CN114910578B - 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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- CN114910578B CN114910578B CN202210371767.XA CN202210371767A CN114910578B CN 114910578 B CN114910578 B CN 114910578B CN 202210371767 A CN202210371767 A CN 202210371767A CN 114910578 B CN114910578 B CN 114910578B
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- 238000000034 method Methods 0.000 title claims abstract description 49
- 229940087603 grape seed extract Drugs 0.000 title claims abstract description 45
- 235000002532 grape seed extract Nutrition 0.000 title claims abstract description 45
- 239000001717 vitis vinifera seed extract Substances 0.000 title claims abstract description 45
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 75
- 239000000523 sample Substances 0.000 claims abstract description 40
- 238000010828 elution Methods 0.000 claims abstract description 24
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims abstract description 17
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 235000019253 formic acid Nutrition 0.000 claims abstract description 10
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 claims abstract description 10
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims abstract description 10
- 239000012488 sample solution Substances 0.000 claims abstract description 9
- 239000003960 organic solvent Substances 0.000 claims abstract description 7
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- MOJZMWJRUKIQGL-FWCKPOPSSA-N Procyanidin C2 Natural products O[C@@H]1[C@@H](c2cc(O)c(O)cc2)Oc2c([C@H]3[C@H](O)[C@@H](c4cc(O)c(O)cc4)Oc4c3c(O)cc(O)c4)c(O)cc(O)c2[C@@H]1c1c(O)cc(O)c2c1O[C@@H]([C@H](O)C2)c1cc(O)c(O)cc1 MOJZMWJRUKIQGL-FWCKPOPSSA-N 0.000 claims description 54
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical group CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 46
- MOJZMWJRUKIQGL-XILRTYJMSA-N procyanidin C1 Chemical compound C1([C@@H]2[C@H](O)[C@H](C3=C(O)C=C(O)C=C3O2)C2=C3O[C@@H]([C@H](O)[C@H](C3=C(O)C=C2O)C=2C(O)=CC(O)=C3C[C@H]([C@H](OC3=2)C=2C=C(O)C(O)=CC=2)O)C=2C=C(O)C(O)=CC=2)=CC=C(O)C(O)=C1 MOJZMWJRUKIQGL-XILRTYJMSA-N 0.000 claims description 44
- MOJZMWJRUKIQGL-FQVWZTFVSA-N Procyanidin C1 Natural products O[C@H]1[C@@H](c2cc(O)c(O)cc2)Oc2c([C@@H]3[C@@H](O)[C@@H](c4cc(O)c(O)cc4)Oc4c3c(O)cc(O)c4)c(O)cc(O)c2[C@H]1c1c(O)cc(O)c2c1O[C@H]([C@H](O)C2)c1cc(O)c(O)cc1 MOJZMWJRUKIQGL-FQVWZTFVSA-N 0.000 claims description 39
- 229920001554 Procyanidin C1 Polymers 0.000 claims description 38
- MOJZMWJRUKIQGL-WNCKYJNFSA-N Catechin-(4alpha->8)-gallocatechin-(4alpha->8)-catechin Chemical compound C1([C@@H]2[C@@H](O)[C@@H](C3=C(O)C=C(O)C=C3O2)C2=C3O[C@@H]([C@@H](O)[C@@H](C3=C(O)C=C2O)C=2C(O)=CC(O)=C3C[C@@H]([C@H](OC3=2)C=2C=C(O)C(O)=CC=2)O)C=2C=C(O)C(O)=CC=2)=CC=C(O)C(O)=C1 MOJZMWJRUKIQGL-WNCKYJNFSA-N 0.000 claims description 15
- 229920002956 Procyanidin C2 Polymers 0.000 claims description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000002245 particle Substances 0.000 claims description 6
- 238000002137 ultrasound extraction Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 5
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 238000010992 reflux Methods 0.000 claims description 2
- 238000001819 mass spectrum Methods 0.000 claims 1
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- 150000008442 polyphenolic compounds Chemical class 0.000 abstract description 13
- 235000013824 polyphenols Nutrition 0.000 abstract description 13
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- 238000001514 detection method Methods 0.000 description 15
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- 238000003908 quality control method Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- 238000000926 separation method Methods 0.000 description 7
- 102100020865 EKC/KEOPS complex subunit LAGE3 Human genes 0.000 description 5
- 101001137983 Homo sapiens EKC/KEOPS complex subunit LAGE3 Proteins 0.000 description 5
