CN114577936B - Separation detection method of beta-nicotinamide mononucleotide in capsule - Google Patents
Separation detection method of beta-nicotinamide mononucleotide in capsule Download PDFInfo
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- FZAQROFXYZPAKI-UHFFFAOYSA-N anthracene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC3=CC(S(=O)(=O)Cl)=CC=C3C=C21 FZAQROFXYZPAKI-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 239000002775 capsule Substances 0.000 title claims abstract description 60
- 238000001514 detection method Methods 0.000 title claims abstract description 52
- 238000000926 separation method Methods 0.000 title claims abstract description 50
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 69
- 238000000034 method Methods 0.000 claims abstract description 53
- 239000013558 reference substance Substances 0.000 claims abstract description 41
- 239000003085 diluting agent Substances 0.000 claims abstract description 37
- 150000002500 ions Chemical class 0.000 claims abstract description 24
- 239000012488 sample solution Substances 0.000 claims abstract description 18
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 15
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 15
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 15
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 42
- 239000000523 sample Substances 0.000 claims description 18
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 238000002604 ultrasonography Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 238000005349 anion exchange Methods 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 4
- 229920002307 Dextran Polymers 0.000 claims description 2
- 240000007594 Oryza sativa Species 0.000 claims description 2
- 235000007164 Oryza sativa Nutrition 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 235000013312 flour Nutrition 0.000 claims description 2
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 claims description 2
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 claims description 2
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 claims description 2
- 229960003943 hypromellose Drugs 0.000 claims description 2
- 238000010829 isocratic elution Methods 0.000 claims description 2
- 235000019359 magnesium stearate Nutrition 0.000 claims description 2
- 239000001814 pectin Substances 0.000 claims description 2
- 235000010987 pectin Nutrition 0.000 claims description 2
- 229920001277 pectin Polymers 0.000 claims description 2
- 235000009566 rice Nutrition 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 229920000642 polymer Polymers 0.000 claims 1
- DAYLJWODMCOQEW-TURQNECASA-N NMN zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP(O)([O-])=O)O2)O)=C1 DAYLJWODMCOQEW-TURQNECASA-N 0.000 abstract description 52
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 5
- 238000002360 preparation method Methods 0.000 abstract description 5
- 238000005342 ion exchange Methods 0.000 abstract description 2
- 238000000825 ultraviolet detection Methods 0.000 abstract description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 14
- 239000011550 stock solution Substances 0.000 description 9
- 238000005303 weighing Methods 0.000 description 9
- 239000012490 blank solution Substances 0.000 description 8
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 7
- 235000005152 nicotinamide Nutrition 0.000 description 7
- 239000011570 nicotinamide Substances 0.000 description 7
- 229960003966 nicotinamide Drugs 0.000 description 7
- 238000007872 degassing Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 4
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 4
- 235000001968 nicotinic acid Nutrition 0.000 description 4
- 239000011664 nicotinic acid Substances 0.000 description 4
- 229960003512 nicotinic acid Drugs 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 238000005251 capillar electrophoresis Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000010812 external standard method Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- YABIFCKURFRPPO-IVOJBTPCSA-N 1-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyridin-1-ium-3-carboxamide;chloride Chemical compound [Cl-].NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 YABIFCKURFRPPO-IVOJBTPCSA-N 0.000 description 2
- 241000026010 Dendrobium candidum Species 0.000 description 2
- BAWFJGJZGIEFAR-NNYOXOHSSA-O NAD(+) 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-O 0.000 description 2
- 239000003792 electrolyte Substances 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- SHFJWMWCIHQNCP-UHFFFAOYSA-M hydron;tetrabutylazanium;sulfate Chemical compound OS([O-])(=O)=O.CCCC[N+](CCCC)(CCCC)CCCC SHFJWMWCIHQNCP-UHFFFAOYSA-M 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 240000006499 Flammulina velutipes Species 0.000 description 1
- 235000016640 Flammulina velutipes Nutrition 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
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical group [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036995 brain health Effects 0.000 description 1
- 230000005189 cardiac health Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002013 hydrophilic interaction chromatography Methods 0.000 description 1
- 150000008040 ionic compounds Chemical class 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 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
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000013441 quality evaluation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—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 using ion-exchange
-
- 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
- G01N2030/065—Preparation using different phases to separate parts of sample
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- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Pathology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- General Physics & Mathematics (AREA)
- Library & Information Science (AREA)
- Engineering & Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a separation and detection method of beta-nicotinamide mononucleotide (NicotinamideMononucleotide, NMN) in a capsule. The detection method comprises the following steps: (1) preparation of a solution: dissolving the content of the capsule in a diluent to prepare a sample solution; dissolving a beta-nicotinamide mononucleotide reference substance in a diluent to prepare a reference substance solution; (2) And respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of the beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph. The ion chromatography of the invention directly adopts the ion exchange chromatographic column for separation, the mobile phase only uses simple ammonium acetate and acetonitrile, no ion pair reagent is required to be added, and the method for ultraviolet detection of NMN is rapid and simple, has low cost and is convenient for popularization.
