CN105181829A - Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives - Google Patents
Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives Download PDFInfo
- Publication number
- CN105181829A CN105181829A CN201510527402.1A CN201510527402A CN105181829A CN 105181829 A CN105181829 A CN 105181829A CN 201510527402 A CN201510527402 A CN 201510527402A CN 105181829 A CN105181829 A CN 105181829A
- Authority
- CN
- China
- Prior art keywords
- folic acid
- acid
- formyl
- tetrahydrofolic
- folic
- 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.)
- Granted
Links
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 title claims abstract description 297
- 239000011724 folic acid Substances 0.000 title claims abstract description 165
- 235000019152 folic acid Nutrition 0.000 title claims abstract description 159
- 229960000304 folic acid Drugs 0.000 title claims abstract description 140
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 title claims abstract description 139
- 150000002224 folic acids Chemical class 0.000 title claims abstract description 82
- 230000001360 synchronised effect Effects 0.000 title claims abstract description 10
- 238000004445 quantitative analysis Methods 0.000 title abstract 2
- 238000000034 method Methods 0.000 claims abstract description 81
- 239000000284 extract Substances 0.000 claims abstract description 33
- 102100021023 Gamma-glutamyl hydrolase Human genes 0.000 claims abstract description 32
- 108010062699 gamma-Glutamyl Hydrolase Proteins 0.000 claims abstract description 32
- ZNOVTXRBGFNYRX-ABLWVSNPSA-N levomefolic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-ABLWVSNPSA-N 0.000 claims abstract description 31
- MSTNYGQPCMXVAQ-KIYNQFGBSA-N 5,6,7,8-tetrahydrofolic acid Chemical compound N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 MSTNYGQPCMXVAQ-KIYNQFGBSA-N 0.000 claims abstract description 25
- AUFGTPPARQZWDO-YPMHNXCESA-N 10-formyltetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1)N)N(C=O)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 AUFGTPPARQZWDO-YPMHNXCESA-N 0.000 claims abstract description 24
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 claims abstract description 24
- UGWUWNVTCLDEOG-ZDUSSCGKSA-N (2s)-2-[[4-[(2-amino-4-oxo-1h-pteridin-6-yl)methyl-formylamino]benzoyl]amino]pentanedioic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CN(C=O)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 UGWUWNVTCLDEOG-ZDUSSCGKSA-N 0.000 claims abstract description 20
- 210000000496 pancreas Anatomy 0.000 claims abstract description 16
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 15
- 210000002966 serum Anatomy 0.000 claims abstract description 14
- 239000011578 levomefolic acid Substances 0.000 claims description 33
- 235000007635 levomefolic acid Nutrition 0.000 claims description 32
- 238000001514 detection method Methods 0.000 claims description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 21
- 229940014144 folate Drugs 0.000 claims description 20
- 241001597008 Nomeidae Species 0.000 claims description 18
- 239000008363 phosphate buffer Substances 0.000 claims description 17
- 241000287828 Gallus gallus Species 0.000 claims description 15
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 12
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000001819 mass spectrum Methods 0.000 claims description 11
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 claims description 11
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 claims description 10
- 238000005259 measurement Methods 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 8
- WQABCVAJNWAXTE-UHFFFAOYSA-N dimercaprol Chemical compound OCC(S)CS WQABCVAJNWAXTE-UHFFFAOYSA-N 0.000 claims description 8
- 230000008569 process Effects 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000010812 external standard method Methods 0.000 claims description 7
- 238000004949 mass spectrometry Methods 0.000 claims description 7
- 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 6
- 229910052786 argon Inorganic materials 0.000 claims description 6
- 239000003480 eluent Substances 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 6
- 235000019253 formic acid Nutrition 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000007921 spray Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 229960001051 dimercaprol Drugs 0.000 claims description 4
- 150000002500 ions Chemical class 0.000 claims description 4
- CIWBSHSKHKDKBQ-DUZGATOHSA-N D-araboascorbic acid Natural products OC[C@@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-DUZGATOHSA-N 0.000 claims description 3
- 230000004913 activation Effects 0.000 claims description 3
- 238000007405 data analysis Methods 0.000 claims description 3
- 229920000831 ionic polymer Polymers 0.000 claims description 3
- 238000002552 multiple reaction monitoring Methods 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 2
- 125000003929 folic acid group Chemical group 0.000 claims description 2
- 238000005457 optimization Methods 0.000 claims description 2
- 239000000047 product Substances 0.000 claims description 2
- 239000007788 liquid Substances 0.000 abstract description 6
- 238000000132 electrospray ionisation Methods 0.000 abstract 2
- QYNUQALWYRSVHF-OLZOCXBDSA-N (6R)-5,10-methylenetetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1C1)N)N1C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QYNUQALWYRSVHF-OLZOCXBDSA-N 0.000 abstract 1
- 229940105150 5-methyltetrahydrofolic acid Drugs 0.000 abstract 1
- UEZVMMHDMIWARA-UHFFFAOYSA-N Metaphosphoric acid Chemical compound OP(=O)=O UEZVMMHDMIWARA-UHFFFAOYSA-N 0.000 abstract 1
- 239000000872 buffer Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 54
- 241000196324 Embryophyta Species 0.000 description 51
- 244000221633 Brassica rapa subsp chinensis Species 0.000 description 26
- 235000010149 Brassica rapa subsp chinensis Nutrition 0.000 description 26
- 238000012360 testing method Methods 0.000 description 21
- 239000000243 solution Substances 0.000 description 15
- 239000003643 water by type Substances 0.000 description 15
- 239000013558 reference substance Substances 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 13
- 238000011084 recovery Methods 0.000 description 11
- 235000013311 vegetables Nutrition 0.000 description 10
- 230000035945 sensitivity Effects 0.000 description 9
- 235000000318 Bindesalat Nutrition 0.000 description 7
- 244000106835 Bindesalat Species 0.000 description 7
- 241000209094 Oryza Species 0.000 description 7
- 235000007164 Oryza sativa Nutrition 0.000 description 7
- 241000219315 Spinacia Species 0.000 description 7
- 235000009337 Spinacia oleracea Nutrition 0.000 description 7
- 235000009566 rice Nutrition 0.000 description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- 238000004760 accelerator mass spectrometry Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000012925 reference material Substances 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- -1 5-MTHF sodium salt Chemical class 0.000 description 2
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000021615 conjugation Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- VLKFYBLZIAZYPW-MMFRDWCLSA-M sodium (4S)-4-[[4-[(2-amino-4-oxo-5,6,7,8-tetrahydro-3H-pteridin-6-yl)methylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound [Na+].N1C=2C(=O)NC(N)=NC=2NCC1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 VLKFYBLZIAZYPW-MMFRDWCLSA-M 0.000 description 2
- UDDUKFYJPZYIPC-ZEDZUCNESA-M sodium folinate Chemical compound [Na+].C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C(O)=O)C=C1 UDDUKFYJPZYIPC-ZEDZUCNESA-M 0.000 description 2
- CIWBSHSKHKDKBQ-SZSCBOSDSA-N 2-[(1s)-1,2-dihydroxyethyl]-3,4-dihydroxy-2h-furan-5-one Chemical compound OC[C@H](O)C1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-SZSCBOSDSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000282894 Sus scrofa domesticus Species 0.000 description 1
- OMOVVBIIQSXZSZ-UHFFFAOYSA-N [6-(4-acetyloxy-5,9a-dimethyl-2,7-dioxo-4,5a,6,9-tetrahydro-3h-pyrano[3,4-b]oxepin-5-yl)-5-formyloxy-3-(furan-3-yl)-3a-methyl-7-methylidene-1a,2,3,4,5,6-hexahydroindeno[1,7a-b]oxiren-4-yl] 2-hydroxy-3-methylpentanoate Chemical compound CC12C(OC(=O)C(O)C(C)CC)C(OC=O)C(C3(C)C(CC(=O)OC4(C)COC(=O)CC43)OC(C)=O)C(=C)C32OC3CC1C=1C=COC=1 OMOVVBIIQSXZSZ-UHFFFAOYSA-N 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 208000007502 anemia Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000007698 birth defect Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004401 flow injection analysis Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 1
