CN113138238A - Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds - Google Patents
Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds Download PDFInfo
- Publication number
- CN113138238A CN113138238A CN202110405210.9A CN202110405210A CN113138238A CN 113138238 A CN113138238 A CN 113138238A CN 202110405210 A CN202110405210 A CN 202110405210A CN 113138238 A CN113138238 A CN 113138238A
- Authority
- CN
- China
- Prior art keywords
- camelina sativa
- reference substance
- components
- catechin
- content
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 244000197813 Camelina sativa Species 0.000 title claims abstract description 67
- 235000014595 Camelina sativa Nutrition 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 39
- 150000007965 phenolic acids Chemical class 0.000 title claims abstract description 20
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 title claims description 14
- 229930003944 flavone Natural products 0.000 title claims description 14
- 150000002212 flavone derivatives Chemical class 0.000 title claims description 14
- 235000011949 flavones Nutrition 0.000 title claims description 14
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 title claims description 14
- 239000013558 reference substance Substances 0.000 claims abstract description 55
- 229950001002 cianidanol Drugs 0.000 claims abstract description 41
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 40
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 40
- 235000005487 catechin Nutrition 0.000 claims abstract description 40
- 238000012937 correction Methods 0.000 claims abstract description 20
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 claims description 50
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 45
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 38
- 229940074393 chlorogenic acid Drugs 0.000 claims description 28
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 claims description 27
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 claims description 27
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 claims description 27
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 claims description 27
- 235000001368 chlorogenic acid Nutrition 0.000 claims description 27
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 claims description 27
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 claims description 27
- 229960004555 rutoside Drugs 0.000 claims description 27
- PFTAWBLQPZVEMU-ZFWWWQNUSA-N (+)-epicatechin Natural products C1([C@@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-ZFWWWQNUSA-N 0.000 claims description 26
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 claims description 26
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims description 26
- GQODBWLKUWYOFX-UHFFFAOYSA-N Isorhamnetin Natural products C1=C(O)C(C)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 GQODBWLKUWYOFX-UHFFFAOYSA-N 0.000 claims description 26
- 235000012734 epicatechin Nutrition 0.000 claims description 26
- LPTRNLNOHUVQMS-UHFFFAOYSA-N epicatechin Natural products Cc1cc(O)cc2OC(C(O)Cc12)c1ccc(O)c(O)c1 LPTRNLNOHUVQMS-UHFFFAOYSA-N 0.000 claims description 26
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 26
- IZQSVPBOUDKVDZ-UHFFFAOYSA-N isorhamnetin Chemical compound C1=C(O)C(OC)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 IZQSVPBOUDKVDZ-UHFFFAOYSA-N 0.000 claims description 26
- 235000008800 isorhamnetin Nutrition 0.000 claims description 26
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims description 26
- 235000005493 rutin Nutrition 0.000 claims description 26
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims description 26
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims description 26
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 claims description 26
- OVSQVDMCBVZWGM-IDRAQACASA-N Hirsutrin Natural products O([C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1)C1=C(c2cc(O)c(O)cc2)Oc2c(c(O)cc(O)c2)C1=O OVSQVDMCBVZWGM-IDRAQACASA-N 0.000 claims description 23
- FVQOMEDMFUMIMO-UHFFFAOYSA-N Hyperosid Natural products OC1C(O)C(O)C(CO)OC1OC1C(=O)C2=C(O)C=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 FVQOMEDMFUMIMO-UHFFFAOYSA-N 0.000 claims description 23
- OVSQVDMCBVZWGM-QSOFNFLRSA-N quercetin 3-O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-QSOFNFLRSA-N 0.000 claims description 23
- OVSQVDMCBVZWGM-QCKGUQPXSA-N isoquercetin Natural products OC[C@@H]1O[C@@H](OC2=C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)[C@H](O)[C@@H](O)[C@@H]1O OVSQVDMCBVZWGM-QCKGUQPXSA-N 0.000 claims description 21
- 229960001285 quercetin Drugs 0.000 claims description 20
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 19
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 19
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 19
- 235000005875 quercetin Nutrition 0.000 claims description 19
- 150000002500 ions Chemical class 0.000 claims description 18
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 18
- 239000011550 stock solution Substances 0.000 claims description 17
- 239000012085 test solution Substances 0.000 claims description 17
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 16
- 238000005303 weighing Methods 0.000 claims description 16
- 238000001514 detection method Methods 0.000 claims description 15
- 239000012086 standard solution Substances 0.000 claims description 15
- 239000007864 aqueous solution Substances 0.000 claims description 14
- 238000004704 ultra performance liquid chromatography Methods 0.000 claims description 14
- 238000005259 measurement Methods 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 11
- 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 description 9
- 235000019253 formic acid Nutrition 0.000 claims description 9
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 claims description 6
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 6
- 239000005695 Ammonium acetate Substances 0.000 claims description 6
- 235000019257 ammonium acetate Nutrition 0.000 claims description 6
- 229940043376 ammonium acetate Drugs 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000003208 petroleum Substances 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- 239000012488 sample solution Substances 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 4
- 238000004451 qualitative analysis Methods 0.000 claims description 4
- 238000004445 quantitative analysis Methods 0.000 claims description 4
- OVSQVDMCBVZWGM-LQSBFMDOSA-N 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-3-[(2r,3s,4r,5r,6s)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxychromen-4-one Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OVSQVDMCBVZWGM-LQSBFMDOSA-N 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 230000010354 integration Effects 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 238000000889 atomisation Methods 0.000 claims description 2
- 238000004885 tandem mass spectrometry Methods 0.000 claims 2