Description
Technical Field
The invention belongs to the technical field of drug detection and analysis, and particularly relates to a separation and detection method of beta-nicotinamide mononucleotide in a capsule.
Background
Beta-Nicotinamide Mononucleotide (NMN) is a key intermediate for the synthesis of coenzyme I-NAD+ and is present in various organisms. As NAD+ is widely involved in various reactions in the body, the nicotinamide mononucleotide is very important for human health, and the NAD+ level in the body can be improved by taking the nicotinamide mononucleotide, so that the health-care functions of resisting aging, promoting heart and brain health and the like are achieved.
The structural formula of NMN is shown as follows:
beta-Nicotinamide Mononucleotide (NMN) belongs to a strong polar compound, and is basically not reserved on a conventional C18 chromatographic column when analyzed by liquid chromatography, and is easily influenced by a matrix. The retention of NMN on the column can be slightly enhanced by adding an ion pair reagent such as tetrabutylammonium bisulfate in the mobile phase, but is generally only slightly longer than the dead volume retention, with very limited ability to exclude interference.
In order to reduce interference of coexisting substances and improve specificity of a method, liquid chromatography-mass spectrometry is mostly adopted to detect NMN in a sample in the current research work, but mass spectrometers are very expensive in millions, and have high requirements on technical level of operators and high maintenance cost. The mass spectrometry method can obviously greatly improve the detection difficulty and cost, and is inconvenient to popularize.
Document 1 (Liu Xiaoqian et al UPLC-MS/MS) discloses a separation and detection method of beta-nicotinamide mononucleotide in dendrobium candidum and homologous dendrobium candidum varieties thereof, wherein the content of nicotinamide mononucleotide and nicotinamide adenine dinucleotide is 2021,8:4034-4039, the document adopts an LC-MSMS method, C18 column separation is carried out, NMN peak-out time is within 1min, the time is too short, the influence of sample matrixes is extremely easy, the cost of an MS/MS instrument is high, and the method popularization is inconvenient.
Document 2 (Liu Xiaofang et al, high performance liquid chromatography-tandem mass spectrometry to determine the content of nicotinamide mononucleotide in food materials, food science and technology 2021, 46 (08): 251-256, 262) discloses a separation and detection method of beta-nicotinamide mononucleotide, which also adopts an expensive LC-MSMS method, compares a C18 column, an HILIC column and a WAX column, and finds that NMN on the former two chromatographic columns is not reserved, the peak time is within 2min, and the NMN peak time on the WAX chromatographic column is about 7 min. The WAX chromatographic column is a weak anion exchange chromatographic column with a silica gel matrix, has strong retention time for monovalent ionic compounds, is difficult to elute for multivalent ions, has short service life due to narrow tolerance pH range and the like, and has high overall detection cost.