- 150000002306 glutamic acid derivatives Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000002652 macrocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 201000010193 neural tube defect Diseases 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- CPNGPNLZQNNVQM-UHFFFAOYSA-N pteridine Chemical compound N1=CN=CC2=NC=CN=C21 CPNGPNLZQNNVQM-UHFFFAOYSA-N 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- IFBHEXSRPLJLIG-ZOWNYOTGSA-M sodium (4S)-4-[[4-[(2-amino-4-oxo-3H-pteridin-6-yl)methyl-formylamino]benzoyl]amino]-5-hydroxy-5-oxopentanoate Chemical compound [Na+].C(=O)N(C1=CC=C(C(N[C@@H](CCC(=O)[O-])C(=O)O)=O)C=C1)CC1=CN=C2N=C(N)NC(=O)C2=N1 IFBHEXSRPLJLIG-ZOWNYOTGSA-M 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229960002098 sodium folate Drugs 0.000 description 1
- 230000005477 standard model Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 208000035581 susceptibility to neural tube defects Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000007669 thermal treatment Methods 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000004704 ultra 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
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
A disclosed rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives successively comprises the following steps: 1) extracting folic acid in plant leaf by using a 0.05 M phosphoric acid buffer; 2) adding mouse serum conjugase and chick pancreas conjugase into the folic acid extract of the step 1); 3) purifying the deconjugation extract by using an ultrafiltration centrifuge tube; 4) separating and determining tetrahydrofolic acid, 5-methyl-tetrahydrofolic acid, 5-formyl-tetrahydrofolic acid, 5,10-methylenetetrahydrofolic acid, 10-formyl-folic acid, 10-formyl- tetrahydrofolic acid, and folic acid by using high performance liquid chromatography-tandem quadrupole mass spectrometer for normal-voltage electrospray ionization (ESI); and 5) performing quantitative determination on folic acid. The method is capable of performing rapid sensitive synchronous batch determination on low-folic-acid-content plant leaf to obtain the total folic acid content and the content of seven folic acid derivates containing folic acid.
Description
Technical field
The present invention relates to Ultra Performance Liquid Chromatography-tandem mass spectrum (UPLC-MS/MS) fast and high sensitivity synchronously detects the method for the total folic acid of plant leaf blade and seven kinds of folic acid derivatives content.
Background technology
Folic acid is a kind of water-soluble B vitamin with important nutritive value, and refer to tetrahydrofolic acid and this class material of derivant thereof, it is made up of pteridine, p-aminobenzoic acid and paddy acyl chain.As a carbon donor, the synthesis of folic acid participation purine, pyrimidine and amino acid whose mutual conversion.Human body lacks folic acid can cause a series of chronic disease, such as neural tube defects, birth defect, macrocyte anaemia, angiocardiopathy and cancer etc.But the mankind and mammal can not synthesize folic acid voluntarily, can only be supplemented by food.But, the folate content that people are taken in by diet is lower than health standards (400 μ g every day), therefore, global many countries are all being devoted to the folate content being increased plant by biological reinforced means, thus make the mankind take in more folic acid by diet.Vegetables are the main meals of the mankind, the biological reinforced of vegetables folic acid is also the whole world one of study hotspot at present, but the content of vegetables Folic Acid is very low, and the derivant containing multiple instability, therefore sets up a kind of high sensitivity, the efficiently and stably method of detection plant leaf blade Folic Acid very necessary.
Because plant Folic Acid has many analogues, and unstable, and the folate content therefore analyzing plant leaf blade is a kind of challenge, and the leaching process of folic acid is crucial.Because folic acid is all very responsive to light, oxygenant and pH, therefore the pH of damping fluid and thermal treatment all can have influence on the stability of folic acid.Crop Folic Acid mainly exists with the form of many glutamates, even if exist with single glutamate form, is also often that multiple derivant coexists, therefore needs to reach conjugation with folic acid conjugase.Folic acid conjugase can, with the serum of the mankind or rat, Ren sus domestica and chicken pancreas etc., select suitable folic acid conjugase could give full play to conjugation in leaching process.
The method of current mensuration folic acid has microbial method, but it can only measure total folic acid, can not distinguish folic acid derivatives, and experimental implementation is loaded down with trivial details time-consuming.The present folic acid also having in the method mensuration plant and food getting more and more and use gas chromatography or liquid chromatography, but gas chromatography can not distinguish different folic acid derivatives.The physiological function of different folic acid derivatives is different, therefore effectively distinguishes that they are extremely important.Liquid chromatography can effectively distinguish multi-form folic acid derivatives, but current more report utilizes efficient liquid phase UV-detector method to detect the sample that composition is single, folate content is high, be not suitable for detecting the low sample of folate content, and, this kind of method is longer for detection time, if batch detection, folic acid is easily degraded in testing process, therefore can only detect a small amount of sample.So, set up stable extract plant leaf blade rapidly folic acid and fast, the method that efficient, high sensitivity, batch synchronization detect plant leaf blade Folic Acid and different folic acid derivatives content thereof is significant to the relevant area research of plant biological strengthening.
The invention of 2011100064527 method of total folic acid and derivative content thereof " in the synchronous quantitative measurement vegetables " also exists that folic acid sample preparation time is longer, Measuring Time long, synchronously can only detect the technological deficiency of folic acid and four kinds of folic acid derivatives.And this method folic acid sample preparation time is short, detection time only needs 5 minutes, synchronously quantitatively can detect folic acid and seven kinds of folic acid derivatives.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of method of Ultra Performance Liquid Chromatography-tandem mass spectrum (UPLC-MS/MS) fast high-sensitive degree total folic acid of Simultaneous Determination plant leaf blade and derivative content thereof.Apply this kind of method and can measure the content of total folic acid and multiple folic acid derivatives in plant leaf blade by Fast synchronization.
In order to solve the problems of the technologies described above, the invention provides the method that fast high-sensitive synchronously quantitatively detects total folic acid and derivative content thereof in vegetables, comprising the following steps successively:
1) folic acid in plant leaf blade and folic acid derivatives, is extracted:
Utilize 0.05M phosphate buffer to boil and extract the folic acid of plant leaf blade, cooling and centrifugal after, obtain supernatant, supernatant is folic acid extract (comprising folic acid and folic acid derivatives);
2), two kinds of folic acid conjugases are added
Toward step 1) in folic acid extract add mouse serum conjugase and chicken pancreas conjugase, hatch 2 hours for 37 DEG C after adding argon gas, 100 DEG C of water-baths stop enzyme for 5 minutes and live, and get supernatant after centrifugal, and supernatant is that extract is closed in folic acid outspan;
3) outspan of ultra-filtration centrifuge tube purifying plant leaf blade folic acid, is used to close extract
Use 10kDa500 μ L ultra-filtration centrifuge tube (Millipore, the U.S.), after activation, add 500 μ L steps 2 inward) folic acid outspan close extract, 12000g4 DEG C centrifugal 15 minutes, obtains the folic acid refined solution after purifying (being the solution of gained after post);
4), Ultra Performance Liquid Chromatography mass spectrometry is separated folic acid derivatives mass spectrometry method
Ultra Performance Liquid Chromatography mass spectrometry detection system is quadrupole rod tandem mass spectrum (WatersXevoTQ-S, Waters, the U.S.) normal pressure electron spray ionisation (ESI) positive pole pattern.WatersIntelliStartTechnology is by polyion reaction monitoring (multiplereactionmonitoring, MRM) that is automatic and the manually all folic acid derivatives of optimization.Each folic acid derivatives uses two or three daughter ions to determine, result is as table 1.MassLynx4.1 (Waters, the U.S.) software is used for system cloud gray model, data acquisition and data analysis.