- 101100122529 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) GON7 gene Proteins 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000012490 blank solution Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 239000011550 stock solution Substances 0.000 description 4
- RQBBFKINEJYDOB-UHFFFAOYSA-N acetic acid;acetonitrile Chemical compound CC#N.CC(O)=O RQBBFKINEJYDOB-UHFFFAOYSA-N 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 244000037364 Cinnamomum aromaticum Species 0.000 description 2
- 235000014489 Cinnamomum aromaticum Nutrition 0.000 description 2
- 244000130270 Fagopyrum tataricum Species 0.000 description 2
- 235000014693 Fagopyrum tataricum Nutrition 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- CWEZAWNPTYBADX-UHFFFAOYSA-N Procyanidin Natural products OC1C(OC2C(O)C(Oc3c2c(O)cc(O)c3C4C(O)C(Oc5cc(O)cc(O)c45)c6ccc(O)c(O)c6)c7ccc(O)c(O)c7)c8c(O)cc(O)cc8OC1c9ccc(O)c(O)c9 CWEZAWNPTYBADX-UHFFFAOYSA-N 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229920002414 procyanidin Polymers 0.000 description 2
- XFZJEEAOWLFHDH-NFJBMHMQSA-N procyanidin B2 Chemical compound C1([C@@H]2[C@H](O)[C@H](C3=C(O)C=C(O)C=C3O2)C=2C(O)=CC(O)=C3C[C@H]([C@H](OC3=2)C=2C=C(O)C(O)=CC=2)O)=CC=C(O)C(O)=C1 XFZJEEAOWLFHDH-NFJBMHMQSA-N 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000010998 test method Methods 0.000 description 2
- 238000009210 therapy by ultrasound Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- XFZJEEAOWLFHDH-UHFFFAOYSA-N (2R,2'R,3R,3'R,4R)-3,3',4',5,7-Pentahydroxyflavan(48)-3,3',4',5,7-pentahydroxyflavan Natural products C=12OC(C=3C=C(O)C(O)=CC=3)C(O)CC2=C(O)C=C(O)C=1C(C1=C(O)C=C(O)C=C1O1)C(O)C1C1=CC=C(O)C(O)=C1 XFZJEEAOWLFHDH-UHFFFAOYSA-N 0.000 description 1
- SMMIDVLUFMPWFN-UHFFFAOYSA-N 4-nitro-n-[(4-phenyldiazenylphenyl)diazenyl]aniline Chemical compound C1=CC([N+](=O)[O-])=CC=C1NN=NC1=CC=C(N=NC=2C=CC=CC=2)C=C1 SMMIDVLUFMPWFN-UHFFFAOYSA-N 0.000 description 1
- 241000723347 Cinnamomum Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000219051 Fagopyrum Species 0.000 description 1
- 235000009419 Fagopyrum esculentum Nutrition 0.000 description 1
- 229920002350 Procyanidin B2 Polymers 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000017803 cinnamon Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- SVWLIIFHXFGESG-UHFFFAOYSA-N formic acid;methanol Chemical compound OC.OC=O SVWLIIFHXFGESG-UHFFFAOYSA-N 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
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- 239000012086 standard solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N2030/042—Standards
- G01N2030/047—Standards external
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
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Abstract
The invention discloses a method for determining procyanidine C1 in grape seed extract, which comprises the following steps: (1) Adding an organic solvent into a grape seed extract sample to be detected for extraction to obtain a sample solution; (2) Taking a sample solution, detecting by adopting a liquid chromatography-mass spectrometer, and quantitatively calculating the procyanidine C1 content in a sample by adopting an external standard method; wherein the chromatographic conditions are: gradient elution is carried out by adopting a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column and taking formic acid as a mobile phase A and methanol as a mobile phase B. According to the method, the analysis is carried out by using a liquid chromatography-mass spectrometer analysis technology, and matrix interference of other polyphenols except procyanidine C1 in the grape seed extract, particularly interference of procyanidine C2 which is optical isomer with procyanidine C1, can be eliminated by optimizing chromatographic conditions, so that the content of procyanidine C1 in the grape seed extract can be accurately measured, and the effective control of the quality of the grape seed extract can be realized.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for determining procyanidine C1 in grape seed extract.