CN113295779a discloses a method for rapidly determining beta-nicotinamide mononucleotide in a health-care product, the method also determines by LC-MSMS method, C18 column separation is performed, and the response value and sensitivity of beta-nicotinamide mononucleotide are improved by adjusting parameters. There is also the problem that the chromatographic column is difficult to retain and is susceptible to matrix effects.
CN113189185A discloses a capillary electrophoresis determination method of beta-nicotinamide mononucleotide in flammulina velutipes, the capillary electrophoresis method has higher column efficiency compared with the traditional liquid chromatography, and the use of reagents is relatively safe. However, the capillary electrophoresis method is generally poor in stability, extremely sensitive to the pH value of the solution and the electrolyte consumption, high in requirement on the operation level of experimenters and poor in reproducibility. The electrolyte contained in the sample also affects separation, so that the method has poor universality and is not suitable for detecting samples consisting of different formulas, and therefore, the popularization is difficult.
Therefore, a method for separating and detecting the beta-nicotinamide mononucleotide in the capsule is developed, which is quick, simple, low in cost and convenient to popularize.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a separation detection method of beta-nicotinamide mononucleotide (Nicotinamide Mononucleotide, NMN). The ion chromatography directly adopts a strong anion exchange chromatographic column for separation, does not need to add ion pair reagents, and detects NMN through ultraviolet, so that the method is quick, simple, low in cost and convenient to popularize.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
The invention provides a separation and detection method of beta-nicotinamide mononucleotide in a capsule, which comprises the following steps:
(1) Preparing a solution: dissolving the content of the capsule in a diluent to prepare a sample solution; dissolving a beta-nicotinamide mononucleotide reference substance in a diluent to prepare a reference substance solution;
(2) And respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of the beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph.
In the invention, the ion chromatography is directly adopted to carry out separation by adopting a strong ion exchange chromatographic column, and the invention selects a proper sample treatment mode and a proper reference substance, so that the content of NMN in the content of the capsule can be rapidly and accurately detected, and the precision, accuracy, recovery rate and the like of the NMN detection method are good, and the detection requirement of the sample is met, thus the method can be applied to the quality evaluation of the preparation. Meanwhile, the method has the advantages of being quick, simple, low in cost and convenient to popularize.
Preferably, in step (1), the content of the capsule comprises: beta-nicotinamide mononucleotide, rice flour, hypromellose, magnesium stearate, silica, pectin, and dextran.
Preferably, in step (1), the diluent comprises acetonitrile and water, wherein the volume ratio of acetonitrile to water is (2-4): (1-3), and for example, may be 2:1, 3:1, 4:1, 2:2, 3:2, 2:3, 4:3, and the like, and preferably is 3:2.
Preferably, in step (1), the ratio of the mass of the content to the volume of the diluent in the sample solution is (0.0005-1.0) mg/1.0 mL, for example, 0.0005 mg/1.0 mL, 0.01 mg/1.0 mL, 0.1 mg/1.0 mL, 0.25 mg/1.0 mL, 0.5 mg/1.0 mL, 0.75 mg/1.0 mL, 1.0 mg/1.0 mL, etc., preferably 0.5 mg/1.0 mL.
Preferably, in the step (1), the ratio of the mass of the reference substance to the volume of the diluent (0.0005-1.0) in the reference substance solution may be, for example, 0.0005mg:1.0mL, 0.01mg:1.0mL, 0.1mg:1.0mL, 0.25mg:1.0mL, 0.5mg:1.0mL, 0.75mg:1.0mL, 1.0mg:1.0mL, etc., preferably 0.5mg:1.0mL.
Preferably, in the step (1), the dissolution is performed under ultrasound, and the time of the ultrasound is 10-30min, for example, 10min, 12min, 14min, 16min, 18min, 20min, 22min, 24min, 26min, 28min, 30min, etc., and the power of the ultrasound is 200-600W, for example, 200W, 250W, 300W, 350W, 400W, 450W, 500W, 550W, 600W, etc.