The multiion reaction monitoring of table 1 folic acid and folic acid derivatives and compound index
5), Ultra Performance Liquid Chromatography mass spectrometry is separated folic acid derivatives chromatographic process
Use WatersACQUITYUPLC system (Waters, the U.S.) to carry out the chromatographic resolution of folic acid derivatives, operating software is TargetLynx4.1 (Waters, the U.S.).Column oven maintains 40 DEG C, and automatic sampler maintains 4 DEG C.Chromatographic column is ACQUITYUPLCBEH, C
18column, specification is 2.1mm × 50mm, and particle diameter 1.7 μm (Waters, the U.S.) connects a VanGuard pre-column C18, and specification is 5mm × 2.1mm, particle diameter 1.8 μm (Waters, the U.S.).Flow velocity 0.4mL/min; Sample size 2 μ L, 5 minutes working times.Mobile phase is eluent A (0.1% formic acid is water-soluble) and eluent B (0.1% formic acid is dissolved in acetonitrile) gradient elution, refers to table 2.Syringe needle cleaning solvent is acetonitrile/water mixed liquor (50/50, v/v);
The best gradient elution of table 2UPLC-MS/MS
6), the quantitative measurement of plant leaf blade folic acid derivatives.
As the efficient highly sensitive synchronous improvement quantitatively detecting the method for the total folic acid of plant leaf blade and derivant thereof of the present invention: step 1) in 0.05M phosphate buffer density be often liter of phosphate buffer adding 10gL (+)-ascorbic acid and 10ml dimercaprol dimercaptopropanol.
Further improvements in methods as the present invention's total folic acid of efficient highly sensitive synchronous quantitative measurment plant leaf blade and derivant thereof: described step 2) in every 10mL folic acid extract, add 2mL chicken pancreas conjugase, after shaking up, draw 3mL solution in 50mL centrifuge tube, add 100 μ L mouse serum conjugases, shake up, be filled with argon gas, hatch 2 hours for 37 DEG C, 100 DEG C of water-baths stop enzyme for 5 minutes and live.
As the efficient highly sensitive synchronous further improvements in methods quantitatively detecting the total folic acid of plant leaf blade and derivant thereof of the present invention: described step 6) be: according to step 4) and step 5) condition, quantitative measurement is carried out to plant leaf blade folic acid and derivant thereof, record main peak peak area, automatically tetrahydrofolic acid is calculated with peak area according to external standard method, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5, 10-anhydroleucovorin, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the content of folic acid, again according to tetrahydrofolic acid, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5, 10-anhydroleucovorin, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the concentration of folic acid and relative molecular mass calculate total folate content.
Method of the present invention comprises following: (A) provides a kind of phosphate buffer to boil the method extracting plant leaf blade Folic Acid; (B) method that two kinds of folic acid conjugases are used in combination is provided; (C) a kind of method of ultra-filtration centrifuge tube purifying plant leaf blade folic acid extract is provided; (D) a kind of detection system is provided to be the method for various folic acid derivatives in the mass spectrum separating plant blade of quadrupole rod tandem mass spectrum (WatersXevoTQ-S, Waters, the U.S.) normal pressure electron spray ionisation (ESI) positive pole pattern; (E) method of various folic acid derivatives in a kind of chromatographic column separating plant blade is provided; (F) method of different folic acid derivatives in a kind of Ultra Performance Liquid Chromatography-tandem mass spectrum efficient high sensitivity Simultaneous Determination plant leaf blade is provided.
Different folic acid and derivative content assay method thereof in plant leaf blade of the present invention, have following characteristics:
(1) add mouse serum conjugase and chicken pancreas conjugase, the Oxidative demage of prevention folic acid simultaneously, improve outspan and close efficiency.
(2) in the present invention, use specification 500 μ L10kDa ultra-filtration centrifuge tube purifying folic acid extract, hydro-extractor once can centrifugal 30 samples, can effective impurity screening, greatly accelerate extraction rate again, reduce folic acid and the degraded of derivant in leaching process thereof.
(3) ACQUITYUPLCBEH, C
18column chromatographic column is in conjunction with Ultra Performance Liquid Chromatography quadrupole rod tandem mass spectrum (UPLC-MS/MS) (WatersXevoTQ-S, Waters, the U.S.) can be separated and quantitative measurement plant leaf blade seven kinds of folic acid derivatives in conjunction with normal pressure electron spray ionisation (ESI) positive pole pattern simultaneously, and only need within 5 minutes, can detect complete, sample size only needs 2 μ L, detection method efficient and sensible, can batch detection sample.
Different folic acid and derivative content assay method thereof in plant leaf blade of the present invention, have following beneficial effect:
(1) the present invention is highly sensitive, rapidly and efficiently, can detect complicated component, the folic acid of the plant leaf blade of low folate content and the mensuration of derivative content thereof by batch synchronization.
(2) the present invention can seven kinds of folic acid derivatives in separating plant blade, and can the total folic acid of quantitative test and seven kinds of folic acid derivatives content.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, this method embodiment is described in further detail.
Fig. 1 is the structural drawing of different folic acid derivatives;
Fig. 2 is the chromatogram that UPLC-MS/MS measures multi-form folic acid derivatives.Specimen in use is the pakchoi that market is buied, folic acid extract is obtained according to this method, but in step 1) add 0.05M phosphate buffer and add mixed sample (mixed sample is the potpourri of seven kinds of folic acid derivatives standard items, and often kind of derivant addition is 0.5 μ g/100g) simultaneously.Sample measures according to the condition of this method, and sample size is 2 μ l, 5 minutes detection times.10-CHO-THF, 10-formyl-tetrahydrofolic acid; 5-CHO-THF, 5-formyl-tetrahydrofolic acid; 10-CHO-FA, 10-formyl-folic acid; M-THF, 5-MTHF; 5,10-CH
+tHF, 5,10-anhydroleucovorin; THF, tetrahydrofolic acid; FA, folic acid.
Embodiment
Method provided by the present invention comprises following content:
1. the compound method of reagent
Agents useful for same of the present invention comprises: acetonitrile (liquid chromatography mass coupling level), methyl alcohol (liquid chromatography mass coupling level), and other reagent is for analyzing pure rank.L (+)-ascorbic acid (Sigma, the U.S.), dimercaprol dimercaptopropanol (Sigma, the U.S.), rat blood serum (Sigma, the U.S., EC numbering 3-4-22-12), activated charcoal (Sigma, the U.S.), chicken pancreas (WS301, Shanghai Wujing Chemical Industry Co., Ltd., China, EC numbering 3-4-22-12), potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium acetate, sodium chloride (Merck, Germany), the accurate substance B CR-485 of vegetable leaf acidity scale (standard reference materials and technique study institute, Belgium), water is ultrapure water (Millipore, the U.S.).Folic acid reference substance, tetrahydrofolic acid sodium salt (H
4folate) reference substance, 5-MTHF sodium salt (5-CH
3– H
4folate) reference substance, 5-formyl-tetrahydrofolic acid sodium salt (5-CHO-H
4folate) reference substance, 5,10-anhydroleucovorin (5,10CH
+-H
4folate) reference substance and 10-formyl-tetrahydrofolic acid (10-CHO-H
4folate) all from Merck & Cie (Switzerland) company, 10-formyl-folic acid sodium salt (10-CHO-folicacid) is from Schirck ' s laboratory (Switzerland).Above-mentioned reference substance all needs to be kept at-80 DEG C of conditions.