- 239000000706 filtrate Substances 0.000 claims 1
- 238000011156 evaluation Methods 0.000 abstract description 23
- 229930003935 flavonoid Natural products 0.000 abstract description 9
- 150000002215 flavonoids Chemical class 0.000 abstract description 9
- 235000017173 flavonoids Nutrition 0.000 abstract description 9
- 238000000926 separation method Methods 0.000 abstract description 9
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000005516 engineering process Methods 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 6
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 239000007788 liquid Substances 0.000 abstract description 3
- 238000011002 quantification Methods 0.000 abstract description 3
- 238000005173 quadrupole mass spectroscopy Methods 0.000 abstract 1
- 239000012071 phase Substances 0.000 description 16
- 239000000523 sample Substances 0.000 description 14
- 238000010812 external standard method Methods 0.000 description 13
- 238000002552 multiple reaction monitoring Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- LUJAXSNNYBCFEE-UHFFFAOYSA-N Quercetin 3,7-dimethyl ether Natural products C=1C(OC)=CC(O)=C(C(C=2OC)=O)C=1OC=2C1=CC=C(O)C(O)=C1 LUJAXSNNYBCFEE-UHFFFAOYSA-N 0.000 description 7
- PUTDIROJWHRSJW-UHFFFAOYSA-N Quercitrin Natural products CC1OC(Oc2cc(cc(O)c2O)C3=CC(=O)c4c(O)cc(O)cc4O3)C(O)C(O)C1O PUTDIROJWHRSJW-UHFFFAOYSA-N 0.000 description 7
- OXGUCUVFOIWWQJ-XIMSSLRFSA-N acanthophorin B Natural products O[C@H]1[C@H](O)[C@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-XIMSSLRFSA-N 0.000 description 7
- 150000001875 compounds Chemical class 0.000 description 7
- OEKUVLQNKPXSOY-UHFFFAOYSA-N quercetin 3-O-beta-D-glucopyranosyl(1->3)-alpha-L-rhamnopyranosyl(1->6)-beta-d-galactopyranoside Natural products OC1C(O)C(C(O)C)OC1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OEKUVLQNKPXSOY-UHFFFAOYSA-N 0.000 description 7
- QPHXPNUXTNHJOF-UHFFFAOYSA-N quercetin-7-O-beta-L-rhamnopyranoside Natural products OC1C(O)C(O)C(C)OC1OC1=CC(O)=C2C(=O)C(O)=C(C=3C=C(O)C(O)=CC=3)OC2=C1 QPHXPNUXTNHJOF-UHFFFAOYSA-N 0.000 description 7
- OXGUCUVFOIWWQJ-HQBVPOQASA-N quercitrin Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1OC1=C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1=O OXGUCUVFOIWWQJ-HQBVPOQASA-N 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000011084 recovery Methods 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 241000196324 Embryophyta Species 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 239000012925 reference material Substances 0.000 description 3
- 239000012087 reference standard solution Substances 0.000 description 3
- 241001234745 Camelina Species 0.000 description 2
- 235000016401 Camelina Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- GXMWXESSGGEWEM-UHFFFAOYSA-N isoquercitrin Natural products OCC(O)C1OC(OC2C(Oc3cc(O)cc(O)c3C2=O)c4ccc(O)c(O)c4)C(O)C1O GXMWXESSGGEWEM-UHFFFAOYSA-N 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000219193 Brassicaceae Species 0.000 description 1
- 235000006526 Dillenia Nutrition 0.000 description 1
- 240000003604 Dillenia indica Species 0.000 description 1
- 235000006528 Dillenia indica Nutrition 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000218377 Magnoliaceae Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- -1 phytosterol Chemical class 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- 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
- G01N30/06—Preparation
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
- G01N30/8634—Peak quality criteria
-
- 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
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- 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)
- Engineering & Computer Science (AREA)
- Quality & Reliability (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for determining the content of 8 flavonoids and phenolic acid components in camelina sativa seeds, which is characterized in that by means of a separation analysis technology of an ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry detector and a diode array detector, and by means of a one-time multi-evaluation method, a catechin reference substance which is easy to obtain is used as an internal reference substance, and relative correction factors between the internal reference substance and other 7 components are calculated through the correction factors, so that the content of 8 flavonoids components in the camelina sativa seeds is determined. The method is simple to operate, high in sensitivity, accurate, efficient and low in cost, can quickly and accurately realize simultaneous quantification of multiple index components in the camelina sativa seeds, has good separation degree of 8 components, and lays a theoretical foundation for quality control of the camelina sativa seeds.
Description
Technical Field
The invention belongs to the technical field of chromatographic detection, and particularly relates to a detection method for simultaneously determining 8 flavone and phenolic acid components in camelina sativa seeds by using UPLC-MS-MS, so that a method for determining multiple scores is established.
Background
Camelina sativa (Camelina sativa L.) is an annual herbaceous plant, an ancient oil crop, belonging to magnoliaceae, subclass dillenia, order garrinae, brassicaceae, Camelina (Camelina). Early researches show that camelina sativa seeds contain rich total flavonoids, phytosterol, unsaturated fatty acid, various amino acids and trace elements, and are rich in fatty acid and various natural active ingredients which are necessary for human bodies, and researches show that the content of the flavonoid ingredients in the camelina sativa seeds is high. The camelina sativa seeds are used for squeezing more oil, the research on the degreased seeds is less, early researches show that the camelina sativa seeds contain flavonoid and phenolic acid components, the content of the flavonoid and the phenolic acid components is determined by a multi-evaluation method, the method for simultaneously determining the 8 flavonoid and phenolic acid components is not reported temporarily, and the practical application of multi-index component quality control is limited due to the fact that standard products are expensive.
The method for measuring the content of the multiple components can realize simultaneous quantification of the multiple index components by using a single reference substance which is easy to obtain and low in price and a relative correction factor, and can accurately identify and position a chromatographic peak to be measured by using a relative retention value; the method overcomes the problem of shortage of reference substances, namely only one representative component or components which do not exist in the sample and have similar performance are measured (easy to obtain, cheap, stable and effective), and simultaneously the content of other effective components to be measured can be calculated.
