CN114235986A - High performance liquid chromatography detection method for contents of vitamins B1, B2, B6, nicotinic acid and nicotinamide - Google Patents
High performance liquid chromatography detection method for contents of vitamins B1, B2, B6, nicotinic acid and nicotinamide Download PDFInfo
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- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 title claims abstract description 158
- 239000011570 nicotinamide Substances 0.000 title claims abstract description 90
- 235000005152 nicotinamide Nutrition 0.000 title claims abstract description 87
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 229960003966 nicotinamide Drugs 0.000 title claims abstract description 62
- 238000001514 detection method Methods 0.000 title claims abstract description 60
- 229960003512 nicotinic acid Drugs 0.000 title claims abstract description 51
- 239000011691 vitamin B1 Substances 0.000 title claims abstract description 35
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 24
- 235000010374 vitamin B1 Nutrition 0.000 title description 8
- 229930003270 Vitamin B Natural products 0.000 claims abstract description 41
- 235000019156 vitamin B Nutrition 0.000 claims abstract description 41
- 239000011720 vitamin B Substances 0.000 claims abstract description 41
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 34
- 235000001968 nicotinic acid Nutrition 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000008569 process Effects 0.000 claims abstract description 9
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims abstract 5
- 239000000523 sample Substances 0.000 claims description 107
- 239000000243 solution Substances 0.000 claims description 55
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 38
- 238000000605 extraction Methods 0.000 claims description 36
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 238000005303 weighing Methods 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 22
- 239000012086 standard solution Substances 0.000 claims description 20
- 229960000583 acetic acid Drugs 0.000 claims description 19
- 239000012362 glacial acetic acid Substances 0.000 claims description 19
- QWSZRRAAFHGKCH-UHFFFAOYSA-M sodium;hexane-1-sulfonate Chemical compound [Na+].CCCCCCS([O-])(=O)=O QWSZRRAAFHGKCH-UHFFFAOYSA-M 0.000 claims description 19
- 239000012528 membrane Substances 0.000 claims description 14
- 238000004811 liquid chromatography Methods 0.000 claims description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- 238000001816 cooling Methods 0.000 claims description 12
- 238000010438 heat treatment Methods 0.000 claims description 12
- 229940066429 octoxynol Drugs 0.000 claims description 12
- 229920002113 octoxynol Polymers 0.000 claims description 12
- 239000012488 sample solution Substances 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 10
- 229960004543 anhydrous citric acid Drugs 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229940088594 vitamin Drugs 0.000 claims description 6
- 229930003231 vitamin Natural products 0.000 claims description 6
- 235000013343 vitamin Nutrition 0.000 claims description 6
- 239000011782 vitamin Substances 0.000 claims description 6
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 6
- 235000013405 beer Nutrition 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 2
- 238000010828 elution Methods 0.000 claims description 2
- 238000010561 standard procedure Methods 0.000 abstract description 4
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 abstract description 3
- 238000012545 processing Methods 0.000 abstract description 2
- 238000004904 shortening Methods 0.000 abstract description 2
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 19
- 239000011716 vitamin B2 Substances 0.000 description 9
- 239000011726 vitamin B6 Substances 0.000 description 9
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 7
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 6
- 229960003495 thiamine Drugs 0.000 description 5
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 5
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 4
- 229930003451 Vitamin B1 Natural products 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 229960002477 riboflavin Drugs 0.000 description 4
- 235000019164 vitamin B2 Nutrition 0.000 description 4
- 235000019158 vitamin B6 Nutrition 0.000 description 4
- 229940011671 vitamin b6 Drugs 0.000 description 4
- 229930003471 Vitamin B2 Natural products 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000040350 B family Human genes 0.000 description 2
- 108091072128 B family Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- JBYSVQKTMBXLKJ-UHFFFAOYSA-N pyridine-3-carboxamide;pyridine-3-carboxylic acid Chemical compound NC(=O)C1=CC=CN=C1.OC(=O)C1=CC=CN=C1 JBYSVQKTMBXLKJ-UHFFFAOYSA-N 0.000 description 1
- 235000008160 pyridoxine Nutrition 0.000 description 1
- 239000011677 pyridoxine Substances 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007901 soft capsule Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 230000009044 synergistic interaction Effects 0.000 description 1
- 235000019157 thiamine Nutrition 0.000 description 1
- 239000011721 thiamine Substances 0.000 description 1
- 235000019160 vitamin B3 Nutrition 0.000 description 1
- 239000011708 vitamin B3 Substances 0.000 description 1
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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
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- 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)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a vitamin B1、B2、B6The high performance liquid chromatography detection method of the content of the nicotinic acid and the nicotinamide dissolves the sample by the extract liquor and effectively dissolves the vitamin B1、B2、B6Nicotinic acid and nicotinamide are separated out and then the extract is used for fixing the volume of the sample, thereby greatly shortening the sample processing time and providing vitamin B for various contents1、B2、B6The niacin and the nicotinamide are effectively separated, and the vitamin B is measured by applying the high performance liquid chromatography1、B2、B6The method of nicotinic acid and nicotinamide, the selection of the sample treatment process and the chromatographic condition, and finally the vitamin B in the single-component and complex-component samples can be simply and accurately treated1、B2、B6And measuring the content of nicotinic acid and nicotinamide. The invention discloses a methodThe method replaces the traditional national standard method for measuring vitamin B1、B2、B6Nicotinic acid, nicotinamide.
Description
Technical Field
The invention relates to the technical field of chromatographic detection, in particular toVitamin B1、B2、B6And a high performance liquid chromatography detection method for the content of nicotinic acid and nicotinamide.
Background
The vitamin B group includes vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (nicotinic acid), vitamin B6 (pyridoxine), etc. They are classified as a family because of their many common properties (e.g., water solubility, coenzymes, etc.) and the need for synergistic interaction.