Background
Procyanidins are a general term for a wide variety of polyphenols widely existing in plants, and have the functions of resisting oxidation and eliminating free radicals. The institute of nutrition and health of the department of academy of sciences in 2021 has issued that procyanidine C1 in grape seed extract has the effect of directionally removing aging cells, so that development of a detection method for measuring procyanidine C1 in grape seed extract has a certain research significance.
The content detection of procyanidine C1 is related to the UPLC determination of procyanidine B-2 and procyanidine C-1 content in cassia bark and the HPLC simultaneous determination of 6 polyphenol components in buckwheat flakes. Both documents use a liquid phase instrument to measure the content of procyanidine C1 at the wavelength of 280nm/220nm, and the grape seed extract has various polyphenol components with similar structures, and the polyphenol substances have strong ultraviolet absorption at the wavelength of 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 literature UPLC for measuring procyanidine B-2 and procyanidine C-1 content in cassia bark: c18 column (2.0mm.times.100mm, 2 μm), mobile phase acetonitrile-0.2% acetic acid water gradient elution, detector wavelength 280nm, and measurement of procyanidin C1 in grape seed extract, and the result is shown in FIG. 1. The result shows that the grape seed extract can be subjected to a lot of peak interference by using the method, so that the procyanidine C1 and other polyphenols can not be effectively separated, and the retention time of the procyanidine C1 is 11.340min (wherein the procyanidine C1 and the procyanidine C2 are optical isomers and are difficult to separate). The method for simultaneously measuring 6 polyphenol components in the Fagopyrum tataricum tablet by using the HPLC method disclosed by the application comprises the following steps of: procyanidin C1 in grape seed extract was measured using a C18 column (250 mm. Times.4.6 mm,5 μm) with a mobile phase of acetonitrile and phosphoric acid aqueous solution gradient elution, and the detection wavelength of 220nm, and the results are shown in FIG. 2. The results show that the detection of grape seed extract by the method can not effectively separate procyanidine C1 and other polyphenols (the retention time of procyanidine C1 is 9.819 min). In addition, as shown in fig. 1 and 2, the substrate interference and baseline elevation of procyanidin C1 in grape seed extract can be achieved by using the literature method, and procyanidin C1 cannot be accurately quantified due to the insufficient specificity of procyanidin C1, so that a determination method with strong specificity capable of accurately quantifying procyanidin C1 content in grape seed extract needs to be developed.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a method for determining procyanidine C1 in grape seed extract, which can eliminate matrix interference of other polyphenols in grape seed extract, has strong specificity and can accurately quantify procyanidine C1 content in 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 grape seed extract sample to be detected for extraction to obtain a sample solution;
(2) Taking a sample solution, detecting by adopting a liquid chromatography-mass spectrometer, and quantitatively calculating the procyanidine C1 content in a sample by adopting an external standard method; wherein the chromatographic conditions are: carrying out gradient elution by adopting 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 comprises the following procedures:
The initial ratio of the mobile phase A is 80-95%, and the mobile phase A is kept for 1min; then reducing the ratio of the mobile phase A to 70-85% when the mobile phase A is in 3 min; then further reducing the ratio of the mobile phase A to 50-65% when the mobile phase A is in 8min, and then keeping for 5min; the mobile phase A ratio was then rapidly increased to 80-95% for 13.01min and held for 3min.