Therefore, the development of a quality control method (one-test-multiple-evaluation method) which is simple in operation, high in detection sensitivity, low in cost, accurate and efficient and can simultaneously and quantitatively analyze multiple active ingredients has important practical significance for quality control of camelina sativa seeds.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a detection method for simultaneously determining 8 flavone and phenolic acid components in camelina sativa seeds.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds comprises the following steps:
(1) preparation of Standard solutions of Mixed controls
Precisely weighing 8 reference substances including chlorogenic acid, catechin, epicatechin, sinapic acid, rutin, isoquercetin, quercetin and isorhamnetin, preparing single reference substance stock solution with methanol as solvent, mixing a certain amount of the single reference substance stock solution uniformly to obtain mixed reference substance standard solution; simultaneously diluting the catechin reference substance stock solution with methanol to obtain a catechin standard solution; refrigerating at 4 deg.C in dark for use;
(2) preparation of test solution
Weighing 10g of dried camelina sativa seed powder, performing reflux extraction for 6-10h by using 250mL of 150-one solvent, and volatilizing the solvent to obtain degreased camelina sativa seed powder; precisely weighing 1.0g of defatted camelina sativa seed powder, adding methanol water solution, weighing and recording as m, reflux-extracting for 1-3h, cooling, weighing again, adding methanol water solution to balance weight to m, shaking, filtering, and filtering with 0.22-0.45 μm filter membrane to obtain sample solution;
(3) calculation of relative correction factors
Measuring by using UPLC-MS-MS, respectively sampling 1 muL, 3 muL, 4 muL and 5 muL of the mixed reference standard solution prepared in the step (1) of sampling series concentration, obtaining UPLC, TIC and MRM graphs and carrying out peak area integration, respectively calculating relative correction factors fs/i of chlorogenic acid, epicatechin, sinapic acid, rutin, isoquercetin, quercitrin and isorhamnetin according to the formula (1) by using catechin in 8 components in the step (1) as an internal reference substance, and subsequently calculating the mass concentrations of 8 components by using the relative correction factors;
fs/i＝fs/fi＝(Wi×As)/(Ws×Ai) (1)
in the formula (1), As is the peak area of the internal reference substance, Ws is the mass concentration of the internal reference substance, and Ai is the peak area of the component i; wi is the mass concentration of the component i, and fs/i is the relative correction factor of the component i;
(4) determination of 8 components in test solution
Sampling the catechin standard solution prepared in the step (1) and the test solution prepared in the step (2), measuring by using UPLC-MS-MS to obtain UPLC, TIC and MRM graphs, integrating peak areas, and calculating according to a formula (2) to obtain the mass concentrations of chlorogenic acid, epicatechin, sinapic acid, rutin, isoquercetin, quercetin and isorhamnetin in the test solution;
Wi＝(fs/i×Ws×Ai)/As (2)
in the formula (2), As is the peak area of the internal reference substance, Ws is the mass concentration of the internal reference substance, and Ai is the peak area of the component i; fs/i is the relative correction factor for component i.
Specifically, the conditions for UPLC measurement in step (3) and step (4) are as follows: the chromatographic column is a reversed phase C18 chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, a formic acid aqueous solution containing 0.01-0.03 mol/L ammonium acetate is used as the mobile phase A, the volume concentration of the formic acid aqueous solution is 0.1%, acetonitrile is used as the mobile phase B, gradient elution is carried out, the flow rate is 0.4-0.6 mL/min, and the detection wavelength is 280nm +/-2 nm; the procedure for the gradient elution was:
specifically, the conditions for MS measurement in step (3) and step (4) are: the ion source is ESI, the detection mode is MRM multi-reaction monitoring, the scanning mode is that positive ions and negative ions are scanned simultaneously, the pressure of the atomization gas is 275.8-379.2 kPa, the temperature of the drying gas is 350 ℃, the flow rate of the drying gas is 10-12L/min, and the voltage of a capillary is 4.0 kV.
Specifically, the MS measurement conditions further include: ion pairs for quantitative analysis: chlorogenic acid 353.3 → 191.1, catechin 291.1 → 139.0, epicatechin 291.1 → 139.0, sinapinic acid 225.1 → 207.0, rutin 609.0 → 300.0, quercetin 449.1 → 303.0, isoquercetin 465.1 → 302.9, and isorhamnetin 317.2 → 301.9.
Specifically, the MS measurement conditions further include: ion pairs for qualitative analysis: chlorogenic acid 353.3 → 191.1; catechin 291.1 → 123.0, 139.0; epicatechin 291.1 → 123.0, 139.0; sinapic acid 225.1 → 207.0, 175.0; rutin 609.0 → 300.0, 301.0; quercetin 449.1 → 303.0, 71.1; isoquercetin 465.1 → 302.9, 274.0; isorhamnetin 317.2 → 301.9, 153.0.
Further preferably, the solvent in the step (2) is petroleum ether or n-hexane, so as to obtain a good degreasing effect.
Further preferably, the volume concentration of the methanol aqueous solution in the step (2) is 70-85%.
Further preferably, the refluxing time in the step (2) is preferably 1-1.5 h.
The invention provides a method for detecting the content of 8 components in camelina sativa seeds by a multi-evaluation method, wherein a catechin reference substance is used as an internal reference substance to establish the multi-evaluation method, and catechin widely exists in natural plants and has the effects of resisting bacteria, diminishing inflammation, preventing and treating viral diseases, resisting tumors, inhibiting obesity, resisting oxidation and the like. As can be seen, catechin is a natural plant active ingredient of research value, and therefore catechin is ultimately preferred as an internal standard.
Two of the 8 components measured by the invention have low content, the response sensitivity of ultraviolet detection is low, the components enter MS detection analysis through UPLC separation, the sensitivity is high, and by comparing the influence of different mobile phase systems on the separation effect, the formic acid aqueous solution containing ammonium acetate is preferably used as a water phase (mobile phase A), and acetonitrile is used as an organic phase (mobile phase B), so that the separation of the components is more suitable.