Preferably, in step (2), the mobile phase a used in the detection is acetonitrile, and the mobile phase B is an aqueous ammonium acetate solution.
Preferably, the concentration of the aqueous ammonium acetate solution is 80-120mM, for example, 80mM, 85mM, 90mM, 95mM, 100mM, 105mM, 110mM, 115mM, 120mM, etc., preferably 100mM.
Preferably, in step (2), isocratic elution is used in the detection, and the volume ratio of mobile phase a to mobile phase B is (35-45): (65-55), for example, may be 35:65, 36:64, 38:62, 40:60, 42:58, 44:56, 45:55, and preferably is 40:60.
Preferably, in the step (2), the ion chromatograph column is a Dionex IonPac AS16 or a Dionex IonPac AS18 anion exchange column.
Preferably, the length of the chromatographic column is 50-300mm, for example, 50mm, 100mm, 150mm, 200mm, 250mm, 300mm, etc., preferably 250mm; the inner diameter of the column is 2-5mm, preferably 4mm.
Preferably, the column temperature of the chromatographic column is 30-60 ℃, for example, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, etc., preferably 40 ℃.
Preferably, in the step (2), the flow rate of the mobile phase in the detection is 0.8-1.2mL/min, for example, 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, and the like, preferably 1.0mL/min.
Preferably, in the step (2), the sample injection amount in the detection is 5 to 50. Mu.L, for example, 5. Mu.L, 10. Mu.L, 20. Mu.L, 25. Mu.L, 50. Mu.L, etc., preferably 10. Mu.L.
Preferably, in step (2), the wavelength of the detection is 250-280nm, for example 250nm, 255nm, 260nm, 265nm, 270nm, 280nm, etc., preferably 260nm.
Preferably, in step (2), the analysis time of the detection is 6-10min, for example, 6min, 7min, 8min, 9min, 10min, and the like, preferably 8min.
As a preferred technical scheme of the invention, the detection method comprises the following steps:
(1) Preparing a solution: dissolving the content of the capsule in the diluent for 10-30min by ultrasonic treatment to prepare a sample solution, wherein the volume ratio of the mass of the content to the diluent is (1-1.5) mg to 1.0mL;
Dissolving a beta-nicotinamide mononucleotide reference substance in a diluent for 10-30min by ultrasound, and preparing a reference substance solution, wherein the volume ratio (0.1-1) of the reference substance to the diluent is 1.0mL;
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
wherein the chromatographic conditions of the ion chromatography include the following:
The mobile phase A is acetonitrile, the mobile phase B is 80-120mM ammonium acetate aqueous solution, and the volume ratio of the mobile phase A to the mobile phase B is (35-45) (65-55); the chromatographic column is a Dionex IonPac AS16 anion exchange chromatographic column, the length is 50-300mm, the inner diameter is 3-5mm, and the column temperature is 30-60 ℃; the flow rate of the mobile phase is 0.8-1.2mL/min, and the sample injection amount is 5-50 mu L; the wavelength of detection is 250-280nm, and the analysis time is 6-10min.
Compared with the prior art, the invention has the following beneficial effects:
The ion chromatography of the invention directly adopts a strong anion exchange chromatographic column for separation, the mobile phase only uses simple ammonium acetate and acetonitrile, no ion pair reagent is required to be added, the method for ultraviolet detection of NMN is rapid and simple, the cost is low, and the method is convenient to popularize.
Drawings
FIG. 1 is a chromatogram of a blank solution spectrum.
FIG. 2 is a chromatogram of the control solution.
FIG. 3 is a NAM, NA, NMN and NR overlay.
FIG. 4 is a graph of quantitative limiting solutions.
FIG. 5 is a diagram of the limit of detection solution.
Fig. 6 is a standard graph.
Fig. 7 is a chromatogram of sample 1 provided in example 1.