1.1,0.05M phosphate buffer (pH=6.1) configuration: get 41.75ml1mol/L potassium dihydrogen phosphate and 8.1ml1mol/L dipotassium hydrogen phosphate, add 10gL (+)-ascorbic acid and 10ml dimercaprol dimercaptopropanol in 1L beaker, add water to about 980ml, regulate pH to 6.1 with 1mol/L potassium hydroxide, slowly pour in 1L volumetric flask and be settled to 1L.
1.2, standard folic acid derivatives stock solution configures: the folic acid reference substance taking 5mg respectively, tetrahydrofolic acid sodium salt reference substance, 5-MTHF sodium salt reference substance, 5-formyl-tetrahydrofolic acid sodium salt reference substance, 5, 10-anhydroleucovorin reference substance, 10-formyl-tetrahydrofolic acid reference substance, 10-formyl-sodium folate Sodium pteroylgutamate salt reference substance, after dissolving with above-mentioned 0.05M phosphate buffer (pH=6.1) respectively, constant volume is in 25mL volumetric flask respectively, make the storing solution of the standard items of 200 μ g/mL, desired concn is diluted to 0.05M phosphate buffer (pH=6.1) during use.
1.3 two kinds of folic acid conjugase configurations:
Get 1mL mouse serum, add the activated charcoal of 1/10 volume, ice bath stirs 1 hour, centrifugal, filters by the syringe filter after 0.20 μm of sterilizing, loads the sterilized tubule of 0.5ml in-20 DEG C of preservations, obtains mouse serum conjugase.
Get 5mg chicken pancreas and be dissolved in 30ml0.05M phosphate buffered saline(PBS) (pH=6.1), in 4 DEG C of preservations; Obtain chicken pancreas conjugase.
2. test main operating process
Due to the as easy as rolling off a log oxidized destruction of folic acid, the operation steps of the solution and test that therefore configure above-mentioned 1.1-1.3 all need be carried out in the room being provided with yellow fluorescence lamp, the glassware such as volumetric flask, beaker of configuration required for solution all needs to use brown vessel, or wraps up lucifuge with masking foil.Ultra Performance Liquid Chromatography mass spectrometry auto injection bottle also needs to use brown bottle.
2.1 phosphate buffers boil and extract plant leaf blade folic acid
Take the fresh blade of 1g to twist into pieces after liquid nitrogen flash freezer rapidly, then be evenly dispensed into rapidly 3 2.0ml scale spiral cover bacteriums and preserve pipe (TK372-SN-F/Q, BBI, raw work), often pipe adds the stainless shot (opening up conspicuous dynamo-electric science and technology) of 500 μ L0.05M phosphate buffers (pH=6.1) and a diameter 5mm, grinds 6 minutes with SCIENTZ-48 high-flux tissue mill (Ningbo Xin Zhi biotechnology limited company) in 70Hz.After the sample of three pipes grinds, proceed to 50ml plastic centrifuge tube together, and with 0.05M phosphate buffer (pH=6.1) flushing preservation pipe, all samples is all shifted, be filled with argon gas (preventing extract oxygenolysis) and rapid cover lid, in 100 DEG C of water-baths 12 minutes, rear rapid ice bath made sample be cooled to 4 DEG C.Rotating speed 27000g, 4 DEG C centrifugal 20 minutes, and supernatant proceeds to 10ml volumetric flask, is settled to 10ml, as plant leaf blade extract with 0.05M phosphate buffer (pH=6.1).
2.2 add two kinds of folic acid conjugases
2ml chicken pancreas conjugase is added in the 2.1 plant leaf blade extracts obtained, mixing, draw the solution after 3ml mixing in new 50ml centrifuge tube, add 100 μ L mouse serum conjugases, mixing, be filled with the rapid cover lid of argon gas, 37 DEG C of water-baths hatch 2 hours, then within 5 minutes, stop enzymatic activity in 100 DEG C of water-baths, rear rapid ice bath is cooled to 4 DEG C.Draw the cooled solution of 1.5ml in 2ml centrifuge tube, rotating speed 18000g, 4 DEG C centrifugal 20 minutes, and supernatant is plant leaf blade folic acid outspan extract, makes next step purifying.
Remarks illustrate: the volume 1.5ml of supernatant, namely centrifugal caused volume change is ignored.
2.3 ultra-filtration centrifuge tube purifying plant leaf blade folic acid outspan extracts
Ultra-filtration centrifuge tube (10kDa, Millipore, the U.S.) activation after, add the obtained supernatant 500 μ L of step 2.2 in ultra-filtration centrifuge tube, rotating speed 12000g, 4 DEG C centrifugal 15 minutes, obtains the plant leaf blade folic acid extract after purifying (crossing the solution of gained after post), to be measured in 4 DEG C of preservations.
2.4 Ultra Performance Liquid Chromatography mass spectrometry (UPLC-MS/MS) Mass Spectrometry Conditions
Ultra Performance Liquid Chromatography mass spectrometry detection system is quadrupole rod tandem mass spectrum (WatersXevoTQ-S, Waters, the U.S.) normal pressure electron spray ionisation (ESI) positive pole pattern.Before detecting sample, WatersIntelliStartTechnology and MassLynx4.1 software is integrated mutually, automatically adjusts, correct and carry out the systems inspection before analysis of experiments for control machine (UPLC-MS/MS).WatersIntelliStartTechnology optimizes the polyion reaction monitoring (multiplereactionmonitoring, MRM) of all folic acid derivatives by automatic and manual methods, determines the product ion mass-to-charge ratio of each folic acid derivatives.Each folic acid derivatives uses two or three daughter ions to determine, result is as table 1.In Flow Injection Analysis process, Automatic Optimal source dates (by WatersIntelliStartTechnology and MassLynx4.1 software Automatic Optimal), refers to table 2.MassLynx4.1 (Waters, the U.S.) software is used for system cloud gray model, data acquisition and data analysis.
The multiion reaction monitoring of table 1 folic acid and folic acid derivatives and compound index
Table 2 folic acid and folic acid derivatives quadrupole rods tandem mass spectrometry detecting device top condition
2.5 Ultra Performance Liquid Chromatography mass spectrometry (UPLC-MS/MS) chromatographic conditions
Use WatersACQUITYUPLC system (Waters, the U.S.) carry out the chromatographic resolution of folic acid derivatives, this system comprises binary liquid phase pump, thermostated autosampler, constant temperature column compartment, and operating software is TargetLynx4.1 (Waters, the U.S.).Column oven maintains 40 DEG C, and automatic sampler maintains 4 DEG C.Chromatographic column is ACQUITYUPLCBEH, C
18column, specification is 2.1mm × 50mm, and particle diameter 1.7 μm (Waters, the U.S.) connects a VanGuard pre-column C18, and specification is 5mm × 2.1mm, particle diameter 1.8 μm (Waters, the U.S.).Flow velocity 0.4mL/min; Sample size 2 μ L (" the plant leaf blade folic acid extract after purifying " or control sample for above-mentioned steps 2.3 gained), 5 minutes working times.Mobile phase is eluent A (0.1% formic acid is water-soluble) and eluent B (0.1% formic acid is dissolved in acetonitrile) gradient elution, refers to table 3.Syringe needle cleaning solvent is acetonitrile/water mixed liquor (50/50, v/v).Each sample detection terminates with sample introduction syringe needle cleaning solvent, then carries out next sample detection, and syringe needle cleaning solvent sample size is all 2 μ L.
The best gradient elution of table 3UPLC-MS/MS
Remarks illustrate: the % in above-mentioned table 3 is volume %.
2.6 plant leaf blade folic acid and derivant quantitative measurement thereof
Under Mass Spectrometry Conditions of the present invention (as described in 2.4) and chromatographic condition (as described in 2.5), sample introduction reference substance tetrahydrofolic acid (1-100ng/mL) respectively, 5-MTHF (1-100ng/mL), 5-formyl-tetrahydrofolic acid (1-100ng/mL), 5, 10-anhydroleucovorin (0.05-75ng/mL), 10-formyl-folic acid (0.05-75ng/mL), 10-formyl-tetrahydrofolic acid (1-100ng/mL) and folic acid (0.05-75ng/mL), sample size 2 μ L, record main peak peak area, Criterion curve (take concentration as horizontal ordinate, peak area is ordinate).