The separation efficiency of the compounds is greatly influenced by chromatographic columns of different brands, and finally, Eclipse Plus C18 chromatographic columns (100mm multiplied by 4.6mm,3.5 mu m) with higher analysis speed are ideally selected through a large number of experimental screening and an ultra-high performance liquid separation technology, and the flow speed of a mobile phase is screened through a large number of experiments. Researches show that the low flow rate is favorable for improving the ionization efficiency of the compound, improving the detection sensitivity, saving the solvent and reducing the cost, and the flow rate of 0.5mL/min is finally determined to be most suitable by comprehensive consideration.
Aiming at the fact that the flavonoid and phenolic acid components are one of main components contained in camelina sativa seeds, the method for simultaneously determining 8 representative flavonoid and phenolic acid component compounds with relatively high content in camelina sativa seeds is established by adopting a one-test-multiple-evaluation technology. Relative correction factors were calculated as quantitative ion peak areas and sample volumes. According to the structural characteristics of active ingredients and impurity ingredients in camelina sativa seeds, ultrahigh performance liquid chromatography and mass spectrometry analysis conditions are preferably selected through a large number of experiments, an easily obtained catechin reference substance is used as an internal reference substance to establish a one-test multi-evaluation method, relative correction factors of the reference substance and chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercetin, quercetin and isorhamnetin are measured, and the content of 8 ingredients is calculated. The method is simple to operate, high in sensitivity, accurate, efficient and low in cost, can objectively and accurately evaluate the quality of the camelina sativa seeds, is used for quality control, can solve the problem that the quality of the camelina sativa seeds cannot be objectively and reasonably controlled due to the lack of reference substances, and has important significance for controlling the quality and ensuring the curative effect.
The prior literature is consulted to find that the related technology for controlling the quality of the camelina sativa seeds is a planting technology mostly, and no related literature is available for jointly measuring the multi-component content of the camelina sativa seeds. According to the method, the UPLC-MS-MS is adopted to analyze the content of 8 components in camelina sativa seeds in different regions, and a multi-evaluation method is established, so that the quality of the camelina sativa seeds can be better controlled. According to the invention, through screening of a plurality of processes, a plurality of effective components in camelina sativa seeds are separated and detected, the separation degree of each peak is good, and the repeatability of the method is good. The content of camelina sativa seeds in 4 different regions is compared by an external standard method and a one-test-multiple evaluation method. The established one-test-multiple-evaluation method has high accuracy and can effectively characterize the quality of the camelina sativa seeds. Compared with the prior art, the method has the following beneficial effects:
1) the method adopts UPLC-MS-MS to simultaneously determine 8 components in the camelina sativa seeds for the first time;
2) for the first time, catechin is used as an internal reference substance, relative correction factors of other 7 compounds are determined, and a one-test multi-evaluation method is established to jointly determine the content of 8 components;
3) the method has good stability, reproducibility and the like.
Drawings
FIG. 1 is a UPLC chromatogram of a control of 8 compounds;
FIG. 2 is a TIC graph of a 8 compound control; peaks 1-8 in FIGS. 1 and 2 are, respectively: 1-chlorogenic acid, 2-catechin, 3-epicatechin, 4-sinapic acid, 5-rutin, 6-isoquercetin, 7-quercetin, 8-isorhamnetin;
FIG. 3 is a MRM chart of 8 compound controls.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
In the following examples, the apparatus used included: ME204 ten-thousandth electronic balance (shanghai mettler toledo), AUW220D ten-thousandth electronic balance (shimadzu, japan), Agilent QQQ 6460C-1290 ultra performance liquid chromatography tandem triple quadrupole mass spectrometer (Agilent technologies ltd., usa), pipette: 100 mu L, 200 mu L, 1000 mu L (Eppendorf), FW-80 high-speed universal pulverizer (Beijing Yongguang), HH-6 digital display constant temperature water bath (Changzhou Zhi Borui apparatus manufacturing Co., Ltd.).
Materials used in the following examples include: 4 batches of camelina sativa seeds (collected from Shaanxi, Qinghai, Gansu and Henan), methanol, petroleum ether, n-hexane and the like are all commercially analyzed, acetonitrile, ammonium acetate and formic acid are mass spectrometrically purified (Fisher Scientific), ultrapure water, control chlorogenic acid (batch No. 110753-.
Example 1
A method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds comprises the following steps:
(1) preparation of Standard solutions of Mixed controls
Precisely weighing 8 reference substances including chlorogenic acid, catechin, epicatechin, sinapic acid, rutin, quercetin, isoquercetin and isorhamnetin, dissolving and diluting with methanol to obtain stock solutions of the single reference substances; wherein the concentration of the chlorogenic acid reference stock solution is 92.4 mug/mL, the concentration of the catechin reference stock solution is 776.16 mug/mL, the concentration of the epicatechin reference stock solution is 40.50 mug/mL, the concentration of the sinapic acid reference stock solution is 42.40 mug/mL, the concentration of the rutin reference stock solution is 970.02 mug/mL, the concentration of the quercetin reference stock solution is 30.0 mug/mL, the concentration of the isoquercetin reference stock solution is 48.01 mug/mL, and the concentration of the isorhamnetin reference stock solution is 75.0 mug/mL; then taking a certain amount of each single reference substance stock solution and uniformly mixing to prepare a mixed reference substance standard solution; in the mixed reference standard solution, the concentration range of a chlorogenic acid reference substance is 0.31-9.24 mu g/mL, the concentration range of a catechin reference substance is 25.87-388.08 mu g/mL, the concentration range of an epicatechin reference substance is 0.68-20.25 mu g/mL, the concentration range of a sinapic acid reference substance is 0.28-8.48 mu g/mL, the concentration range of a rutin reference substance is 32.34-646.68 mu g/mL, the concentration range of an isoquercitrin reference substance is 0.10-6.0 mu g/mL, the concentration range of a quercitrin reference substance is 0.25-7.50 mu g/mL, and the concentration range of an isorhamnetin reference substance is 0.15-15.0 mu g/mL; meanwhile, diluting the catechin reference substance stock solution with methanol to obtain a catechin standard solution with the concentration of 25.87 mu g/mL; and (4) refrigerating at 4 ℃ in dark for later use.