Fig. 8 is a chromatogram of sample 2 provided in example 2.
Fig. 9 is a chromatogram of sample 3 provided in example 3.
Fig. 10 is a comparative chromatogram of examples 4 and 5.
FIG. 11 is a comparative chromatogram of examples 6 and 7.
Fig. 12 is a comparative chromatogram of examples 8 and 9.
Fig. 13 is a partial enlarged view of fig. 12.
Fig. 14 is a comparative chromatogram of examples 10 and 11.
Fig. 15 is a partial enlarged view of embodiment 10 of fig. 2.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The sources of the components of the following examples and comparative examples are shown below:
| Name of the name | Manufacturer' s | Make/model specification |
| Ion chromatograph | Thermo Scientific | Dionex ICS-5000 |
| Dionex IonPac AS16 | Thermo Scientific | 250mm×4mm |
| Beta-nicotinamide mononucleotide control | Aladdin | Purity: 98.80% |
Example 1
The embodiment provides a separation detection method of beta-nicotinamide mononucleotide in a capsule, which comprises the following steps:
(1) Preparation of the solution:
Mobile phase a: acetonitrile, ultrasonic degassing for 10min;
mobile phase B: performing ultrasonic degassing on 100mM ammonium acetate aqueous solution for 10min;
Blank solution: acetonitrile-water (3:2 by volume) was also used as diluent. (FIG. 1 is a chromatogram of a blank solution spectrum, as shown in FIG. 1, baseline plateau);
Control stock solution: weighing about 100mg of NMN reference substance, placing in a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance stock solution with concentration of 10 mg/mL;
control solution: weighing 0.5mL of reference substance stock solution, placing into a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance solution with concentration of 0.5mg/mL (FIG. 2 is a chromatogram of the reference substance solution, as shown in FIG. 2, NMN shows a peak at 4.563min position, and 2.330min small peak is predicted to be nicotinamide);
Test solution: weighing 30mg (corresponding to 12.5mg containing NMN) of capsule content, placing into 25mL measuring flask, adding diluent, dissolving with ultrasound for 20min, diluting to scale, shaking, filtering to obtain sample solution, and preparing two parts in parallel.
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
wherein the chromatographic conditions of the ion chromatography include the following:
(3) Detection result
(A) Anti-interference test:
FIG. 3 is a plot of NAM, NA, NMN superimposed on NR, as shown in FIG. 3, nicotinamide riboside chloride NR, nicotinic acid NA, and nicotinamide NAM chromatograms without interference with nicotinamide mononucleotide NMN detection. Wherein, the nicotinic acid NA can synthesize nicotinamide NAM, then synthesize nicotinamide riboside chloride NR, and then synthesize nicotinamide mononucleotide NMN. Whereas nicotinamide mononucleotide NMN degradation also produces nicotinamide NAM and niacin NA.
(B) Quantitative limiting solution:
FIG. 4 is a graph of quantitative limiting solution, as shown in FIG. 4, the control solution is diluted stepwise to have a concentration of 0.5026 μg/mL, 6 needles are continuously injected, the RSD of the peak area is 5.1%, and the signal-to-noise ratio is between 41.0 and 55.9 and is not less than 10.
(C) Detection limit solution:
FIG. 5 is a graph of the limit of detection solution, as shown in FIG. 5, the control solution is diluted stepwise to a concentration of 0.2010 μg/mL, and the signal-to-noise ratio is 18.3, not less than 3.
(D) Linearity and measurement range (0.0005 mg/mL-1.00 mg/mL):
The control solution was diluted stepwise, and peak areas of the corresponding concentrations were measured by ion chromatography, respectively, and standard working curves were made, the linearity and range of which are shown in table 1 below, and the standard curves are shown in fig. 6:
TABLE 1
| Numbering device | NMN concentration (mg/mL) | NMN peak area (mAU min) |
| LOQ | 0.0005026 | 0.0489 |
| L2 | 0.01005 | 0.9521 |
| L3 | 0.1005 | 9.5291 |
| L4 | 0.2513 | 30.8494 |
| L5 | 0.5026 | 62.0800 |
| L6 | 0.7538 | 95.2373 |
| L7 | 1.0051 | 124.8955 |
Fig. 6 is a standard graph, where y= 125.9103x-0.9676, r=0.9997.
