CN114047267B - Method for analyzing nicotinic acid in tobacco root system by derivatization-gas chromatography-mass spectrometry - Google Patents
Method for analyzing nicotinic acid in tobacco root system by derivatization-gas chromatography-mass spectrometry Download PDFInfo
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- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 title claims abstract description 148
- 229960003512 nicotinic acid Drugs 0.000 title claims abstract description 81
- 235000001968 nicotinic acid Nutrition 0.000 title claims abstract description 78
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 42
- 241001002356 Valeriana edulis Species 0.000 title claims abstract description 40
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 title claims abstract description 23
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 32
- 238000000605 extraction Methods 0.000 claims abstract description 28
- 238000001212 derivatisation Methods 0.000 claims abstract description 27
- 150000002500 ions Chemical class 0.000 claims abstract description 27
- 239000000243 solution Substances 0.000 claims abstract description 24
- 239000000047 product Substances 0.000 claims abstract description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 16
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 16
- 239000002904 solvent Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000002414 normal-phase solid-phase extraction Methods 0.000 claims abstract description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000011780 sodium chloride Substances 0.000 claims abstract description 8
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000000126 substance Substances 0.000 claims abstract description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000004913 activation Effects 0.000 claims abstract description 4
- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 4
- 238000001035 drying Methods 0.000 claims abstract description 4
- 238000002386 leaching Methods 0.000 claims abstract description 4
- 239000002244 precipitate Substances 0.000 claims abstract description 4
- TWBYWOBDOCUKOW-UHFFFAOYSA-N isonicotinic acid Chemical group OC(=O)C1=CC=NC=C1 TWBYWOBDOCUKOW-UHFFFAOYSA-N 0.000 claims description 27
- 238000004853 microextraction Methods 0.000 claims description 10
- 239000006185 dispersion Substances 0.000 claims description 8
- 239000007788 liquid Substances 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000001819 mass spectrum Methods 0.000 claims description 6
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical group ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000010438 heat treatment Methods 0.000 claims description 5
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 239000007789 gas Substances 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 3
- 239000012159 carrier gas Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 239000001307 helium Substances 0.000 claims description 3
- 229910052734 helium Inorganic materials 0.000 claims description 3
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000010813 internal standard method Methods 0.000 claims description 3
- 238000005360 mashing Methods 0.000 claims description 3
- 239000012071 phase Substances 0.000 claims description 3
- 238000002137 ultrasound extraction Methods 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims 1
- 238000001514 detection method Methods 0.000 abstract description 10
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000000694 effects Effects 0.000 abstract description 3
- 239000011159 matrix material Substances 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 14
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 11
- 235000002637 Nicotiana tabacum Nutrition 0.000 description 10
- 241000208125 Nicotiana Species 0.000 description 9
- 235000005152 nicotinamide Nutrition 0.000 description 9
- 239000011570 nicotinamide Substances 0.000 description 9
- 239000000203 mixture Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- SNICXCGAKADSCV-JTQLQIEISA-N (-)-Nicotine Chemical compound CN1CCC[C@H]1C1=CC=CN=C1 SNICXCGAKADSCV-JTQLQIEISA-N 0.000 description 6
- 229960003966 nicotinamide Drugs 0.000 description 6
- SNICXCGAKADSCV-UHFFFAOYSA-N nicotine Natural products CN1CCCC1C1=CC=CN=C1 SNICXCGAKADSCV-UHFFFAOYSA-N 0.000 description 6
- 229960002715 nicotine Drugs 0.000 description 6
- 238000011084 recovery Methods 0.000 description 6
- 238000000746 purification Methods 0.000 description 5