(2) Preparation of test solution
Crushing the dried camelina sativa seeds by a crusher, sieving the crushed camelina sativa seeds with a No. 3 sieve (50 meshes), weighing 10g of camelina sativa seed powder, placing the powder into a Soxhlet extractor, adding 200mL of petroleum ether, performing reflux extraction for 8 hours, volatilizing the petroleum ether from the grease-removed powder to obtain degreased camelina sativa seed powder. Precisely weighing 1.0g of degreased camelina sativa seed powder, adding 50mL of 80% methanol aqueous solution, weighing and recording as m, reflux-extracting for 1h, cooling, weighing again, adding 80% methanol aqueous solution to make up the weight to m, shaking up, filtering, and filtering with 0.45 μm filter membrane to obtain the sample solution.
(3) Calculation of relative correction factors
And (2) measuring by using UPLC-MS-MS, carrying out sample injection on the mixed reference standard solution prepared in the step (1) of the series concentration, wherein the sample injection volumes are 1 muL, 3 muL, 4 muL and 5 muL respectively, obtaining UPLC, TIC and MRM graphs, carrying out peak area integration (shown in figures 1, 2 and 3), carrying out linear regression on 8 components by taking the mass concentration X of the reference solution as a horizontal coordinate and the peak area Y as a vertical coordinate, and obtaining a linear equation, a correlation coefficient and a detection limit shown in the following table 1. Selecting catechin as an internal reference substance, respectively calculating relative correction factors fs/i of chlorogenic acid, epicatechin, sinapic acid, rutin, isoquercetin, quercetin and isorhamnetin according to the following formula by combining peak area data,
fs/i＝fs/fi＝(Wi×As)/(Ws×Ai)
in the formula, As is the peak area of the internal reference, Ws is the mass concentration of the internal reference, Ai is the peak area of the component i, Wi is the mass concentration of the component i, and fs/i is the relative correction factor of the component i. In one embodiment of the present invention, the relative correction factors calculated by the one-test-multiple evaluation method are shown in table 2 below.
Table 18 linear equations, correlation coefficients and detection limits for the standards
TABLE 2 relative correction factor fs/i of catechin to chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercetin, quercitrin, and isorhamnetin
When UPLC-MS-MS (ultra performance liquid chromatography tandem triple quadrupole mass spectrometer) is adopted for measurement, the parameters are set as follows:
1) UPLC Condition
A chromatographic column: eclipse Plus C18 column (100 mm. times.4.6 mm,3.5 μm)
Flow rate: 0.5 mL/min; column temperature: 35 ℃; detection wavelength: 280nm +/-2 nm
Mobile phase: the method comprises the following steps of (1) preparing a mobile phase A and a mobile phase B, wherein a formic acid aqueous solution containing 0.03mol/L of ammonium acetate is used as the mobile phase A, the volume concentration of the formic acid aqueous solution is 0.1%, and acetonitrile is used as the mobile phase B, namely, a 0.1% formic acid aqueous solution (A) -acetonitrile (B) containing 0.03mol/L of ammonium acetate; performing gradient elution, wherein the procedure of the gradient elution is as follows:
2) MS Condition
An ion source: ESI, detection mode: multiple Reaction Monitoring (MRM), positive and negative ion mode scanning simultaneously; temperature of the drying gas: 350 ℃; atomizing gas pressure: 275.8 kPa; flow rate of drying gas: 10L/min; capillary voltage: 4.0 kV. Ion pairs for qualitative and quantitative analysis of 8 components of chlorogenic acid, catechin, epicatechin, sinapic acid, rutin, quercetin, isoquercetin and isorhamnetin detected by MRM method, residence time (millisecond/ms), collision energy and fragmentation voltage, and ionization mode are shown in the following table:
wherein, the ion pair for quantitative analysis: chlorogenic acid ([ M-H)]-)353.3 → 191.1 Catechin ([ M + H ]]+)291.1 → 139.0, epicatechin ([ M + H)]+)291.1 → 139.0, sinapic acid ([ M + H)]+)225.1 → 207.0, rutin ([ M-H)]-)609.0 → 300.0, Quercetin ([ M + H)]+)449.1 → 303.0, isoquercitrin ([ M + H)]+)465.1 → 302.9, isorhamnetin ([ M + H)]+)317.2 → 301.9. Ion pairs for qualitative analysis: chlorogenic acid 353.3 → 191.1; catechin 291.1 → 123.0, 139.0; epicatechin 291.1 → 123.0, 139.0; sinapic acid 225.1 → 207.0, 175.0; rutin 609.0 → 300.0, 301.0; quercetin 449.1 → 303.0, 71.1; isoquercetin 465.1 → 302.9, 274.0; isorhamnetin 317.2 → 301.9, 153.0.
A large number of tests and analyses show that [ M-H ] is easily obtained in the optimization process of chlorogenic acid and rutin]-Not easy to obtain [ M + H]+Ion peak, so negative ionization mode is selected, and catechin, epicatechin, sinapic acid, quercetin, isoquercetin and isorhamnetin are easily obtained [ M + H]+Ion peaks, so the positive ionization mode is selected.
(4) Determination of 8 component contents in test solution
External standard method (ESTD) measurement: and (3) taking the mixed reference substance standard solution prepared in the step (1) and the test solution prepared in the step (2), sampling 5 mu L of the mixed reference substance standard solution, measuring by using UPLC-MS-MS to obtain UPLC, TIC and MRM graphs, integrating peak areas, calculating the mass concentrations of catechin, chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercetin, quercetin and isorhamnetin according to a standard curve, and finally calculating the content (mu g/g) of 8 components in the sample.
One test multiple evaluation method (QAMS) assay: sampling 5 mu L of the catechin standard solution prepared in the step (1) and the test solution prepared in the step (2), measuring by using UPLC-MS-MS to obtain UPLC, TIC and MRM chromatograms, integrating peak areas, and calculating the mass concentrations of chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercetin, quercetin and isorhamnetin in the test solution according to the following formula.