The method adopts an organic solvent to extract a grape seed extract sample to be detected, the extracted sample solution is analyzed by a liquid chromatography-mass spectrometer, 0.05-0.5% formic acid and methanol are used as mobile phases to carry out gradient elution, a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column is used for carrying out procyanidine isomer separation, and an external standard method is used for quantifying to obtain the procyanidine C1 content in the grape seed extract.
According to the invention, through optimizing the chromatographic column, the mobile phase and the elution program, not only other polyphenols in the grape seed extract can be separated, but also procyanidine C1 and procyanidine C2 which are optical isomers can be separated, and matrix interference of other polyphenols except procyanidine C1 is eliminated, so that accurate quantification of procyanidine C1 is realized.
Preferably, in step (2), the gradient elution procedure is:
| 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, wherein the temperature of ultrasonic extraction is 25-35 ℃ and the extraction time is 5-15min.
Preferably, in the step (2), the column length of the chromatographic column is 15cm, the inner diameter is 4.6mm, and the particle size is 2.6 μm.
Preferably, in step (2), the formic acid concentration is preferably 0.1%.
Preferably, in the step (2), the column temperature of the chromatographic column is 25-40 ℃ and the flow rate is 0.2-0.6mL/min.
Compared with the prior art, the invention has the following advantages:
According to the method, the analysis is carried out by using a liquid chromatography-mass spectrometer analysis technology, and matrix interference of other polyphenols except procyanidine C1 in the grape seed extract, particularly interference of procyanidine C2 which is optical isomer with procyanidine C1, can be eliminated by optimizing chromatographic conditions, so that the content of procyanidine C1 in the grape seed extract can be accurately measured, and the effective control of the quality of the grape seed extract can be realized.
The detection method has strong specificity, and the linearity, precision, durability test (stability), specificity test (blank), detection limit, quantitative limit and accuracy (recovery rate) test 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 procyanidin C1 content of the glucose seed extract.
Drawings
FIG. 1 is a chromatogram of a sample of grape seed extract analyzed according to the method of literature UPLC determination of procyanidin B-2 and procyanidin C-1 content in cinnamon.
FIG. 2 is a chromatogram of a sample of grape seed extract in the liquid phase analyzed according to the method of reference HPLC method for simultaneous determination of 6 polyphenols in Fagopyrum Tataricum meal.
FIG. 3 is a graph showing the working standard of procyanidin C1.
Fig. 4 is a chromatogram of a C18 column analysis procyanidin C1 and procyanidin C2 mixed control.
Fig. 5 is a control chromatogram of a mixture of procyanidin C1 and procyanidin C2 for pentafluorophenyl column analysis.
FIG. 6 is a chromatogram of a phenyl column analysis procyanidin C1 and procyanidin C2 mixed control.
FIG. 7 is a chromatogram of a C30 column assay procyanidin C1 and procyanidin C2 mixed control.
FIG. 8 is a chromatogram of an analytical grape seed extract sample from acetonitrile-acetic acid water gradient elution procedure.
FIG. 9 is a chromatogram of a sample of grape seed extract analyzed by methanol-formic acid aqueous gradient elution procedure.
FIG. 10 is a chromatogram of a gradient elution procedure-analytical procyanidin C1 control.
FIG. 11 is a chromatogram of a gradient elution procedure-analytical procyanidin C2 control.
FIG. 12 is a chromatogram of a gradient elution procedure three analytical procyanidin C1 control.
Fig. 13 is a chromatogram of a gradient elution procedure three analytical procyanidin C2 control.