- WKBALTUBRZPIPZ-UHFFFAOYSA-N 2,6-di(propan-2-yl)aniline Chemical group CC(C)C1=CC=CC(C(C)C)=C1N WKBALTUBRZPIPZ-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- KIDHWZJUCRJVML-UHFFFAOYSA-N putrescine Chemical compound NCCCCN KIDHWZJUCRJVML-UHFFFAOYSA-N 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 230000002194 synthesizing effect Effects 0.000 description 4
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000013048 microbiological method Methods 0.000 description 3
- BUJMKGAQHPJMRF-UHFFFAOYSA-N 2-(methylamino)butanal Chemical compound CCC(NC)C=O BUJMKGAQHPJMRF-UHFFFAOYSA-N 0.000 description 2
- TVCXVUHHCUYLGX-UHFFFAOYSA-N 2-Methylpyrrole Chemical group CC1=CC=CN1 TVCXVUHHCUYLGX-UHFFFAOYSA-N 0.000 description 2
- 102100032252 Antizyme inhibitor 2 Human genes 0.000 description 2
- 230000005526 G1 to G0 transition Effects 0.000 description 2
- 101000798222 Homo sapiens Antizyme inhibitor 2 Proteins 0.000 description 2
- RMIVMBYMDISYFZ-UHFFFAOYSA-N N-methylputrescine Chemical compound CNCCCCN RMIVMBYMDISYFZ-UHFFFAOYSA-N 0.000 description 2
- 102000052812 Ornithine decarboxylases Human genes 0.000 description 2
- 108700005126 Ornithine decarboxylases Proteins 0.000 description 2
- 239000005700 Putrescine Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- XHVUVQAANZKEKF-UHFFFAOYSA-N bromine pentafluoride Chemical compound FBr(F)(F)(F)F XHVUVQAANZKEKF-UHFFFAOYSA-N 0.000 description 2
- 238000004737 colorimetric analysis Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000004817 gas chromatography Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 1
- 244000061176 Nicotiana tabacum Species 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 108091022910 Putrescine N-methyltransferase Proteins 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- RWRDLPDLKQPQOW-UHFFFAOYSA-N Pyrrolidine Chemical group C1CCNC1 RWRDLPDLKQPQOW-UHFFFAOYSA-N 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- -1 and in addition Chemical compound 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 235000008452 baby food Nutrition 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- KVYGGMBOZFWZBQ-UHFFFAOYSA-N benzyl nicotinate Chemical class C=1C=CN=CC=1C(=O)OCC1=CC=CC=C1 KVYGGMBOZFWZBQ-UHFFFAOYSA-N 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 239000003501 hydroponics Substances 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 125000000627 niacin group Chemical group 0.000 description 1
- 150000002814 niacins Chemical class 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 150000006636 nicotinic acid Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 150000003222 pyridines Chemical class 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- GJAWHXHKYYXBSV-UHFFFAOYSA-N quinolinic acid Chemical compound OC(=O)C1=CC=CN=C1C(O)=O GJAWHXHKYYXBSV-UHFFFAOYSA-N 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N30/08—Preparation using an enricher
-
- 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
- G01N30/14—Preparation by elimination of some components
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- 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)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention discloses a method for analyzing nicotinic acid in tobacco root systems by a derivatization-gas chromatography-mass spectrometry method, which comprises the following steps: adding an internal standard substance and an extraction solvent into a tobacco root system sample to obtain an extraction solution; sequentially adding methanol and HCl to activate the solid-phase extraction column, slowly adding an extraction solution after activation, sequentially adding HCl and methanol for leaching, eluting with ammonia water and methanol solution, and drying with nitrogen to obtain purified nicotinic acid; adding acetone and a derivatization reagent into purified nicotinic acid, adding a buffer solution, uniformly mixing, and then carrying out pentafluorobromide derivatization; adding water and extraction solvent, mixing with NaCl, stirring again to dissolve NaCl completely, centrifuging, and collecting precipitate; the products are separated and quantified by adopting a gas chromatography-mass spectrometry-selective ion scanning mode. The invention can solve the technical problems of relatively low sensitivity, poor specificity and repeatability and high matrix effect in the detection of nicotinic acid in the prior art.
Description
Technical Field
The invention relates to a method for analyzing nicotinic acid in a tobacco root system by a derivatization-gas chromatography-mass spectrometry method, and belongs to the field of methods for measuring nicotinic acid which is a precursor for synthesizing nicotine in the tobacco root system.