Wi＝(fs/i×Ws×Ai)/As
In the formula, As is the peak area of the internal reference material, Ws is the mass concentration of the internal reference material, and Ai is the peak area of the component i; fs/i is the relative correction factor for component i.
Taking 4 camelina sativa seeds of different producing areas respectively, comparing with the one-test-multiple evaluation method (QAMS) of the above example 1 and the conventional external standard method, and determining the peak contents of 8 components of catechin, chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercitin, quercitrin and isorhamnetin in the camelina sativa seeds, wherein the test results are shown in tables 3 and 4. As a result, it was found that: compared with the content value calculated by the one-test-multiple-evaluation method of the invention, the content value measured by the conventional external standard method has no significant difference (P is more than 0.05) through t test, which indicates that the one-test-multiple-evaluation method of the invention can be used for the multi-component quality evaluation research of camelina sativa seeds.
TABLE 3 determination of 8 components (. mu.g/g) in camelina sativa seeds by external standard method (ESTD) and one-test-multiple evaluation method (QAMS)
TABLE 4 determination of 8 components (. mu.g/g) in camelina sativa seeds by external standard method (ESTD) and one-test-multiple evaluation method (QAMS)
Example 2 blank test
And (3) taking 5 mu L of 80% methanol, injecting the methanol into UPLC-MS-MS (ultra high performance liquid chromatography-tandem triple quadrupole mass spectrometer) for determination, and recording a chromatogram and a multi-reaction monitoring graph. The results show that: the solvent peak of 80% methanol aqueous solution is concentrated before 3min, and the detection is not interfered.
Example 3
A method for measuring the content of 8 components in camelina sativa seeds comprises the following steps of example 1, except that in the step (2), during preparation of a test solution, petroleum ether extraction is changed into n-hexane extraction, and the results are shown in tables 5 and 6. The experimental results show that: the change of extraction solvent did not constitute a significant difference to the measurement results.
TABLE 5 determination of 8 components (. mu.g/g) in camelina sativa seeds by external standard method (ESTD) and one-test-multiple evaluation method (QAMS)
TABLE 6 determination of 8 components (. mu.g/g) in camelina sativa seeds by external standard method (ESTD) and one-test-multiple evaluation method (QAMS)
Example 4 precision test
And (3) precision investigation, namely taking 5 mu L of mixed reference substance solution with one concentration, injecting the mixed reference substance solution into UPLC-MS-MS, continuously and repeatedly injecting samples for 6 times, recording peak areas of 8 components, and indicating that: the peak areas RSD of the catechin, the chlorogenic acid, the sinapic acid, the epicatechin, the rutin, the isoquercetin, the quercitrin and the isorhamnetin are respectively 0.95%, 1.10%, 1.05%, 1.27%, 1.14%, 1.47%, 1.59% and 1.78%, which indicates that the precision of the instrument is good.
EXAMPLE 5 repeatability test
The method comprises the steps of performing repeatability inspection, taking the same camelina sativa seed sample, preparing 6 parts of test solution in parallel, sequentially analyzing and measuring, measuring the contents of catechin, chlorogenic acid, sinapic acid, epicatechin, rutin, isoquercetin, quercitrin and isorhamnetin in the 6 parts of sample by referring to the one-measurement-multiple-evaluation method in example 1, and calculating the RSD contents of 8 index component samples to be 0.98%, 1.35%, 1.43%, 1.98%, 1.86%, 1.77%, 1.64% and 2.01% respectively, thereby indicating that the method has good repeatability.
Example 6 stability test
And (3) stability inspection, namely, taking the same test solution, and carrying out sample injection analysis for 0, 4, 8, 12 and 24 hours, wherein the peak areas RSD of the catechin, the chlorogenic acid, the sinapic acid, the epicatechin, the rutin, the isoquercitin, the quercitrin and the isorhamnetin are respectively 1.05%, 1.12%, 1.75%, 1.68%, 0.99%, 1.56%, 1.22% and 1.98%, and the stability of the test solution in 24 hours is proved to be good.
Example 7 recovery test
Sample adding recovery rate, precisely weighing 6 parts of camelina sativa seed sample with known content, wherein each part is about 0.50g, precisely adding single reference substance solutions with different concentrations respectively, preparing the sample solution according to the preparation method, precisely sucking 5 mu L of the sample solution, performing measurement according to the one-measurement and multi-evaluation method described in the example 1, performing sample injection analysis, calculating the sample adding recovery rate according to the following formula, and obtaining results shown in table 7, which indicates that the measurement method is good.
The sample recovery rate (%) was [ (/. measured amount/(original amount + added amount) ] × 100%
Table 7, sample recovery test results for 8 components (n ═ 6)
In summary, it can be seen that: the invention establishes an analysis method for the content of 8 components in camelina sativa seeds, adopts a UPLC-MS-MS liquid chromatography-mass spectrometry method to simultaneously determine the content of 8 components in camelina sativa seeds, has reliable method and good reproducibility, realizes the simultaneous quantification of multiple index components in camelina sativa seeds, has good separation degree of 8 index components, and lays a theoretical foundation for the quality control of camelina sativa seeds; the method takes catechin as an internal reference material, simultaneously measures the content of 8 components in the camelina sativa seeds by a one-measurement-multiple-evaluation method, has the advantages of simplicity, rapidness, accuracy, relatively stable chemical property of catechin, higher content, low price and easy obtainment, avoids quantitative obstruction caused by shortage of reference substances or high cost of the reference substances, and simultaneously measures 8 index components in the camelina sativa seeds by means of a single reference substance.
In order to meet the requirement of conciseness, each part of the specification focuses on the difference from other parts, and the same and similar parts among the parts can be mutually referred. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention.
