1.25 mg/g.
3.3.2 quantitative limits
When the signal to noise ratio (S/N) is 10, the limit of the procyanidin C1 quantification is 0.04 mu g/mL; when the sample sampling amount is 0.2g and the constant volume is 25000mL, the limit of the method for the determination of the procyanidin C1 is 4.17 mg/g.
Precision test
3.4.1 test methods
6 parts of sample is weighed, the sample is processed according to the preparation method of 2.4 samples, the content of the sample is detected, and the RSD (%) of the sample is calculated.
3.4.2 test data are shown in Table 5 below [ test samples are: glucose seed extract
Table 5:
3.4.3 conclusion of the test
The content of the procyanidin C1 in 6 samples is 2.4 percent, which shows that the method has better precision and meets the requirement of GB/T27404-2008 laboratory quality control Specification (GB/T27404-2008 requires RSD to be less than or equal to 2.7 percent).
Accuracy test (recovery rate)
3.5.1 test methods
Adding a standard: precisely weighing about 0.2g of sample 9 parts (the content of procyanidine C1 in the known sample is 5.61g/100 g), dividing into 3 groups, placing each group of 3 parts in a 50mL volumetric flask, adding 70% ethanol solution, performing ultrasonic treatment at room temperature to fully dissolve for about 5-15min, taking out, fixing the volume to the scale of the volumetric flask by using 70% ethanol solution, and shaking up to obtain sample added standard intermediate solution 1. Precisely transferring 1mL of the sample labeling intermediate solution 1 into a 50mL volumetric flask, adding 0.6mL, 0.9mL and 1.2mL of the control stock solution (0.247 mg/mL) into each group, adding 70% ethanol solution to the volumetric flask to reach the constant volume, and shaking up to obtain the sample labeling intermediate solution 2. Precisely transferring 1mL of sample and the labeled intermediate solution 2, placing the sample and the labeled intermediate solution in a 10mL volumetric flask, adding 70% ethanol solution to a constant volume until the volume is scaled by the volumetric flask, shaking up, and filtering through a filter membrane to obtain a sample labeled test solution with the sample injection amount of 2 mu L.
3.5.2 test data are shown in table 6 below [ test samples are: glucose seed extract
Table 6:
measured add = measured amount of spiked sample-measured amount of sample;
recovery (%) = measured addition/theoretical addition × 100%.
3.5.3 conclusion of the test
The average recovery rate is: 100.1 percent and RSD is 2.6 percent; the method meets the requirements of GB/T27404-2008 laboratory quality control Specification (GB/T27404-2008 requires that the recovery rate is 95-105%).
Conclusion
The content determination method of the procyanidin C1 is subjected to linear, precision, durability tests (stability), specificity tests (blank), detection limit, quantification limit and accuracy (recovery rate) tests, meets the requirements of GB/T27404-2008 'laboratory quality control Specification', proves that the content determination method is scientific and effective, and can achieve the purpose of quality control on the procyanidin C1 content of the glucose seed extract.
Optimization of chromatographic column
The difficulty in detecting procyanidin C1 in grape seed extract lies in the separation of procyanidin C1 (PCC 1) and procyanidin C2 (PCC 2) which are optical isomers of each other. The present inventors have found that, in order to solve the above technical problems, the selection of a chromatographic column is one of the key factors.
The invention compares a C18 column, a pentafluorophenyl column, a phenyl column, and a C30 column.
C18 column: the chromatographic column used in the conventional chromatographic analysis experiment is a C18 chromatographic column, the mixed control of PCC1 and PCC2 is detected by using a C18 chromatographic column (4.6 mm × 150mm, 2.6 μm) in the invention, and the rest of the detection conditions are the same as those in example 1, and the chromatogram is shown in FIG. 3. Wherein 8.25min is PCC2,8.74min is PCC1, and as can be seen from the figure, the C18 chromatographic column can not effectively separate the procyanidin C1 from the procyanidin C2. The method tries to regulate the popular phase gradient proportion for multiple times, but the C18 chromatographic column cannot effectively separate the popular phase gradient proportion from the popular phase gradient proportion, so that the proanthocyanidin C1 cannot be quantitatively analyzed.
Pentafluorophenyl chromatographic column: the experiment was carried out using a pentafluorophenyl column (column length 15cm, inner diameter 4.6mm, particle size 2.6 μm) instead of the C18 column, and the chromatogram is shown in FIG. 4, wherein 10.56min is PCC2 and 11.91min is PCC 1. As can be seen from the figure, the pentafluorophenyl chromatographic column can effectively separate the procyanidin C1 from the procyanidin C2, and the separation degree is better.
Phenyl chromatographic column: the test was carried out by using a Phenyl column (Agent XDB-Phenyl column length 15cm, inner diameter 4.6mm, particle diameter 2.6 μm) instead of the C18 column for detection, and its chromatogram was as shown in FIG. 5. Wherein 9.24min is PCC2, and 9.86min is PCC 1. As can be seen from the figure, the phenyl chromatographic column can effectively separate the procyanidin C1 from the procyanidin C2, and the separation degree is better.
C30 column: the test was carried out using a C30 column (Shimadzu C30 column 15cm in length, 4.6mm in inner diameter, 2.6 μm in particle diameter) in place of the C18 column, and the chromatogram thereof is shown in FIG. 6. Wherein, PCC2 is obtained at 11.38min, PCC1 is obtained at 12.86min, and as can be seen from the figure, the C30 chromatographic column can effectively separate the procyanidin C1 from the procyanidin C2, and the separation degree is better.