Background
Niacin in the broad sense is an important class of vitamins in the vitamin B group, which comprises both niacin and niacinamide. In organisms, niacin and niacinamide can react with various dehydrogenases to produce nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, and in addition, niacin and niacinamide can also be combined with polysaccharides or polypeptides to form niacin in a chemical combination state. Thus, niacin and niacinamide can be divided into 2 types, free and bound. The free nicotinic acid is usually extracted with dilute acidic aqueous solution for detection and analysis, and the bound nicotinic acid is required to be hydrolyzed into the free nicotinic acid by heating with strong alkalinity or acidic aqueous solution for detection and analysis.
The free nicotinic acid is one of key precursors for synthesizing nicotine in tobacco root systems, the synthesis precondition of the nicotine is mainly 2, one is methyl pyrrole ring, the other is nicotinic acid, the methyl pyrrole ring is mainly used for synthesizing putrescine from arginine and ornithine through Arginine Decarboxylase (ADC) and Ornithine Decarboxylase (ODC), then N-methyl putrescine is formed by putrescine N-methyltransferase, N-methyl amino butyraldehyde is further obtained by oxidizing putrescine N-methyl oxidase, and the N-methyl amino butyraldehyde is further spontaneously converted into pyrrolidine ring necessary for synthesizing the nicotine; nicotinic acid is mainly formed by aspartic acid through pyridine nucleotide circulation, and then participates in the synthesis of nicotine; alternatively, tryptophan may be converted to quinolinic acid and then to niacin. Therefore, the qualitative and quantitative analysis of the nicotinic acid in the tobacco root system can be used for deeply knowing the synthesis path of the nicotine, and a certain theoretical basis is provided for guiding the analysis of the nitrogen/alkaloid metabolic path of the tobacco.
The existing method for measuring the nicotinic acid comprises a colorimetric method, a microbiological method, a capillary electrophoresis method and a high performance liquid chromatography. Wherein, the microbiological method and the high performance liquid chromatography are methods recommended by national standards of China for detecting nicotinic acid and nicotinamide in infant foods and dairy products. The principle of colorimetry is thatThe reaction needs to use high-toxicity cyanogen bromide, has relatively low sensitivity and poor specificity, and all substances containing pyridine rings are developed. The microbiological method is a classical method for determining nicotinic acid and nicotinamide, and the free nicotinic acid content or the total nicotinic acid content in food can be determined by using different microorganisms, but the experimental period of the method is long and the repeatability is poor. The ultra-high performance liquid chromatography-tandem mass spectrometry is another powerful means for detecting nicotinic acid and nicotinamide in foods, but the method has a remarkable matrix effect and is easy to be interfered by other impurities, so that the accuracy of results is affected.
Disclosure of Invention
Based on the above, the invention provides a method for analyzing nicotinic acid in tobacco root systems by using a derivatization-gas chromatography-mass spectrometry method, which aims to solve the technical problems of relatively low sensitivity, poor specificity and repeatability and high matrix effect in the detection of nicotinic acid in the prior art.
The technical scheme of the invention is as follows: a method for analyzing nicotinic acid in tobacco root systems by a derivatization-gas chromatography-mass spectrometry method, comprising the following steps of:
s1, extracting tobacco root system samples: adding an internal standard substance and an extraction solvent into a tobacco root system sample, mashing, homogenizing, performing ultrasonic extraction, and centrifugally filtering to obtain a tobacco root system extraction solution;
s2, purifying tobacco root system extraction solution: sequentially adding methanol and HCl to activate the solid-phase extraction column, slowly adding an extraction solution after activation, sequentially adding HCl and methanol for leaching, then adding an elution solution for eluting, collecting the elution solution, and then drying with nitrogen to obtain purified nicotinic acid;
s3 derivatization of nicotinic acid: adding acetone and a derivatization reagent into purified nicotinic acid, adding a buffer solution, uniformly mixing, and carrying out pentafluorobromide benzyl derivatization under the condition of water bath heating reaction to obtain a nicotinic acid derivatization product;
and S4, carrying out dispersion liquid microextraction on the nicotinic acid derivative: adding water and an extraction solvent into the nicotinic acid derivative product, mixing uniformly by vortex, adding NaCl, vortex again to completely dissolve the NaCl, centrifuging, and taking a precipitate phase to perform gas chromatography-mass spectrometry analysis in a micro-chromatographic bottle;
determination of S5 niacin derivatization products: separating and quantifying the nicotinic acid derivative product by adopting a gas chromatography-mass spectrometry-selective ion scanning mode, and qualitatively determining by comparing the retention time with a mass spectrum and quantifying by an internal standard method.