Claims (8)
1. A method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds is characterized by comprising the following steps:
(1) preparation of Standard solutions of Mixed controls
Precisely weighing 8 reference substances including chlorogenic acid, catechin, epicatechin, sinapic acid, rutin, isoquercetin, quercetin and isorhamnetin, preparing single reference substance stock solution with methanol as solvent, mixing a certain amount of the single reference substance stock solution uniformly to obtain mixed reference substance standard solution; simultaneously diluting the catechin reference substance stock solution with methanol to obtain a catechin standard solution;
(2) preparation of test solution
Weighing 10g of dried camelina sativa seed powder, performing reflux extraction for 6-10h by using 250mL of 150-one solvent, and volatilizing the solvent to obtain degreased camelina sativa seed powder; precisely weighing 1.0g of degreased camelina sativa seed powder, adding a methanol aqueous solution, weighing and recording as m, performing reflux extraction for 1-3h, cooling, weighing again, complementing the weight to m with the methanol aqueous solution, shaking up, filtering, and filtering the filtrate with a 0.22-0.45 mu m filter membrane to obtain a sample solution;
(3) calculation of relative correction factors
Measuring by using UPLC-MS-MS, sampling a standard solution of the mixed reference substance prepared in the step (1) of the series concentration, sampling 1 muL, 3 muL, 4 muL and 5 muL respectively, obtaining UPLC, TIC and MRM graphs and carrying out peak area integration, taking catechin in 8 components in the step (1) as an internal reference substance, and calculating relative correction factors of chlorogenic acid, epicatechin, sinapic acid, rutin, isoquercitin, quercetin and isorhamnetin according to the formula (1)fs/i；
fs/i＝fs/fi＝(Wi×As)/(Ws×Ai) （1）
In the formula (1), As is the peak area of the internal reference substance, Ws is the mass concentration of the internal reference substance, and Ai is the peak area of the component i; wi is the mass concentration of the component i;
(4) determination of 8 components in test solution
Sampling the catechin standard solution prepared in the step (1) and the test solution prepared in the step (2), measuring by using UPLC-MS-MS to obtain UPLC, TIC and MRM graphs, integrating peak areas, and calculating according to a formula (2) to obtain the mass concentrations of chlorogenic acid, epicatechin, sinapic acid, rutin, isoquercetin, quercetin and isorhamnetin in the test solution;
Wi＝( fs/i×Ws×Ai) / As （2）
in the formula (2), As is the peak area of the internal reference substance, Ws is the mass concentration of the internal reference substance, and Ai is the peak area of the component i;fs/i is the relative correction factor for component i.
2. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds according to claim 1, wherein the conditions for UPLC determination in the steps (3) and (4) are as follows: the chromatographic column is a reversed phase C18 chromatographic column, the mobile phase consists of a mobile phase A and a mobile phase B, a formic acid aqueous solution containing 0.01-0.03 mol/L ammonium acetate is used as the mobile phase A, the volume concentration of the formic acid aqueous solution is 0.1%, acetonitrile is used as the mobile phase B, gradient elution is carried out, the flow rate is 0.4-0.6 mL/min, and the detection wavelength is 280nm +/-2 nm; the procedure for the gradient elution was:
3. the method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds according to claim 1 or 2, wherein the conditions for MS measurement in the steps (3) and (4) are as follows: the ion source is ESI, the detection mode is MRM multi-reaction monitoring, the scanning mode is that positive ions and negative ions are scanned simultaneously, the pressure of the atomization gas is 275.8-379.2 kPa, the temperature of the drying gas is 350 ℃, the flow rate of the drying gas is 10-12L/min, and the voltage of a capillary is 4.0 kV.
4. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seed according to claim 3, wherein the MS measurement conditions further comprise: ion pairs for quantitative analysis: chlorogenic acid 353.3 → 191.1, catechin 291.1 → 139.0, epicatechin 291.1 → 139.0, sinapinic acid 225.1 → 207.0, rutin 609.0 → 300.0, quercetin 449.1 → 303.0, isoquercetin 465.1 → 302.9, and isorhamnetin 317.2 → 301.9.
5. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seed according to claim 3, wherein the MS measurement conditions further comprise: ion pairs for qualitative analysis: chlorogenic acid 353.3 → 191.1; catechin 291.1 → 123.0, 139.0; epicatechin 291.1 → 123.0, 139.0; sinapic acid 225.1 → 207.0, 175.0; rutin 609.0 → 300.0, 301.0; quercetin 449.1 → 303.0, 71.1; isoquercetin 465.1 → 302.9, 274.0; isorhamnetin 317.2 → 301.9, 153.0.
6. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds according to claim 1, which is characterized in that: in the step (2), the solvent is petroleum ether or n-hexane.
7. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds according to claim 1, which is characterized in that: in the step (2), the volume concentration of the methanol water solution is 70-85%.