According to extracting method of the present invention (as 2.1, 2.2, described in 2.3), extract folic acid and the derivant thereof of plant leaf blade, measure test sample folic acid, sample size 2 μ L, record main peak peak area, TargetLynx4.1 (Waters, the U.S.) automatically calculate the concentration (ng/mL) of corresponding each folic acid derivatives with peak area according to external standard method, then tetrahydrofolic acid in test sample (μ g/100g) is gone out according to following formula scales, 5-MTHF (μ g/100g), 5-formyl-tetrahydrofolic acid (μ g/100g), 5, 10-anhydroleucovorin (μ g/100g), 10-formyl-folic acid (μ g/100g), 10-formyl-tetrahydrofolic acid (μ g/100g), folic acid (μ g/100g) content.
Owing to containing a certain amount of 5-MTHF in mouse serum, the 5-MTHF concentration of blank solution when therefore calculating test sample 5-MTHF concentration, first need be deducted.Blank solution is not for add plant leaf blade, and all the other are all by 2.1,2.2,2.3,2.4,2.5 operation gained.
1) tetrahydrofolic acid content (μ g/100g)=tetrahydrofolic acid (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
2) 5-MTHF content (μ g/100g)=(5-MTHF measured value-blank sample) (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
3) 5-formyl-tetrahydrofolic acid content (μ g/100g)=5-formyl-tetrahydrofolic acid (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
4) 5,10-anhydroleucovorin content (μ g/100g)=5,10-anhydroleucovorins (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
5) 10-formyl-folate content (μ g/100g)=10-formyl-folic acid (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
6) 10-formyl-tetrahydrofolic acid content (μ g/100g)=10-formyl-tetrahydrofolic acid (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
7) folate content (μ g/100g)=folic acid (ng/mL) × 12 (mL) × 10
-3× 100g/ plant leaf blade weight (g)
8) total folate content (μ g/100g)=441.4 × (tetrahydrofolic acid content (μ g/100g) ÷ 445.4+5-methyl-tetrahydro folate content (μ g/100g) ÷ 459.5+5-formyl-tetrahydrofolic acid content (μ g/100g) ÷ 473.5+5,10-anhydroleucovorin content (μ g/100g) ÷ 457.1+10-formyl-folate content (μ g/100g) ÷ 469.41+10-formyl-tetrahydrofolic acid content (μ g/100g) ÷ 473.44+ folate content (μ g/100g) ÷ 441.4)
Note: 445.4 is tetrahydrofolic acid molecular weight, 459.5 be 5-MTHF molecular weight, 473.5 is 5-formyl-tetrahydrofolic acid molecular weight, 457.1 is 5,10-anhydroleucovorin molecular weight, 469.41 is 10-formyl-folate molecule amounts, and 473.44 is 10-formyl-tetrahydrofolic acid molecular weight, and 441.4 is folate molecule amounts.
Embodiment 1. just the present invention detects the test example explanation of the accurate substance C RM485 of vegetable leaf acidity scale (from standard reference materials and technique study institute, Belgium).
1. linear relationship is investigated and sensitivity testing
At mass spectrum of the present invention, under chromatographic condition, selection standard folic acid derivatives solution respectively: tetrahydrofolic acid (1, 15, 30, 50, 75, 90, 100ng/mL), 5-MTHF (1, 15, 30, 50, 75, 90, 100ng/mL), 5-formyl-tetrahydrofolic acid (1, 15, 30, 50, 75, 90, 100ng/mL), 5, 10-anhydroleucovorin (0.05, 0.25, 1, 15, 30, 50, 75ng/mL), 10-formyl-folic acid (0.05, 0.25, 1, 15, 30, 50, 75ng/mL), 10-formyl-tetrahydrofolic acid content (1, 15, 30, 50, 75, 90, 100ng/mL), folic acid (0.05, 0.25, 1, 15, 30, 50, 75ng/mL), sample size 2 μ l, record peak area, take concentration as horizontal ordinate, peak area is ordinate, drawing standard curve, regression coefficient, slope is in table 4.From table 4, the regression coefficient R of each derivant
2all be greater than 0.99, visible regression coefficient is good.
Standard folic acid derivatives tetrahydrofolic acid (1-100ng/mL), 5-MTHF (1-100ng/mL), 5-formyl-tetrahydrofolic acid (1-100ng/mL), 5, 10-anhydroleucovorin (0.05 – 75ng/mL), 10-formyl-folic acid (0.05 – 75ng/mL), 10-formyl-tetrahydrofolic acid (1-100ng/mL), folic acid (0.05 – 75ng/mL) calculates tetrahydrofolic acid with counter sample concentration under 3 times of signal to noise ratio (S/N ratio) concentration, 5-Jia Ji – tetrahydrofolic acid, 5, 10-anhydroleucovorin, 5-Jia Xian – tetrahydrofolic acid, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the detectability of folic acid, under 10 times of signal to noise ratio (S/N ratio) concentration, counter sample concentration calculates tetrahydrofolic acid, 5-Jia Ji – tetrahydrofolic acid, 5-Jia Xian – tetrahydrofolic acid, 5, 10-anhydroleucovorin, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the quantitative limit of folic acid, the results are shown in Table 4.The detectability of the tetrahydrofolic acid that this method measures, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5,10-anhydroleucovorins, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, folic acid is respectively 0.03,0.06,0.10,0.10,0.07,0.20,0.11ng/ml, quantitative limit is respectively 0.11,0.20,0.30,0.32,0.23,0.40,0.35ng/ml, visible this method sensitivity is high.
Table 4 standard model linear relationship is investigated, the measurement result of detectability and quantitative limit
2. degree of accuracy test
Take 1g greengrocery standard substance BCR-485 (article No.: BCR-485, Belgium), according to " 2.1, 2.2, 2.3 " item method obtains the plant leaf blade folic acid extract after purifying, preparation standard folic acid derivatives solution simultaneously, " 2.4 " are referred to and chromatographic condition refers to " 2.5 " according to the Mass Spectrometry Conditions of this method, sample feeding amount 2 μ L, 5 minutes working times, record main peak peak area, obtain the content (ng/mL) of each form folic acid derivatives with peak area according to external standard method, again according to the formulae discovery tetrahydrofolic acid of method " 2.6 ", 5-MTHF, the content (μ g/100g) of 5-formyl-tetrahydrofolic acid and total folic acid, refer to table 5.Because Norme Belge reference material and technique study institute think that 5-MTHF is the principal mode of vegetables Folic Acid, therefore the data of tetrahydrofolic acid and 5-formyl-tetrahydrofolic acid do not provide.Result is visible, the main folic acid derivatives of BCR-485 is 5-MTHF, the 5-MTHF of the BCR-485 that UPLC-MS/MS method of the present invention measures and total folate content, within the scope of standard reference value, illustrate the inventive method measurement result accurately and reliably.
Table 5 greengrocery standard substance BCR-485 degree of accuracy is tested
a5-MTHF standard reference value in the BCR-485 that HPLC measures
bthe total folic acid standard reference value of BCR-485 of microbioassay
3. recovery test
Take the pakchoi that 1g market is buied, folic acid extract is obtained according to this method, take this pakchoi of 1g simultaneously, but in step 1) add 0.05M phosphate buffer and add the mixed sample of two kinds of variable concentrations simultaneously respectively (mixed sample is the potpourri of seven kinds of folic acid derivatives, low concentration mixed sample is 0.5 μ g/100g, high concentration mixed sample is 7.5 μ g/100g), other steps are identical.Sample measures according to the condition of this method, and sample size is 2 μ l, 5 minutes detection times.Absolute recovery=(pakchoi mix with mixed sample surveyed concentration-pakchoi survey concentration) ÷ adds concentration × 100% of mixed sample, the results are shown in Table 6.The absolute recovery of 5-MTHF, 5-formyl-tetrahydrofolic acid, 10-formyl-folic acid and folic acid is greater than 90%, and the recovery of all the other folic acid derivatives is also greater than 70%, and visible this method recovery is higher, and data are reliable.