Optionally, in the extraction of the tobacco root system sample, the internal standard substance is isonicotinic acid.
Optionally, in the extraction of the tobacco root system sample, the extraction solvent is a dilute HCl solution.
Optionally, in the purification of the tobacco root system extraction solution, the solid phase extraction column is a PCX solid phase extraction column.
Optionally, in the purification of the tobacco root system extraction solution, the eluting solution is a mixed solution of ammonia water and methanol.
Alternatively, in the derivatization of niacin, the derivatizing agent is 2,3,4,5, 6-pentafluorobromide.
Optionally, in the derivatization of niacin, the buffer solution is phosphate buffer.
Optionally, in the derivatization of nicotinic acid, the derivatization reaction condition is that water bath heating reaction is carried out for 30-60min at 50-80 ℃.
Alternatively, in the liquid-liquid microextraction of the dispersion of the niacin derivative product, the extraction solvent is carbon tetrachloride.
Alternatively, the conditions for gas chromatography-mass spectrometry-selective ion scanning are: chromatographic column: DB-1701; sample injection amount: 1 μl; split ratio: 20:1, a step of; carrier gas: helium gas; flow rate: 1.0mL/min; sample inlet temperature: 280 ℃; programming temperature: the initial temperature is 50 ℃ for 3min, then the temperature is increased to 160 ℃ at the speed of 18 ℃ per minute for 4min, and then the temperature is increased to 260 ℃ at the speed of 20 ℃ for 5min; ion source temperature: 230 ℃, four-stage bar temperature: 150 ℃; ionization energy: 70eV, transmission line temperature: 280 ℃, full scan mass number range 35-400aum, solvent delay: 8.20min; acquisition mode: the full scan is acquired simultaneously with the selective ion scan.
The beneficial effects of the invention are as follows: the invention discloses a pretreatment method for solid phase extraction purification-pentafluorobromide derivatization-dispersion liquid microextraction, and adopts a gas chromatography-mass spectrum selective ion scanning method to qualitatively and quantitatively detect nicotinic acid in tobacco root systems. The method comprises the steps of carrying out pretreatment of solid-phase extraction purification, pentafluorobromine derivatization and dispersion liquid microextraction on a sample, and then adopting a gas chromatography-mass spectrometry selective ion scanning method to qualitatively and quantitatively detect nicotinic acid in tobacco root systems, and effectively combining three technologies of solid-phase extraction, pentafluorobromine derivatization and dispersion liquid microextraction.
The invention also has the following advantages: 1) The PCX solid phase extraction column (similar to a stationary phase) purification can effectively reduce the interference of impurities and improve the accuracy of the method; 2) The use of the isomer internal standard isonicotinic acid can improve the stability of the method; 3) 2,3,4,5, 6-pentafluorobromide derivatization is insensitive to water, and the derivatization product has high volatility and good gas chromatography detection adaptability; 4) The high enrichment factor of the dispersion liquid microextraction improves the sensitivity of nicotinic acid detection; 5) The detection method of gas chromatography-mass spectrometry selective ion scanning has less interference of impurity peaks, and further improves the accuracy.