Therefore, a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column is selected as the chromatographic column of the invention.
Fifthly, optimization of chromatographic conditions
The selection of mobile phase and elution procedure is also one of the key technologies to achieve effective separation of procyanidin C1 and procyanidin C2.
Selection of mobile phase:
the grape seed extract was examined using acetonitrile-acetic acid water as the mobile phase under the same conditions as in example 1, and the chromatogram thereof is shown in FIG. 7 (PCC 1 retention time 6.26 min). The results show that acetonitrile-acetic acid water does not separate well the procyanidin C1 peak from other interfering component peaks in the sample.
The grape seed extract was examined using 0.1% formic acid solution and methanol as mobile phase, and the chromatogram of example 1 is shown in fig. 8 (PCC 1 retention time 11.87 min), which shows that 0.1% formic acid solution and methanol can separate the procyanidin C1 peak from other interfering component peaks in the sample.
Optimization of gradient elution procedure:
gradient elution procedure one: the initial 10% methanol, the methanol was increased from 10% to 80% over 5 minutes, the 80% methanol was held for 3 minutes, and the 10% methanol was immediately restored for 2 minutes. The procyanidin C1 control is shown in FIG. 9-1, procyanidin C2 control is shown in FIG. 9-2, and the retention times of procyanidin C1 (retention time 5.97 min) and procyanidin C2 (retention time 5.95 min) coincide. Therefore, too large a gradient of methanol elution makes separation of procyanidin C1 and procyanidin C2 impossible.
Gradient elution procedure two: adjusting the gradient elution procedure to start with 5% methanol, hold for 1 minute, increase to 15% methanol after 3 minutes, increase to 35% methanol after 8 minutes, resume 5% methanol for 2 minutes after 5 minutes with 35% methanol, as shown in fig. 4, it was possible to effectively separate procyanidin C1 (retention time 11.91 min) and procyanidin C2 (retention time 10.56 min).
Gradient elution procedure three: the gradient elution procedure was adjusted to start with 20% methanol, hold for 1 minute, increase to 30% methanol after 3 minutes, increase to 50% methanol after 8 minutes, and recover 20% methanol immediately after 5 minutes with 50% methanol for 2 minutes as shown in fig. 10-1 and fig. 10-2, which effectively separated procyanidin C1 (retention time 8.91 min) and procyanidin C2 (retention time 5.95 min).
Claims (9)
1. A method for determining procyanidine C1 in a grape seed extract is characterized by comprising the following steps:
(1) adding a to-be-detected sample of the grape seed extract into an organic solvent for extraction to obtain a test solution;
(2) taking a test sample solution, detecting by using a liquid chromatograph-mass spectrometer, and quantitatively calculating the content of procyanidine C1 in the sample by using an external standard method; wherein the chromatographic conditions are as follows: performing gradient elution by using a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column and using formic acid with the mass concentration of 0.05-0.5% as a mobile phase A and methanol as a mobile phase B, wherein the gradient elution procedure is as follows:
the initial ratio of the mobile phase A is 80-95%, and the mobile phase A is kept for 1 min; then reducing the proportion of the mobile phase A to 70-85% in 3 min; then further reducing the proportion of the mobile phase A to 50-65% in 8min, and then keeping for 5 min; then the ratio of mobile phase A is increased to 80-95% in 13.01min and kept for 3 min.
2. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in the step (2), the mass concentration of formic acid is 0.1%.
3. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in the step (2), the procedure of gradient elution is as follows:
。
4. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in step (1), the organic solvent is ethanol solution, methanol solution or acetonitrile solution, preferably 50% -80% ethanol solution.
5. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in step (1), the extraction manner is ultrasonic extraction or heating reflux extraction, preferably ultrasonic extraction.
6. The method for determining procyanidin C1 in grape seed extract as claimed in claim 5, wherein the ultrasonic extraction temperature is 25-35 deg.C, and the extraction time is 5-15 min.
7. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in step (2), the chromatographic column has a column length of 15cm, an inner diameter of 4.6mm and a particle size of 2.6 μm.
8. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in step (2), the column temperature of the chromatographic column is 25-40 ℃ and the flow rate is 0.2-0.6 mL/min.
9. The method for determining procyanidin C1 in grape seed extract as claimed in claim 1, wherein in the step (2), the mass spectrometry conditions are as follows:
collecting in a multi-reaction monitoring MRM scanning mode, an electrospray ionization source ESI and a positive ion mode; the main parameters are as follows: ion voltage: 4000 + 5500V, 40-60PSI of atomizing gas, 40-60PSI of auxiliary heating gas, the heating temperature is 300 + 450 ℃, and the collision voltage is 20-40V; the analytically quantitative ion pair is 868.0/716.0; the analytical qualitative ion pair was 868.0/698.3.
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| KR102064813B1 (en) * | 2019-07-02 | 2020-01-10 | 주식회사 바이오메디앙 | Method for analyzing Vitis vinifera extract |
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| US20180346731A1 (en) * | 2017-06-06 | 2018-12-06 | Ohio State Innovation Foundation | Formation of Stable Pyranoanthocyanins, and Uses Thereof as Sources of Natural Color |
| CN108918711A (en) * | 2018-07-16 | 2018-11-30 | 中国烟草总公司郑州烟草研究院 | The detection method of polyphenol compound in a kind of tobacco leaf |
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