Remarks illustrate: the mixed sample of above-mentioned interpolation two kinds of variable concentrations, be specially for low concentration: the potpourri adding seven kinds of folic acid derivatives at 500 μ L0.05M phosphate buffers (pH=6.1), thus make the concentration of these seven kinds of folic acid derivatives be 0.5 μ g/100g.
Table 6 folic acid derivatives recovery test
4. relative standard deviation test
Take the pakchoi that 1g market is purchased, according to " 2.1, 2.2, 2.3 " item method obtains the plant leaf blade folic acid extract after purifying, add 0.5 μ g/100g hybrid standard sample and (in " the folic acid refined solution after purifying " of step 2.3 gained, add the potpourri of seven kinds of folic acid derivatives, thus make the concentration of these seven kinds of folic acid derivatives be 0.5 μ g/100g), 6 repetitions, preparation standard folic acid derivatives solution simultaneously, " 2.4 " are referred to and chromatographic condition refers to " 2.5 " according to the Mass Spectrometry Conditions of this method, sample feeding amount 2 μ L, 5 minutes working times, record main peak peak area, obtain the content (ng/mL) of each form folic acid derivatives with peak area according to external standard method, again according to the formulae discovery tetrahydrofolic acid of method " 2.6 ", 5-MTHF, 5-formyl-tetrahydrofolic acid, 5, 10-anhydroleucovorin, 10-formyl-tetrahydrofolic acid, the content (μ g/100g) of 10-formyl-folic acid and total folic acid, be designated as A, then to these 6 samples sample introduction again, calculate total folic acid and folic acid derivatives content, be designated as B, the relative standard deviation value that the same day then calculating corresponding folic acid derivatives according to formula │ B-A │/A is detected.After same day sample detection, place 4 DEG C of automatic samplers, again loading after 24 hours, sample feeding amount 2 μ L, 5 minutes working times, record main peak peak area, obtain the content (ng/mL) of each form folic acid derivatives with peak area according to external standard method, again according to formulae discovery folic acid and the folic acid derivatives content (μ g/100g) of method " 2.6 ", be designated as C, then calculate the value of detection relative standard deviation every other day of corresponding folic acid derivatives according to formula │ C-A │/A.The results are shown in Table 7.From table 7, the folic acid derivatives relative standard deviation that the same day is detected is in 1.6 to 4.1% scopes, detect folic acid derivatives relative standard deviation every other day in 2.8 to 7.8% scopes, show that sample at least keeps stablizing for 24 hours in automatic sampler, illustrate that this method at least can continuous detecting 24 hours, can batch detection sample.
Table 7 folic acid derivatives relative standard deviation is tested
Embodiment 1 is investigated from linear relationship, detectability and quantitative limit, degree of accuracy test, the recovery and relative standard deviation test prove this method have that sensitivity is high, degree of accuracy is high, the recovery is high, can the feature of batch detection sample.The folic acid derivatives lowest detection that this method measures is limited to tetrahydrofolic acid 0.03ng/ml, highest detection is limited to 10-formyl tetrahydrofolic acid 0.20ng/ml, minimumly quantitatively be limited to tetrahydrofolic acid 0.11ng/ml, be the highlyest quantitatively limited to 10-formyl-tetrahydrofolic acid 0.40ng/ml, visible this method sensitivity is high.This method adopts greengrocery standard substance BCR-485 to carry out degree of accuracy test, and the 5-MTHF of detection and total folate content are all within the scope of standard reference value, and visible this method degree of accuracy is high, and result accurately and reliably.The absolute recovery of this method 5-MTHF, 5-formyl-tetrahydrofolic acid, 10-formyl-folic acid and folic acid is greater than 90%, and all the other relatively unstable folic acid derivatives absolute recoveries are also greater than 70%, and the data that visible this method measures are accurate.This method sample detection time is 5 minutes, and sample is placed in 4 DEG C of automatic samplers and at least can be kept stablizing 24 hours, and therefore this method can batch detection sample, improves detection efficiency.
Embodiment 2. with regard to the present invention detect buy in market pakchoi, spinach, romaine lettuce and chamber planting rice leaf in total folic acid and the explanation of folic acid derivatives content test examples
Get the pakchoi that market is bought, spinach, the rice leaf sample of romaine lettuce and chamber planting, obtain folic acid according to 2.1-2.3 method and extract solution, preparation standard folic acid derivatives solution simultaneously, according to Mass Spectrometry Conditions and the chromatographic condition mensuration of the Ultra Performance Liquid Chromatography mass spectrometry of above-mentioned 2.4-2.5, sample size 2 μ L, record peak area, the typical curve of obtained each folic acid derivatives, and obtain folic acid derivatives concentration (ng/mL) according to external standard method with peak area, again according to 2.6 total folate content of formulae discovery test sample and each folic acid derivatives content (μ g/100g), the results are shown in Table 8.From table 8, the total folate content of spinach is the highest, reaches 223.74 μ g/100g, is secondly pakchoi, total folate content 207.851 μ g/100g, and romaine lettuce is close with the total folate content of rice leaf, is 117.458 μ g/100g and 118.303 μ g/100g respectively.In romaine lettuce, main folic acid derivatives is 5-MTHF and tetrahydrofolic acid; The main folic acid derivatives of spinach is 5-MTHF, 5-formyl-tetrahydrofolic acid and 5,10-anhydroleucovorin; The main folic acid derivatives of pakchoi is 5-MTHF and 5-formyl-tetrahydrofolic acid; The main folic acid derivatives of rice leaf is 5-MTHF and 5-formyl-tetrahydrofolic acid.
Table 8 romaine lettuce, spinach, pakchoi and rice leaf folic acid and derivative content thereof
This tests proof, this method can effectively be extracted and seven kinds of folic acid of Fast synchronization detection romaine lettuce, spinach, pakchoi, rice leaf and folic acid derivatives, tetrahydrofolic acid, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5,10-anhydroleucovorins, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid and folic acid respectively.And, this method is highly sensitive, folic acid and the derivant thereof of the plant leaf blade that folate content is low can be detected, can effectively detect romaine lettuce, spinach, pakchoi and rice leaf 5, the content of 10-anhydroleucovorin, 10-formylpropionic acid, 10-formyl tetrahydrofolic acid and folic acid, still belongs to the first time at home.
Comparative example 1, made into " 6 μ L AMS " by " the 2mL chicken pancreas conjugase " in step 2.2, all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
Comparative example 2, made into " 15 μ L deproteinized enzyme " by " the 2mL chicken pancreas conjugase " in step 2.2, all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
Comparative example 3, made into " 6 μ L AMSs and 15 μ L deproteinized enzymes " by " the 2mL chicken pancreas conjugase " in step 2.2, all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
Comparative example 4, step 2.2 to be made into: add 2ml chicken pancreas conjugase, after drawing 3ml to new centrifuge tube, then add " 6 μ L AMSs and 15 μ L deproteinized enzymes " and " 100 μ L mouse serum conjugase ".All the other are equal to embodiments of the invention 2 (only detecting pakchoi).
The testing result of above-mentioned all comparative examples is as shown in table 9.Table 9 is visible, and comparative example 4 is all best with the result of use of embodiment 2, but the former need add 6 μ L AMSs and 15 μ L deproteinized enzymes, therefore the chicken pancreas conjugase taked of method of the present invention and mouse serum conjugase economical and efficient the most used in combination.