Detailed Description
The present invention is further illustrated by the following specific embodiments, but the embodiments of the present invention are not limited by the following embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the following embodiments, and all the modifications, substitutions, combinations, and simplifications should be made in the scope of the present invention.
Example 1:
1. Instrument and reagent
1 Instrument
An electronic balance; an ultrasonic oscillator; liquid phase mass spectrometer: LC-30A type liquid chromatograph (Shimadzu, japan) was serially connected to TRIPLE QUAD type 4500 mass spectrometer (AB SCIEX, USA).
2 Reagent
Unless otherwise indicated, all reagents were analytically pure.
Reagent: formic acid (chromatographic purity), primary water, methanol (chromatographic purity), ethanol.
Control: procyanidin C1 (source: dupusi organism, lot number: PS011257, purity: 95.11%), procyanidin C2 (source: ZZ STANARDS, lot number: 22B200-A1, purity: 99.0%).
2. Analysis method
2.1 Instrument conditions and parameters:
2.1.1 chromatographic conditions
Chromatographic column: filomide F5 filler (column length 15cm, inner diameter 4.6mm, particle size 2.6 μm);
Column temperature: 40 ℃;
Flow rate: 0.4mL/min;
Mobile phase: a is 0.1% formic acid solution, B is methanol, and the gradient elution is carried out according to the following table 1;
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:
| Curtain gas CUR | Collision gas CAD | Ion voltage IS | Atomization temperature TEM | Atomizer GS1 | Auxiliary heating gas GS2 |
| 15 | 8 | 5500 | 400 | 50 | 50 |
Table 3:
| Name of the name | 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 solution
Accurately weighing 12.98mg of procyanidine C1 reference substance, placing in a 50mL volumetric flask, adding 70% ethanol solution, ultrasonically dissolving to volume to scale, and shaking to obtain 0.247mg/mL of reference stock solution.
And precisely transferring 1.00mL of control stock solution, placing the control stock solution into a 50mL volumetric flask, adding 70% ethanol solution to a scale, and shaking uniformly to obtain 4.94 mug/mL of control intermediate solution.
And precisely transferring 1.00mL of the contrast intermediate solution, placing the contrast intermediate solution into a 10mL volumetric flask, adding 70% ethanol solution to a scale, and shaking uniformly to obtain 0.494 mug/mL of contrast working solution.
2.3 Standard working Curve drawing
Precisely sucking the sample injection amount of the working standard liquid: sample injection amount of 0.5 μl,1 μl,2 μl,4 μl,8 μl and sample solution: 2 μl was injected into a liquid chromatograph-mass spectrometer to establish a standard curve equation.
2.4 Sample preparation of test solution
Precisely weighing about 0.2g of uniform sample, placing in a 50mL volumetric flask, adding 70% ethanol solution, performing ultrasonic treatment at room temperature until the solution is fully dissolved for 5-15min, taking out, fixing the volume to the scale of the volumetric flask with 70% ethanol solution, and shaking uniformly to obtain sample intermediate liquid 1. And precisely transferring 1mL of the sample intermediate liquid 1, placing the sample intermediate liquid 1 into a 50mL volumetric flask, adding 70% ethanol solution to fix the volume to the scale of the volumetric flask, and shaking uniformly to obtain a sample intermediate liquid 2. And precisely transferring 1mL of sample intermediate solution 2, placing the sample intermediate solution in a 10mL volumetric flask, adding 70% ethanol solution to the scale of the volumetric flask, shaking uniformly, and filtering by a filter membrane to obtain a sample test solution.
2.5 Calculation of results:
Wherein: the content of procyanidine C1 in the X-sample is g/100g;
c-concentration of procyanidin C1 in the sample solution, μg/mL;
M-mass of sample, g;
v-volume of sample dilution, mL;
k-unit conversion coefficient, k=0.0001.