(E) Content determination of test sample
Fig. 7 is a chromatogram of sample 1 provided in example 1, as shown in fig. 7, NMN shows a peak at 4.560min, the peak area is 60.3530mau min, and the mass content of NMN in the content of sample 1 is 39.3% calculated by the reference peak area external standard method.
Example 2
The embodiment provides a separation detection method of beta-nicotinamide mononucleotide in a capsule, which comprises the following steps:
(1) Preparation of the solution:
Mobile phase a: acetonitrile, ultrasonic degassing for 10min;
mobile phase B: performing ultrasonic degassing on 100mM ammonium acetate aqueous solution for 10min;
Blank solution: acetonitrile-water (3:2 by volume) was also used as diluent. (FIG. 1 is a chromatogram of a blank solution spectrum, as shown in FIG. 1, baseline plateau);
Control stock solution: weighing about 100mg of NMN reference substance, placing in a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance stock solution with concentration of 10 mg/mL;
control solution: weighing 0.5mL of reference substance stock solution, placing into a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance solution with concentration of 0.5mg/mL (FIG. 2 is a chromatogram of the reference substance solution, as shown in FIG. 2, NMN shows a peak at 4.563min position, and 2.330min small peak is predicted to be nicotinamide);
Test solution: weighing 30mg (corresponding to 12.5mg containing NMN) of capsule content, placing into 25mL measuring flask, adding diluent, dissolving with ultrasound for 20min, diluting to scale, shaking, filtering to obtain sample solution, and preparing two parts in parallel.
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
wherein the chromatographic conditions of the ion chromatography include the following:
(3) Detection result
Fig. 8 is a chromatogram of the test article 2 provided in example 2, as shown in fig. 8, the test article 2 contains β -nicotinamide mononucleotide NMN, nicotinic acid NA, and nicotinamide NAM at the same time, wherein the NMN peak area is 38.1928mau×min, and the NMN mass content in the content of the test article 2 is 24.6% calculated by the reference peak area external standard method.
Example 3
The embodiment provides a separation detection method of beta-nicotinamide mononucleotide in a capsule, which comprises the following steps:
(1) Preparation of the solution:
Mobile phase a: acetonitrile, ultrasonic degassing for 10min;
mobile phase B: performing ultrasonic degassing on 100mM ammonium acetate aqueous solution for 10min;
Blank solution: acetonitrile-water (3:2 by volume) was also used as diluent. (FIG. 1 is a chromatogram of a blank solution spectrum, as shown in FIG. 1, baseline plateau);
Control stock solution: weighing about 100mg of NMN reference substance, placing in a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance stock solution with concentration of 10 mg/mL;
control solution: weighing 0.5mL of reference substance stock solution, placing into a 10mL measuring flask, adding diluent to dissolve and dilute to scale, and shaking to obtain reference substance solution with concentration of 0.5mg/mL (FIG. 2 is a chromatogram of the reference substance solution, as shown in FIG. 2, NMN shows a peak at 4.563min position, and 2.330min small peak is predicted to be nicotinamide);
Test solution: weighing 30mg (corresponding to 12.5mg containing NMN) of capsule content, placing into 25mL measuring flask, adding diluent, dissolving with ultrasound for 20min, diluting to scale, shaking, filtering to obtain sample solution, and preparing two parts in parallel.
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
wherein the chromatographic conditions of the ion chromatography include the following:
(3) Detection result
Fig. 9 is a chromatogram of sample 3 provided in example 3, as shown in fig. 9, NMN shows a peak at 4.557min, the peak area is 44.8099mau min, and the mass content of NMN in the content of sample 3 is 28.7% calculated by the reference peak area external standard method.