The different conjugase collocation of table 9 uses the impact of mensuration pakchoi folic acid total content
In comparative example 5, step 2.2, " 37 DEG C of water-baths hatch 2 hours, " makes into " 37 DEG C of water-baths hatch 0.5 hour, ", and all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
In comparative example 6, step 2.2, " 37 DEG C of water-baths hatch 2 hours, " makes into " 37 DEG C of water-baths hatch 1 hour, ", and all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
In comparative example 7, step 2.2, " 37 DEG C of water-baths hatch 2 hours, " makes into " 37 DEG C of water-baths hatch 4 hours, ", and all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
In comparative example 8, step 2.2, " 37 DEG C of water-baths hatch 2 hours, " makes into " 37 DEG C of water-baths hatch 6 hours, ", and all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
In comparative example 9, step 2.2, " 37 DEG C of water-baths hatch 2 hours, " makes into " 37 DEG C of water-baths hatch 24 hours, ", and all the other are equal to embodiments of the invention 2 (only detecting pakchoi).
The testing result of above-mentioned all comparative examples is as shown in table 10.Table 10 is visible, and water-bath is hatched the impact of duration on the total folate content of pakchoi and reach maintenance level from after 2 hours, and in order to save sample processing time, this method adopts water-bath in 2 hours to hatch duration the best.
Table 10 water-bath hatches duration to the impact of mensuration pakchoi folic acid total content
In comparative example 10, step 2.3, " ultra-filtration centrifuge tube (10kDa; Millipore, the U.S.) " makes that " ultra-filtration centrifuge tube (5kDa, Millipore; the U.S.), all the other are equal to embodiments of the invention 2 (only detecting pakchoi) into.Testing result is as shown in table 11.As seen from Table 11, the ultra-filtration centrifuge tube effect of 10kDa specification is better, and the ultra-filtration centrifuge tube price of 10kDa specification is lower.
The different ultra-filtration centrifuge tube specification of table 11 is on the impact of mensuration pakchoi folic acid total content
More than illustrating is only two instantiations of the present invention.Can prove according to above example, the method of Ultra Performance Liquid Chromatography-tandem mass spectrum (UPLC-MS/MS) total folic acid of Simultaneous Determination vegetables of the present invention and folic acid derivatives content be efficient fast, sensitivity is high, degree of accuracy is high, the recovery is high, can quantitatively detect 7 kinds of folic acid of low content folic acid plant leaf blade and the method for folic acid derivatives by batch synchronization.This method can be applied to the biological reinforced field of vegetables, also can be applied to the research of researcher to plant folic acid and derivant aspect thereof.Other distortion that are that others skilled in the art's direct derivation from content disclosed by the invention in this field goes out or that associate, all should think protection scope of the present invention.
Claims (4)
1. fast high-sensitive synchronously quantitatively detects the method for the total folic acid of blade and folic acid derivatives, it is characterized in that comprising the following steps successively:
1) folic acid in plant leaf blade and folic acid derivatives, is extracted:
Utilize 0.05M phosphate buffer to boil and extract the folic acid of plant leaf blade, cooling and centrifugal after, obtain supernatant, supernatant is folic acid extract;
2), two kinds of folic acid conjugases are added:
In step 1) in folic acid extract in add mouse serum conjugase and chicken pancreas conjugase, hatch 2 hours for 37 DEG C after being filled with argon gas, 100 DEG C of water-baths stop enzyme for 5 minutes and live, and get supernatant after centrifugal, and supernatant is that extract is closed in folic acid outspan;
3) outspan of ultra-filtration centrifuge tube purifying plant leaf blade folic acid, is used to close extract:
Using 10kDa500 μ L ultra-filtration centrifuge tube, after activation, add 500 μ L steps 2 inward) the folic acid outspan of gained closes extract, and 12000g4 DEG C is centrifugal 15 minutes, obtains the folic acid refined solution after purifying;
4), Ultra Performance Liquid Chromatography mass spectrometry is separated folic acid derivatives mass spectrometry method:
Ultra Performance Liquid Chromatography mass spectrometry detection system is quadrupole rod tandem mass spectrum normal pressure electron spray ionisation (ESI) positive pole pattern; WatersIntelliStartTechnology is by polyion reaction monitoring (multiplereactionmonitoring, MRM) that is automatic and the manually all folic acid derivatives of optimization; Each folic acid derivatives uses two or three product ions to determine, MassLynx4.1 software is used for system cloud gray model, data acquisition and data analysis, as following table 1;
The multiion reaction monitoring of table 1 folic acid and folic acid derivatives and compound index
5), Ultra Performance Liquid Chromatography mass spectrometry is separated folic acid derivatives chromatographic process:
Use WatersACQUITYUPLC system to carry out the chromatographic resolution of folic acid derivatives, operating software is TargetLynx4.1; Column oven maintains 40 DEG C, and automatic sampler maintains 4 DEG C; Chromatographic column is ACQUITYUPLCBEH, C
18column, specification is 2.1mm × 50mm, and particle diameter 1.7 μm connects a VanGuard pre-column C18, and specification is 5mm × 2.1mm, particle diameter 1.8 μm; Flow velocity 0.4mL/min; Sample size 2 μ L, 5 minutes working times; Mobile phase is eluent A (0.1% formic acid is water-soluble) and eluent B (0.1% formic acid is dissolved in acetonitrile) gradient elution, refers to table 2; Syringe needle cleaning solvent is acetonitrile/water mixed liquor (50/50, v/v);
The best gradient elution of table 2, UPLC-MS/MS
6) quantitative measurement of plant leaf blade folic acid derivatives.
2. the efficient highly sensitive synchronous method quantitatively detecting the total folic acid of plant leaf blade and derivant thereof according to claim 1, is characterized in that: described step 1) in 0.05M phosphate buffer be often liter of phosphate buffer adding 10gL (+)-ascorbic acid and 10ml dimercaprol dimercaptopropanol.
3. the method for the total folic acid of efficient highly sensitive synchronous quantitative measurment plant leaf blade according to claim 2 and derivant thereof, it is characterized in that: described step 2) in every 10mL folic acid extract, add 2mL chicken pancreas conjugase, after shaking up, draw 3mL solution in 50mL centrifuge tube, add 100 μ L mouse serum conjugases, shake up, be filled with argon gas, hatch 2 hours for 37 DEG C, 100 DEG C of water-baths stop enzyme for 5 minutes and live.