3. Methodological verification
3.1 Specificity test (blank)
Treating the blank solution according to a preparation method of a 2.4 sample, and determining the blank solution according to a 2.1 chromatographic condition, and comparing the blank solution with the peak time of the procyanidine C1 working standard solution. The results showed that the blank solution was not peaked at the time of peak emergence of procyanidin C1, indicating that the blank was substantially non-interfering with the assay results.
3.2 Linear Range validation
3.2.1 Working standard fluid test data are shown in table 4 below:
Table 4:
3.2.2 Standard working graph
The standard working curve is plotted as shown in fig. 3 with the concentration on the abscissa and the peak area on the ordinate.
3.2.3 Linear conclusion of the test
Linear evaluation: the correlation coefficient R of the procyanidine C1 is 0.9999 respectively, so the procyanidine C1 measured by the method shows 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 the correlation coefficient R not less than 0.99 ').
3.3 Detection and quantification limits
The detection limit DL and the quantification limit QL of the analysis method are calculated from the signal-to-noise ratio (S/N). DL is defined as the corresponding concentration to be analyzed when S/n=3, and QL is defined as the corresponding concentration to be analyzed when S/n=10.
3.3.1 Detection Limit
When the signal-to-noise ratio (S/N) is 3, the detection limit of procyanidine C1 is 0.01 mug/mL; the detection limit of procyanidine C1 in the method is 1.25mg/g when the sample sampling amount is 0.2g and the volume is 25000 mL.
3.3.2 Quantitative limit
When the signal-to-noise ratio (S/N) is 10, the quantification limit of procyanidine C1 is 0.04 mug/mL; the sample sampling amount is 0.2g, and the procyanidine C1 quantitative limit of the method is 4.17mg/g when the volume is 25000 mL.
3.4 Precision test
3.4.1 Test methods
6 Samples were weighed, treated according to the 2.4 sample preparation method, the sample content was measured, and the RSD (%) was calculated.
3.4.2 Test data are shown in table 5 below [ test samples are: glucose seed extract
Table 5:
3.4.3 test conclusion
The RSD of the content of the procyanidine C1 in 6 samples is 2.4%, which shows that the method has better precision and meets the requirements of GB/T27404-2008 'laboratory quality control Specification' (the RSD is less than or equal to 2.7 percent as required by GB/T27404-2008).
3.5 Accuracy test (recovery)
3.5.1 Test methods
And (3) marking: 9 parts of a sample (the content of procyanidine C1 of a known sample is 5.61g/100 g) of about 0.2g is precisely weighed, the sample is divided into 3 groups, 3 parts of each group are placed in a 50mL volumetric flask, 70% ethanol solution is added, the mixture is subjected to ultrasonic treatment at room temperature until the mixture is fully dissolved for about 5-15min, the mixture is taken out, the volume of the mixture is fixed to the scale of the volumetric flask by using the 70% ethanol solution, and the sample is uniformly shaken to obtain a sample-added intermediate liquid 1. Precisely transferring 1mL of sample adding standard intermediate liquid 1, placing the sample adding standard intermediate liquid 1 into a 50mL volumetric flask, adding 0.6mL, 0.9mL and 1.2mL of control stock solution (0.247 mg/mL) into each group, adding 70% ethanol solution to fix the volume to the scale of the volumetric flask, and shaking uniformly to obtain sample adding standard intermediate liquid 2. And precisely transferring 1mL of sample, adding the standard intermediate solution 2, placing in a 10mL volumetric flask, adding 70% ethanol solution to the scale of the volumetric flask, shaking uniformly, and filtering by a filter membrane to obtain a sample-adding standard test solution, wherein the sample injection amount is 2 mu L.
3.5.2 Test data as follows table 6 [ test samples are: glucose seed extract
Table 6:
Measured addition = measured addition of labeled sample-measured sample;
Recovery (%) = measured addition/theoretical addition x 100%.