Examples 4 to 5
This example provides a method for the separation and detection of β -nicotinamide mononucleotide in two capsules, differing from example 1 only in the volume ratio of acetonitrile to water in the blank solution (diluent), as shown in table 2 below:
TABLE 2
FIG. 10 is a graph comparing examples 4 and 5, as shown in FIG. 10, the retention behavior and peak area effect of the volume ratio of acetonitrile to water in the diluent is less, preferably 3:2.
Examples 6 to 7
This example provides a method for the separation and detection of β -nicotinamide mononucleotide in two capsules, differing from example 1 only in the volume ratio of acetonitrile to 100mM aqueous ammonium acetate in the mobile phase, as shown in table 3 below:
TABLE 3 Table 3
FIG. 11 is a graph comparing examples 6 and 7, wherein the volume ratio of acetonitrile to water in the diluent is preferably 40:60, as shown in FIG. 11; the lower acetonitrile content and the higher acetonitrile content can lead to the longer overall analysis time; more acetonitrile and less water can cause distortion of the NMN peak shape.
Examples 8 to 9
This example provides a method for the separation and detection of β -nicotinamide mononucleotide in two capsules, differing from example 1 only in the selection of mobile phase a and mobile phase B, as shown in table 4 below:
TABLE 4 Table 4
FIG. 12 is a graph comparing examples 8 and 9, wherein NMN shows a significantly blunted peak shape when mobile phase A is methanol and mobile phase B is ammonium acetate, and the theoretical plate number is only 40%; when mobile phase a is acetonitrile and mobile phase B is ammonium formate, NMN cannot be separated from the previously unknown impurities, and fig. 13 is a partial enlarged view; according to the application, the mobile phase A is acetonitrile, and the mobile phase B is an ammonium acetate aqueous solution, so that the aim of well separating and detecting the beta-nicotinamide mononucleotide can be achieved.
Examples 10 to 11
This example provides a method for the separation and detection of β -nicotinamide mononucleotide in two capsules, differing from example 1 only in the chromatographic column, as shown in table 5 below:
TABLE 5
FIG. 14 is a graph comparing examples 10 and 11, AS shown in FIG. 14, the NMN chromatographic peak obtained by the AS18 chromatographic column has good column effect, but the peak is too fast, and the separation capacity from impurities is relatively limited, AS shown in FIG. 15; the NMN chromatographic peak column effect obtained by adopting the AS15 chromatographic column is lower, the retention time is slightly short, and the separation capability is poor; the Dionex IonPac AS16 can be used for separating and detecting beta-nicotinamide mononucleotide.
The applicant states that the present invention describes the method of separation detection of β -nicotinamide mononucleotide in the capsules of the present invention by the above examples, but the present invention is not limited to the above process steps, i.e. it does not mean that the present invention must be carried out in dependence on the above process steps. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of selected raw materials, addition of auxiliary components, selection of specific modes, etc. fall within the scope of the present invention and the scope of disclosure.
Claims (27)
1. A method for separating and detecting beta-nicotinamide mononucleotide in a capsule, which is characterized by comprising the following steps of:
(1) Preparing a solution: dissolving the content of the capsule in a diluent to prepare a sample solution; dissolving a beta-nicotinamide mononucleotide reference substance in a diluent to prepare a reference substance solution;
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
In the step (2), the chromatographic column for the ion chromatograph is a Dionex IonPac AS16 strong anion exchange polymer matrix chromatographic column; the mobile phase A used for detection is acetonitrile, and the mobile phase B is an ammonium acetate aqueous solution; isocratic elution is adopted in the detection, and the volume ratio of the mobile phase A to the mobile phase B is (35-45) (65-55).
2. The method for the separation and detection of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in step (1), the content of the capsule comprises: beta-nicotinamide mononucleotide, rice flour, hypromellose, magnesium stearate, silica, pectin, and dextran.
3. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein in the step (1), the diluent comprises acetonitrile and water, and the volume ratio of acetonitrile to water is (2-4): 1-3.
4. A method for the separation and detection of β -nicotinamide mononucleotide in a capsule according to claim 3, wherein the volume ratio of acetonitrile to water is 3:2.
5. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in the step (1), the ratio of the mass of the content to the volume of the diluent in the sample solution is (0.0005-1.0) mg/1.0 mL.
6. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 5, wherein the volume ratio of the mass of the content to the diluent is 0.5 mg/1.0 ml.
7. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in the step (1), the ratio of the mass of the reference substance to the volume of the diluent in the reference substance solution is (0.0005-1.0) mg/1.0 mL.
8. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 7, wherein the ratio of the mass of the reference substance to the volume of the diluent is 0.5 mg/1.0 ml.
9. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in the step (1), the dissolution is performed under ultrasound for 10 to 30 minutes, and the power of the ultrasound is 200 to 600W.
10. The method for separating and detecting beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the concentration of the ammonium acetate aqueous solution is 80-120mM.
11. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 10, wherein the concentration of the ammonium acetate aqueous solution is 100mM.
12. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the volume ratio of mobile phase A to mobile phase B is 40:60.
13. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the length of the chromatographic column is 50-300mm.
14. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 13, wherein the length of the chromatographic column is 250mm.
15. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the inner diameter of the chromatographic column is 2-5mm.
16. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 15, wherein the inner diameter of the chromatographic column is 4mm.
17. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the column temperature of the chromatographic column is 30-60 ℃.
18. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 17, wherein the column temperature of the chromatographic column is 40 ℃.
19. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in step (2), the flow rate of the mobile phase in the detection is 0.8-1.2mL/min.
20. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 19, wherein the flow rate of a mobile phase in the detection is 1.0mL/min.
21. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in the step (2), the sample injection amount in the detection is 5 to 50 μl.
22. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 21, wherein the sample injection amount in the detection is 10 μl.
23. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in step (2), the wavelength of the detection is 250 to 280nm.
24. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 23, wherein the wavelength of detection is 260nm.
25. The method for detecting the separation of β -nicotinamide mononucleotide in a capsule according to claim 1, wherein in step (2), the analysis time of the detection is 6 to 10min.
26. The method for detecting the separation of beta-nicotinamide mononucleotide in a capsule according to claim 25, wherein the analysis time of the detection is 8min.
27. The method for the separation and detection of beta-nicotinamide mononucleotide in a capsule according to claim 1, wherein the detection method comprises the following steps:
(1) Preparing a solution: dissolving the content of the capsule in the diluent for 10-30min by ultrasonic treatment to prepare a sample solution, wherein the volume ratio of the mass of the content to the diluent is (0.0005-1.0) mg/1.0 mL;
dissolving a beta-nicotinamide mononucleotide reference substance in a diluent in an ultrasonic manner for 10-30min to prepare a reference substance solution, wherein the volume ratio (0.0005-1.0) of the reference substance to the diluent is 1.0mL;
(2) Respectively injecting the sample solution and the reference substance solution into an ion chromatograph, and detecting the content of beta-nicotinamide mononucleotide in the capsule by using the ion chromatograph;
wherein the chromatographic conditions of the ion chromatography include the following:
The mobile phase A is acetonitrile, the mobile phase B is 80-120mM ammonium acetate aqueous solution, and the volume ratio of the mobile phase A to the mobile phase B is (35-45) (65-55); the chromatographic column is a Dionex IonPac AS16 strong anion exchange chromatographic column, the length is 50-300mm, the inner diameter is 2-5mm, and the column temperature is 30-60 ℃; the flow rate of the mobile phase is 0.8-1.2mL/min, and the sample injection amount is 5-50 mu L; the wavelength of detection is 250-280nm, and the analysis time is 6-10min.
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