Drawings
FIG. 1 is a flow chart of a method for analyzing nicotinic acid in tobacco root systems by a derivatization-gas chromatography-mass spectrometry method;
FIG. 2 is an overlay chromatogram (A) of isonicotinic acid and niacin in tobacco root samples and standards; amplified chromatograms (B) of isonicotinic acid and nicotinic acid pentafluorobromide derivative products, wherein 1 is isonicotinic acid and 2 is nicotinic acid, the nicotinic acid concentration of a root system sample is 1.2 mug/g, and the nicotinic acid concentration of a standard sample is 1.0 mug/mL;
FIG. 3 mass spectrum (A) of the pentafluorobromide derivative product of isonicotinic acid; a mass spectrum diagram (B) of a pentafluorobromide benzyl nicotinate derivative product, wherein m/z303 is a quantitative ion, and m/z181 and m/z106 are qualitative ions;
figure 4 nicotinic acid monograph linear correlation standard curve.
Detailed Description
Examples: derivatization-gas chromatography-mass spectrometry method analysis of nicotinic acid in tobacco root system
Referring to fig. 1, a method for analyzing nicotinic acid in tobacco root systems by using a derivatization-gas chromatography-mass spectrometry method comprises the following steps:
s1, extracting tobacco root system samples: weighing 0.5g of tobacco root system sample, putting the tobacco root system sample into a 50mL centrifuge tube, adding 10 mu L of 1mg/mL isonicotinic acid (prepared by water) and 10mL of 0.1MHCl extraction solution, homogenizing at a high speed, mashing at 20000rpm for 2min, performing ultrasonic extraction for 40min, centrifuging for 5min, and filtering by a water-based filter membrane to obtain the tobacco root system extraction solution.
S2, purifying tobacco root system extraction solution: sequentially adding 5mL of methanol and 5mL of 0.1MHCl to activate the PCX solid phase extraction column (similar to a stationary phase), slowly adding 5mL of tobacco root system extraction solution after activation, sequentially adding 1mL of 0.1MHCl and 1mL of methanol for leaching, then adding 3.5mL (v/v 1: 6) of ammonia water for eluting, collecting the eluting solution, and then drying with nitrogen to obtain purified nicotinic acid.
S3 derivatization of nicotinic acid: 1.2ml of acetone and 10 mu L of 2,3,4,5, 6-pentafluorobromide benzyl are added into nicotinic acid, the mixture is uniformly mixed after 0.2ml of phosphorus buffer solution with pH value of 6.8 is added, and the mixture is heated in a water bath at 60 ℃ to react for 40min for derivatization, so as to obtain a nicotinic acid derivatization product.
And S4, carrying out dispersion liquid microextraction on the nicotinic acid derivative: 5mL of water and 80. Mu.L of carbon tetrachloride microextraction solvent are added, vortex mixing is carried out, 0.3g of NaCl is added, vortex is carried out again to completely dissolve NaCl, centrifugation is carried out at 3000rpm for 5min, and then a precipitate phase is taken out in a micro-chromatographic bottle for gas chromatography-mass spectrometry analysis.
Determination of S5 niacin derivatization products: separating and quantifying the nicotinic acid derivative product by adopting a gas chromatography-mass spectrometry-selective ion scanning mode, and qualitatively comparing the retention time with a mass spectrum, and quantifying by an internal standard method (isonicotinic acid). The conditions for gas chromatography-mass spectrometry-selective ion scanning are as follows: gas chromatography (7890A-5975C); CTC multifunctional autosampler, chromatographic column: DB-1701 (30 m.times.0.25 mm i.d..times.0.25. Mu.md.f.); sample injection amount: 1 μl; split ratio: 20:1, a step of; carrier gas: helium gas; flow rate: 1.0mL/min; sample inlet temperature: 280 ℃; programming temperature: the initial temperature is 50 ℃ for 3min, then the temperature is increased to 160 ℃ at the speed of 18 ℃ per minute for 4min, and then the temperature is increased to 260 ℃ at the speed of 20 ℃ for 5min; ion source temperature: 230 ℃, four-stage bar temperature: 150 ℃; ionization energy: 70eV, transmission line temperature: 280 ℃, full scan mass number range 35-400aum, solvent delay: 8.20min; acquisition mode: the full scan is acquired simultaneously with the selective ion scan. The quantitative selection ion scanning mode is adopted, and m/z303 with smaller abundance is selected for quantification in consideration of that the ion m/z181 with the largest abundance is nonspecific fragment ion. Thus, the pentafluorobrominated benzyl derivative of isonicotinic acid has a retention time of 16.566min, a quantitative ion of m/z303 and a qualitative ion of m/z181 and m/z106; the penumbromide derivative of niacin has a retention time of 16.834min, a quantitative ion of m/z303 and a qualitative ion of m/z181 and m/z106, and the results are shown in FIGS. 2 and 3.