3.5.3 Conclusion of the test
The average recovery rate is: 100.1% and RSD of 2.6%; meets the requirements of GB/T27404-2008 'laboratory quality control Specification' (GB/T27404-2008 requires a recovery rate of 95-105%).
3.6 Conclusion
The content determination method is proved to be scientific and effective by performing linearity, precision, durability test (stability), specificity test (blank), detection limit, quantitative limit and accuracy (recovery rate) test on the content determination method of the procyanidine C1, and meets the requirements of GB/T27404-2008 'laboratory quality control Specification', so that the quality control purpose of the procyanidine C1 content of the glucose seed extract can be achieved.
4. Optimization of chromatographic columns
The detection difficulty of procyanidin C1 in grape seed extract is the separation of procyanidin C1 (PCC 1) and procyanidin C2 (PCC 2) which are optical isomers from each other. The research of the invention finds that the selection of the chromatographic column is one of key factors for solving the technical problems.
The present invention compares C18, pentafluorophenyl, phenyl and C30 columns.
C18 chromatography column: the chromatographic column used in the conventional chromatographic analysis experiment is a C18 chromatographic column, the invention adopts a C18 chromatographic column (4.6mm multiplied by 150mm,2.6 μm) to detect the mixed reference substance of PCC1 and PCC2, and the rest detection conditions are the same as those of the example 1, and the chromatogram is shown in figure 4. Of these, 8.25min was PCC2 and 8.74min was PCC1, and as can be seen from the figure, the C18 column failed to separate procyanidin C1 and procyanidin C2 effectively. The invention tries to adjust the gradient proportion of the epidemic phase for multiple times, but the C18 chromatographic column cannot effectively separate the two phases, so that the quantitative analysis of procyanidine C1 cannot be performed.
Pentafluorophenyl chromatography column: the test was carried out using a pentafluorophenyl column (column length 15cm, inner diameter 4.6mm, particle size 2.6 μm) instead of the C18 column, the chromatogram of which is shown in FIG. 5, wherein 10.56min was PCC2 and 11.91min was PCC1. From the figure, the pentafluorophenyl chromatographic column can effectively separate procyanidine C1 from procyanidine C2, and the separation degree is good.
Phenyl chromatographic column: the test was carried out by using Phenyl column (Aglent XDB-Phenyl column length 15cm, inner diameter 4.6mm, particle size 2.6 μm) instead of C18 column, and the chromatogram is shown in FIG. 6. Wherein 9.24min is PCC2 and 9.86min is PCC1. From the figure, the phenyl chromatographic column can effectively separate procyanidine C1 from procyanidine C2, and the separation degree is good.
C30 chromatographic column: the test was carried out using a C30 column (Shimadzu C30 column length 15cm, inner diameter 4.6mm, particle size 2.6 μm) instead of a C18 column, and the chromatogram thereof is shown in FIG. 7. The PCC2 is 11.38min and the PCC1 is 12.86min, and the C30 chromatographic column can effectively separate the procyanidine C1 from the procyanidine C2, and has good separation degree.
Therefore, the invention selects a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column as the chromatographic column of the invention.
5. Optimization of chromatographic conditions
The choice of mobile phase and elution procedure is also one of the key technologies to achieve efficient separation of procyanidins C1 and C2.
Selection of mobile phase:
grape seed extract was tested using acetonitrile-acetic acid as mobile phase, the other conditions were the same as in example 1, and the chromatogram thereof was as shown in FIG. 8 (PCC 1 retention time 6.26 min). The results show that acetonitrile-acetic acid water does not separate procyanidin C1 peak from other interfering component peaks in the sample well.
The grape seed extract was examined using 0.1% formic acid solution and methanol as mobile phase, and the other conditions were the same as in example 1, and the chromatogram thereof was shown in FIG. 9 (PCC 1 retention time 11.87 min), which shows that 0.1% formic acid solution and methanol can separate procyanidin C1 peak from other interfering component peaks in the sample.