8. The method for detecting the content of 8 flavone and phenolic acid components in camelina sativa seeds according to claim 1, which is characterized in that: in the step (2), the reflux time is 1-1.5 h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110405210.9A CN113138238A (en) | 2021-04-15 | 2021-04-15 | Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110405210.9A CN113138238A (en) | 2021-04-15 | 2021-04-15 | Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113138238A true CN113138238A (en) | 2021-07-20 |
Family
ID=76813100
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110405210.9A Pending CN113138238A (en) | 2021-04-15 | 2021-04-15 | Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113138238A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252509A (en) * | 2022-09-22 | 2022-11-01 | 河南省纳普生物技术有限公司 | Camelina sativa extract used as raw material of whitening antibacterial cosmetics and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103163260A (en) * | 2011-12-16 | 2013-06-19 | 成都康弘制药有限公司 | Fingerprints of drugs and determination method thereof |
| CN104614479A (en) * | 2015-01-30 | 2015-05-13 | 北京市营养源研究所 | Detection method of vitamin in food |
| CN106153800A (en) * | 2016-06-22 | 2016-11-23 | 中国农业科学院生物技术研究所 | The HPLC MS/MS detection method of transgenic corns secondary metabolites |
| WO2017148426A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining fingerprint of chinese medicine composition |
| WO2017148418A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining component contents of chinese medicine composition |
| CN111103365A (en) * | 2019-12-03 | 2020-05-05 | 山西大学 | Method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on MRM-IDA-EPI mode |
| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
-
2021
- 2021-04-15 CN CN202110405210.9A patent/CN113138238A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103163260A (en) * | 2011-12-16 | 2013-06-19 | 成都康弘制药有限公司 | Fingerprints of drugs and determination method thereof |
| CN104614479A (en) * | 2015-01-30 | 2015-05-13 | 北京市营养源研究所 | Detection method of vitamin in food |
| WO2017148426A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining fingerprint of chinese medicine composition |
| WO2017148418A1 (en) * | 2016-03-03 | 2017-09-08 | 石家庄以岭药业股份有限公司 | Method for determining component contents of chinese medicine composition |
| CN106153800A (en) * | 2016-06-22 | 2016-11-23 | 中国农业科学院生物技术研究所 | The HPLC MS/MS detection method of transgenic corns secondary metabolites |
| CN111103365A (en) * | 2019-12-03 | 2020-05-05 | 山西大学 | Method for simultaneously, qualitatively and quantitatively analyzing 6 components in total flavonoids of wild jujube leaves based on MRM-IDA-EPI mode |
| CN111579701A (en) * | 2020-05-19 | 2020-08-25 | 河南科高中标检测技术有限公司 | Standard fingerprint spectrum establishment method for camelina sativa seeds and method for detecting rutin content |
Non-Patent Citations (5)
| Title |
|---|
| MD. JIAUR RAHMAN: "Phenolic profiles and antioxidant activity of defatted camelina and sophia seeds", 《FOOD CHEMISTRY》 * |
| 刘文静: "超高效液相色谱-串联质谱法同时分析紫薯中15种酚酸类组分", 《福建农业学报》 * |
| 吴有根: "一测多评法测定冠心丹参颗粒中4种成分的含量", 《中国医院药学杂志》 * |
| 姚彤: "HPLC-MS/MS法同时测定毛樱桃叶中7种黄酮类成分的含量", 《沈阳药科大学学报》 * |
| 梁生旺: "《中药制剂分析》", 30 April 2013, 中国中医药出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115252509A (en) * | 2022-09-22 | 2022-11-01 | 河南省纳普生物技术有限公司 | Camelina sativa extract used as raw material of whitening antibacterial cosmetics and preparation method and application thereof |
| CN115252509B (en) * | 2022-09-22 | 2023-06-23 | 河南省纳普生物技术有限公司 | Camelina sativa extract used as raw material of whitening antibacterial cosmetics, and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Li et al. | Simultaneous determination of clenbuterol, salbutamol and ractopamine in milk by reversed-phase liquid chromatography tandem mass spectrometry with isotope dilution | |
| Xu et al. | Rapid determination of 1-deoxynojirimycin in Morus alba L. leaves by direct analysis in real time (DART) mass spectrometry | |
| Liu et al. | Rapid analysis of 27 components of Isodon serra by LC–ESI-MS–MS | |
| Jin et al. | Simultaneous quantification of 19 diterpenoids in Isodon amethystoides by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry | |
| CN106153800B (en) | The HPLC-MS/MS detection methods of transgenic corns secondary metabolites | |
| CN114200038A (en) | Method for detecting compound content in wrinkled gianthyssop herb zhengqi oral liquid by liquid chromatography-mass spectrometry | |
| Meng et al. | Direct analysis in real time coupled with quadrupole-Orbitrap high-resolution mass spectrometry for rapid analysis of pyrethroid preservatives in wooden food contact materials | |
| Liu et al. | Simultaneous determination of pimpinellin, isopimpinellin and phellopterin in rat plasma by a validated UPLC–MS/MS and its application to a pharmacokinetic study after administration of Toddalia asiatica extract | |
| CN113138238A (en) | Method for detecting content of 8 flavone and phenolic acid components in camelina sativa seeds | |
| CN112114079B (en) | Method for simultaneously detecting 9 chemical components in quisqualis indica | |
| CN113156034B (en) | Method for rapidly detecting various coffee flavor substances | |
| Pan et al. | Development and validation of a UPLC-MS/MS method for the simultaneous determination and detection of four neuritogenic compounds in different parts of Gentiana rigescens Franch using multiple reaction monitoring and precursor ion scanning | |
| Shi et al. | Simultaneous determination of eight components in Radix Tinosporae by high-performance liquid chromatography coupled with diode array detector and electrospray tandem mass spectrometry | |
| CN111595983A (en) | Method for measuring chemical component content in veratrum nigrum | |
| CN111638287A (en) | Quantitative detection method for detecting multiple organic acids in human body dry urine filter paper sheet | |
| CN108132309B (en) | Analysis method for determining residual quantity of thiediazole copper in dendrobium officinale | |
| CN114113403B (en) | Method for determining liquid chromatography-mass spectrometry (LC-MS) of Hedan tablets | |
| CN114646690B (en) | Detection method for chemical components in immature bitter orange, longstamen onion bulb and cassia twig decoction and fingerprint establishment method | |
| Li et al. | Chemical profiling and quantification analysis of flavonoids in different varieties of Euryales Semen by ultra‐high‐performance liquid chromatography with tandem mass spectrometry | |
| CN113933435A (en) | Ultra-high performance liquid chromatography tandem mass spectrometry method for rapidly detecting residual quantity of bifenazate and metabolites thereof in citrus | |
| CN109187780B (en) | Detection method of compound motherwort granules | |
| CN107632082B (en) | Method for measuring alkaloid components in zanthoxylum armatum medicinal material | |
| CN111896636A (en) | Liquid chromatography tandem mass spectrometry detection method for emodin and aloe-emodin in mouse urine | |
| CN115166085B (en) | Method for determining ajoene in soft capsule health food | |
| CN115326981B (en) | Method for measuring content of various components in garden balsam medicinal material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210720 |