Verification example: the method of the invention verifies the linearity, recovery rate, precision and sensitivity
Establishing a standard curve of nicotinic acid by using isonicotinic acid as an internal standard, establishing a linear equation of standard curve y=bx+a in a certain concentration range by using the peak area ratio of nicotinic acid to isonicotinic acid as y and the mass ratio of nicotinic acid to isonicotinic acid as x, and when R 2 Above 0.999, linearity is good. The method is characterized in that the signal to noise ratio of the quantitative ion m/z303 in the selected ion scanning mode is larger than 3 and 10, and the method is the lowest detection limit and the lowest quantitative limit. The results are shown in tables 1 and 2 below and in FIG. 4: the linear equation is y= 1.6270x-0.0073 in the mass range of 0.02-2, R 2 The method has the advantages that the method is easy to operate, the linear relation is good, the lowest detection limit and the quantitative limit are respectively 0.006 mug/g and 0.018 mug/g, the detection limit of the method is low, and the method is suitable for detecting the trace nicotinic acid content composition in tobacco root systems.
Table 1 mass ratio of nicotinic acid to isonicotinic acid x and peak area ratio of m/z303 y in standard curve
TABLE 2 nicotinic acid standard curve and correlation coefficient and LOD and LOQ of method
The recovery rate of the method is verified by adopting a standard sample adding method, a certain amount of nicotinic acid is added into the tobacco root system, and the mixture is uniformly mixed and then is placed at 4 ℃ overnight, and then the extraction, purification, derivatization, microextraction and gas chromatography-mass spectrometry analysis are carried out. The recovery is the content of the corresponding target in the added sample minus the content of the target in the blank sample divided by the content of the added target. Two concentration levels of 0.5 and 5.0 μg/g were added to the tobacco root system, respectively, and the recovery was calculated. The precision of the method is that the two concentrations are added 5 times in the same day, the precision is the daily precision, and the two concentrations are repeated 5 times in 5 days, and the daily precision is the daily precision. Stability test of pentafluorobromide-derived products is that peak areas are measured and compared after the products are placed for 0, 6, 12, 18, 24, 36 and 48 hours at normal temperature, and corresponding relative standard deviations are obtained. The results are shown in Table 3 below: the recovery rate of nicotinic acid in the tobacco root system is 96.0% -98.0%, the daily precision is 2.6% -3.1%, the daily precision is 4.1% -5.3%, and the stability of the derivative product in 48h is lower than 2.0%. The above results indicate that the method meets strict quantitative requirements.
TABLE 3 additive recovery, precision and stability of niacin extraction in tobacco root systems
Application example 1: composition of different nitrogen application amounts on content of niacin in tobacco seedling root system
In order to study the variation trend of different nitrogen application amounts on nicotinic acid in tobacco seedling root systems, a variety K326 is used as a study object, a water culture (low-concentration nitrogen and high-concentration nitrogen) mode is adopted for culture, 3 repetitions are set for each treatment, and root systems are collected for measurement after 15d of growth. The content of niacin in the root systems of tobacco seedlings with different nitrogen application amounts was analyzed according to the method in the examples. The measurement results are shown in Table 4, and it can be seen from the following table that the niacin concentration in the low-concentration nitrogen tobacco seedling root system is lower than that in the high-concentration nitrogen tobacco seedling root system, and the niacin increase multiple is greater than that of the nitrogen increase multiple.