Optimization of gradient elution procedure:
Gradient elution procedure one: starting with 10% methanol, methanol was increased from 10% to 80% in 5 minutes, 80% methanol was maintained for 3 minutes, and immediately thereafter 10% methanol was recovered for 2 minutes. Procyanidin C1 control as shown in FIG. 10, procyanidin C2 control as shown in FIG. 11, the retention times of procyanidin C1 (retention time 5.97 min) and procyanidin C2 (retention time 5.95 min) were coincident. From this, it was found that too large a gradient of methanol elution can prevent separation of procyanidin C1 and procyanidin C2.
Gradient elution procedure two: the gradient elution procedure was adjusted to the initial 5% methanol for 1 minute, after 3 minutes, increased to 15% methanol, after 8 minutes, increased to 35% methanol, and after 5 minutes of 35% methanol retention, the horse recovered 5% methanol for 2 minutes, as shown in fig. 5, 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 20% methanol for 1 minute, after 3 minutes, to 30% methanol, after 8 minutes, to 50% methanol, after 5 minutes of 50% methanol retention, and after 20% methanol recovery was performed for 2 minutes, as shown in fig. 12, fig. 13, procyanidin C1 (retention time 8.91 min) and procyanidin C2 (retention time 5.95 min) were efficiently separated.
Claims (8)
1. A method for determining procyanidin C1 in grape seed extract, characterized in that the method is capable of effectively separating procyanidin C1 and procyanidin C2, and specifically comprises the following steps:
(1) Adding an organic solvent into a grape seed extract sample to be detected for extraction to obtain a sample solution;
(2) Taking a sample solution, detecting by adopting a liquid chromatography-mass spectrometer, and quantitatively calculating the procyanidine C1 content in a sample by adopting an external standard method; wherein the chromatographic conditions are: adopting a pentafluorophenyl chromatographic column, a phenyl chromatographic column or a C30 chromatographic column, taking formic acid with the mass concentration of 0.05-0.5% as a mobile phase A, taking methanol as a mobile phase B for gradient elution, wherein the gradient elution comprises the following procedures:
；
the column temperature of the chromatographic column is 25-40 ℃ and the flow rate is 0.2-0.6 mL/min;
The mass spectrum conditions are as follows: adopting a multi-reaction monitoring MRM scanning mode, and collecting in an electrospray ion source ESI and positive ion mode; the main parameters are as follows: ion voltage: 4000-5500V, 40-60PSI of atomizing gas, 40-60PSI of auxiliary heating gas, 300-450 ℃ of heating temperature and 20-40V of collision voltage; analytical quantitative ion pair 868.0/716.0; qualitative ion pair was analyzed to be 868.0/698.3.
2. The method for assaying procyanidin C1 in grape seed extract according to claim 1, wherein the mass concentration of formic acid in the step (2) is 0.1%.
3. The method for assaying procyanidin C1 in grape seed extract according to claim 1, wherein in the step (1), the organic solvent is an ethanol solution, a methanol solution or an acetonitrile solution.
4. A method for assaying procyanidin C1 in grape seed extract according to claim 3 wherein in step (1) the organic solvent is 50% -80% ethanol solution.
5. The method for assaying procyanidin C1 in grape seed extract according to claim 1, wherein in the step (1), the extraction mode is ultrasonic extraction or heating reflux extraction.
6. The method for assaying procyanidin C1 in grape seed extract according to claim 5, wherein in the step (1), the extraction mode is ultrasonic extraction.
7. The method for assaying procyanidin C1 in grape seed extract according to claim 6, wherein the ultrasonic extraction temperature is 25-35 ℃ and the extraction time is 5-15min.
8. The method for assaying procyanidin C1 in grape seed extract according to claim 1, wherein in the step (2), the column length of the chromatographic column is 15cm, the inner diameter is 4.6mm, and the particle size is 2.6 μm.
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