4. the efficient highly sensitive synchronous method quantitatively detecting the total folic acid of plant leaf blade and derivant thereof according to claim 3, it is characterized in that: described step 6) be: according to step 4) and step 5) condition, quantitative measurement is carried out to plant leaf blade folic acid and derivant thereof, record main peak peak area, automatically tetrahydrofolic acid is calculated with peak area according to external standard method, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5, 10-methine-tetrahydrofolic acid, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the content of folic acid, again according to tetrahydrofolic acid, 5-MTHF, 5-formyl-tetrahydrofolic acid, 5, 10-anhydroleucovorin, 10-formyl-folic acid, 10-formyl-tetrahydrofolic acid, the concentration of folic acid and relative molecular mass calculate total folate content.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510527402.1A CN105181829B (en) | 2015-08-25 | 2015-08-25 | Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510527402.1A CN105181829B (en) | 2015-08-25 | 2015-08-25 | Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105181829A true CN105181829A (en) | 2015-12-23 |
| CN105181829B CN105181829B (en) | 2017-04-12 |
Family
ID=54904052
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510527402.1A Expired - Fee Related CN105181829B (en) | 2015-08-25 | 2015-08-25 | Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105181829B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770819A (en) * | 2016-12-05 | 2017-05-31 | 山东省药学科学院 | A kind of method of LC-MS quantitative determination rat plasma middle period acid concentration |
| CN109060996A (en) * | 2018-09-07 | 2018-12-21 | 中国农业科学院生物技术研究所 | The extraction and quantitative approach of folic acid in corn kernel |
| CN109633029A (en) * | 2019-01-11 | 2019-04-16 | 山西农业大学 | A kind of measuring method of millet seed folic acid extracting method and folate content |
| CN115144520A (en) * | 2022-05-31 | 2022-10-04 | 北京豪思生物科技股份有限公司 | Method for detecting folic acid and pentamethyl tetrahydrofolic acid in breast milk |
| CN117214350A (en) * | 2023-11-08 | 2023-12-12 | 中国农业科学院北京畜牧兽医研究所 | Method and kit for detecting total content of polymorphic folic acid |
| CN117987419A (en) * | 2024-04-03 | 2024-05-07 | 中国农业科学院北京畜牧兽医研究所 | Aptamer, sensor, detection kit and application thereof in detecting tetrahydrofolate |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090203145A1 (en) * | 2008-02-07 | 2009-08-13 | Min Huang | Methods for analysis of vitamins |
| CN102175794A (en) * | 2011-01-13 | 2011-09-07 | 浙江大学 | Method for measuring content of total folic acid and derivatives thereof in vegetables synchronously and quantitatively |
| US20120208220A1 (en) * | 2011-02-14 | 2012-08-16 | National Cheng Kung University | Method and kit for detecting foltate |
| CN103063768A (en) * | 2012-12-24 | 2013-04-24 | 内蒙古伊利实业集团股份有限公司 | Detection method of folic acid content in dairy product |
-
2015
- 2015-08-25 CN CN201510527402.1A patent/CN105181829B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090203145A1 (en) * | 2008-02-07 | 2009-08-13 | Min Huang | Methods for analysis of vitamins |
| CN102175794A (en) * | 2011-01-13 | 2011-09-07 | 浙江大学 | Method for measuring content of total folic acid and derivatives thereof in vegetables synchronously and quantitatively |
| US20120208220A1 (en) * | 2011-02-14 | 2012-08-16 | National Cheng Kung University | Method and kit for detecting foltate |
| CN103063768A (en) * | 2012-12-24 | 2013-04-24 | 内蒙古伊利实业集团股份有限公司 | Detection method of folic acid content in dairy product |
Non-Patent Citations (4)
| Title |
|---|
| ACHIM FREISLEBEN等: "Specific and sensitive quantification of folate vitamers in foods by stable isotope dilution assays using high-performance liquid chromatography-tandem mass spectrometry", 《ANAL BIOANAL CHEM》 * |
| CHRISTINE M. PFEIFFER等: "Determination of Folate in Cereal-Grain Food Products Using Trienzyme Extraction and Combined Affinity and Reversed-Phase Liquid Chromatography", 《J. AGRIC. FOOD CHEM.》 * |
| 戴金凤等: "超高效液相串联质谱测定四棱豆中总叶酸和多聚谷氨酸叶酸的含量", 《现代食品科技》 * |
| 郭丽琼等: "样品前处理对检测柑橘中5-甲基四氢叶酸的影响", 《食品科学》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106770819A (en) * | 2016-12-05 | 2017-05-31 | 山东省药学科学院 | A kind of method of LC-MS quantitative determination rat plasma middle period acid concentration |
| CN109060996A (en) * | 2018-09-07 | 2018-12-21 | 中国农业科学院生物技术研究所 | The extraction and quantitative approach of folic acid in corn kernel |
| CN109060996B (en) * | 2018-09-07 | 2021-06-11 | 中国农业科学院生物技术研究所 | Method for extracting and quantifying folic acid in corn kernels |
| CN109633029A (en) * | 2019-01-11 | 2019-04-16 | 山西农业大学 | A kind of measuring method of millet seed folic acid extracting method and folate content |
| CN115144520A (en) * | 2022-05-31 | 2022-10-04 | 北京豪思生物科技股份有限公司 | Method for detecting folic acid and pentamethyl tetrahydrofolic acid in breast milk |
| CN115144520B (en) * | 2022-05-31 | 2023-12-05 | 北京豪思生物科技股份有限公司 | Method for detecting folic acid and pentamethyltetrahydrofolic acid in breast milk |
| CN117214350A (en) * | 2023-11-08 | 2023-12-12 | 中国农业科学院北京畜牧兽医研究所 | Method and kit for detecting total content of polymorphic folic acid |
| CN117214350B (en) * | 2023-11-08 | 2024-04-12 | 中国农业科学院北京畜牧兽医研究所 | Method and kit for detecting total content of polymorphic folic acid |
| CN117987419A (en) * | 2024-04-03 | 2024-05-07 | 中国农业科学院北京畜牧兽医研究所 | Aptamer, sensor, detection kit and application thereof in detecting tetrahydrofolate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105181829B (en) | 2017-04-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105181829A (en) | Rapid high-sensitivity synchronous quantitative determination method for leaf total folic acid and folic acid derivatives | |
| Jakob et al. | Application of a simplified HPLC assay for the determination of phylloquinone (vitamin K1) in animal and plant food items | |
| CN106855545B (en) | Method for simultaneously detecting fat-soluble vitamins and water-soluble vitamins in feed | |
| CN103969362B (en) | Method for quantitatively detecting FQs(fluroquinolones) in chicken manure | |
| CN103543218B (en) | Method for measuring tetracycline antibiotic residue in protein-rich sample | |
| CN102175794B (en) | Method for measuring content of total folic acid and derivatives thereof in vegetables synchronously and quantitatively | |
| CN107957464B (en) | Method for simultaneously detecting multiple glycopeptide antibiotics in animal-derived food | |
| Tayade et al. | Sequential determination of fat-and water-soluble vitamins in Rhodiola imbricata root from trans-Himalaya with rapid resolution liquid chromatography/tandem mass spectrometry | |
| CN109030658B (en) | Method for detecting fructo-oligosaccharide and raffinose in infant milk powder | |
| CN109060996B (en) | Method for extracting and quantifying folic acid in corn kernels | |
| CN110208437A (en) | Vitamin K1 and farnoquinone (MK4) method in blood are detected simultaneously | |
| CN101900711A (en) | High performance liquid chromatography method of oxytetracycline residues in soils | |
| CN113419009B (en) | Liquid chromatography tandem mass spectrometry determination method for fluensulfone metabolite | |
| CN103123345B (en) | Method for rapidly detecting phenoxyacetic acid herbicide in soil | |
| CN108956816B (en) | HPLC analysis method for simultaneously determining curcumin and 5-fluorouracil concentration in plasma | |
| Yeung et al. | Quality evaluation of commercial products of Ganoderma lucidum made from its fruiting body and spore | |
| CN102749394A (en) | Separation and measurement method for reduction-type ascorbic acid and erythorbic acid in fruit and vegetable tissues and related products | |
| RU2633013C2 (en) | Method for levomycetin determination in fodders of animal origin using high-effective liquid chromatography | |
| Shearer | [25] Assay of K vitamins in tissues by high-performance liquid chromatography with special reference to ultraviolet detection | |
| CN103323551A (en) | Method for detecting content of medlar acid | |
| CN102435699B (en) | Method for rapidly determining melamine in milk and dairy products by liquid chromatography-tandem mass spectrometry | |
| CN115718146A (en) | Milk freeze-dried powder amoxicillin matrix standard substance and preparation method thereof | |
| CN113156035B (en) | Method for measuring content of all-trans vitamin A in formula food for special medical application | |
| CN106248825B (en) | Starting material A detection method in hydrochloride landiolol material medicine | |
| CN103926368A (en) | Method for extracting biotin from corn steep liquor and thin layer chromatography (TLC) scanning detection method of biotin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170412 |