TABLE 4 influence of different Nitrogen application hydroponics on Nicotiana tabacum seedling root systems composition
Note that: high nitrogen concentration was 2 times that of low nitrogen concentration, and data were expressed as mean ± standard deviation (n=3)
Application example 2: composition of 2, 6-diisopropylaniline stress on content of nicotinic acid in tobacco seedling root system
4 gradients of 0.00 mug/mL, 1.77 mug/mL, 17.7 mug/mL and 88.5 mug/mL of 2, 6-diisopropylaniline are set to stress tobacco seedlings, 3 replicates are set for each treatment, and root systems are collected and measured after 15d of growth. The results of the measurement are shown in Table 5, and it can be seen from the following table that low concentration (1.77. Mu.g/mL) of 2, 6-diisopropylaniline inhibits nicotinic acid synthesis, the concentration of nicotinic acid increases significantly with increasing concentration, and the nicotinic acid content is significantly higher than that of the blank (0.00. Mu.g/mL) at high concentration (88.5. Mu.g/mL).
TABLE 5 influence of 2, 6-diisopropylaniline stress on the composition of niacin content in tobacco seedling roots
Note that: data are expressed as mean ± standard deviation (n=3)
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (1)
1. A method for analyzing nicotinic acid in tobacco root systems by a derivatization-gas chromatography-mass spectrometry method, which is characterized by comprising the following steps of:
s1, extracting tobacco root system samples: adding an internal standard substance and an extraction solvent into a tobacco root system sample, mashing, homogenizing, performing ultrasonic extraction, and centrifugally filtering to obtain a tobacco root system extraction solution, wherein the internal standard substance is isonicotinic acid, and the extraction solvent is a dilute HCl solution;
s2, purifying tobacco root system extraction solution: sequentially adding methanol and HCl to activate a solid-phase extraction column, slowly adding an extraction solution after activation, sequentially adding HCl and methanol for leaching, then adding an elution solution for eluting, collecting the elution solution, and then drying with nitrogen to obtain purified nicotinic acid, wherein the solid-phase extraction column is a PCX solid-phase extraction column, and the elution solution is a mixed solution of ammonia water and methanol;
s3 derivatization of nicotinic acid: adding acetone and a derivatization reagent into purified nicotinic acid, adding a buffer solution, uniformly mixing, and carrying out pentafluorobromide derivatization under the water bath heating reaction condition to obtain a nicotinic acid derivatization product, wherein the derivatization reagent is 2,3,4,5, 6-pentafluorobromide, the buffer solution is a phosphoric acid buffer solution, and the derivatization reaction condition is that the water bath heating reaction is carried out for 30-60min at 50-80 ℃;
and S4, carrying out dispersion liquid microextraction on the nicotinic acid derivative: adding water and an extraction solvent into the nicotinic acid derivative product, mixing uniformly by vortex, adding NaCl, vortex again to completely dissolve the NaCl, centrifuging, and taking a precipitate phase to perform gas chromatography-mass spectrometry in a micro-chromatographic bottle, wherein the extraction solvent is carbon tetrachloride;
determination of S5 niacin derivatization products: separating and quantifying the nicotinic acid derivative product by adopting a gas chromatography-mass spectrometry-selective ion scanning mode, and comparing and qualifying the nicotinic acid derivative product with a mass spectrum by using a retention time and an internal standard method for quantification, wherein the conditions of the gas chromatography-mass spectrometry-selective ion scanning are as follows: chromatographic column: DB-1701; sample injection amount: 1. mu L; split ratio: 20:1, a step of; carrier gas: helium gas; flow rate: 1.0mL/min; sample inlet temperature: 280 ℃; programming temperature: the initial temperature is 50 ℃ for 3min, then the temperature is increased to 160 ℃ at the speed of 18 ℃ per minute for 4min, and then the temperature is increased to 260 ℃ at the speed of 20 ℃ for 5min; ion source temperature: 230 ℃, four-stage bar temperature: 150 ℃; ionization energy: 70eV, transmission line temperature: 280 ℃, full scanning mass number range 35-400aum, solvent delay: 8.20min; acquisition mode: the full scan is acquired simultaneously with the selective ion scan.
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