If the vitamin B group is regarded as a football team, each vitamin B is like a player, and a complete team is required to get on the field and each player plays own jobs to fight cooperatively. Given that there are insufficient players or that only individual players are present, that is an absolute poor kick. This is true of the vitamin B group supplementation.
The effects of the vitamin B group are complementary, and the single intake of any one or more of the vitamin B group only increases the needed amount of other non-supplemented vitamin B, so that the part with insufficient intake causes physical abnormality due to deficiency and is rather clumsy. The vitamin B family is a large family, and members of these families must act simultaneously, a phenomenon called vitamin B family co-fusion.
The vitamin B group plays an important role in maintaining human health, preventing and treating various diseases.
In the known methods for measuring the contents of the vitamins B1, B2, B6, nicotinic acid and nicotinamide, the contents of the nicotinic acid and the nicotinamide are determined by a GB5009.89-2016 method, a vitamin B1 GB5009.84-2016 method, a vitamin B2 GB5009.85-2016 method and a vitamin B6 GB5009.154-2016 method, but the analysis of the national standard method has limitations, the detection process is complicated, various enzymolysis and pH value adjustment are needed, meanwhile, the contents of the vitamins B1, B2 and B6 are tested by a fluorescence detector, and the contents of the nicotinic acid and the nicotinamide are tested by an ultraviolet detector and cannot be tested simultaneously.
Disclosure of Invention
Based on the technical problems in the prior art, the invention provides vitamin B1、B2、B6High content of nicotinic acid and nicotinamideThe detection method of the liquid chromatography simplifies the detection process, and the content of the vitamin B1, B2, B6, nicotinic acid and nicotinamide is rapidly and accurately analyzed by the high performance liquid chromatography, thereby realizing the vitamin B1、B2、B6Simultaneously detecting the nicotinic acid and the nicotinamide.
The technical scheme adopted by the invention for solving the technical problems is as follows:
vitamin B1、B2、B6The high performance liquid chromatography detection method for the content of nicotinic acid and nicotinamide comprises the following steps:
s1, preparation of an extraction solution:
preparing a BHT dimethyl sulfoxide solution with the mass concentration of 0.2%; weighing 10g of anhydrous citric acid, adding 100ml of water for dissolving, then adding 0.2% BHT dimethyl sulfoxide solution to 1000ml, and uniformly mixing;
s2, preparation of mobile phase sodium n-hexane sulfonate glacial acetic acid:
weighing 1.3g of sodium n-hexane sulfonate, adding water to dissolve and diluting to 1000 ml; 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly to obtain sodium n-hexane sulfonate glacial acetic acid;
s3, preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in a volumetric flask by using an extraction solution, heating in a water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, and fixing the volume to the scale by using an extraction liquid;
s4, 2% octoxynol solution:
weighing 2ml of octoxynol into a 100ml volumetric flask, adding water to dissolve, fixing the volume to a scale, and uniformly mixing to obtain the octoxynol-containing beer.
S5, sample solution preparation:
weighing a proper amount of sample, placing the sample in a 50ml brown volumetric flask, (if the sample contains grease, 5ml of 2% octoxynol solution is needed to be added), dissolving the sample by using an extraction solution, heating the sample in a water bath at 70 +/-2 ℃ to completely dissolve the sample, cooling the sample to room temperature, fixing the volume to the scale by using an extraction liquid, centrifuging and filtering the solution;
s6, detecting by liquid chromatography, and calculating according to the detection resultVitamin B in the sample1、B2、B6And the concentration of nicotinamide content.
The detection method of the invention uses the extraction liquid to dissolve the sample, and effectively dissolves the vitamin B1、B2、B6Nicotinic acid and nicotinamide are separated out and then the extract is used for fixing the volume of the sample, thereby greatly shortening the sample processing time and providing vitamin B for various contents1、B2、 B6The niacin and the nicotinamide are effectively separated, and the vitamin B is measured by applying the high performance liquid chromatography1、B2、B6The method of nicotinic acid and nicotinamide, the selection of the sample treatment process and the chromatographic condition, and finally the vitamin B in the single-component and complex-component samples can be simply and accurately treated1、B2、B6And measuring the content of nicotinic acid and nicotinamide. The method of the invention replaces the traditional national standard method for measuring vitamin B1、B2、B6Nicotinic acid, nicotinamide.
The invention has the advantages that: the invention determines vitamin B by applying high performance liquid chromatography1、B2、B6The content of nicotinic acid and nicotinamide is determined by the sample treatment process (the sample is divided into two types, namely, an oil-free sample which can be directly treated according to the treatment process, an oil-containing sample which is treated according to the treatment process after 2 percent octoxynol solution with the total volume of 10 percent of the sample is added) and the chromatographic conditions, and finally the vitamin B in the single-component and complex-component samples is simply and accurately selected1、B2、B6And measuring the content of nicotinic acid and nicotinamide. The method can replace the traditional national standard method to measure the vitamin B1、B2、B6The contents of nicotinic acid and nicotinamide are simple, convenient, quick and accurate.
Preferably, the centrifugation treatment is: the rotation speed of 2500-.
Preferably, in the standard solution and the sample treatment step, the obtained solutions are filtered by using a filter membrane, wherein the filter membrane is a 0.45um filter membrane.
Preferably, in the liquid chromatography detection, the conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80.
preferably, in the liquid chromatography detection, gradient elution is adopted, and the concentration of methanol and sodium n-hexane sulfonate glacial acetic acid changes along with time:
preferably, in the calculation process of the detection result, the following formula is adopted to calculate the vitamin B in the sample1、B2、B6Concentration of niacin, niacinamide content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)，
In the formula: x: vitamin B in the sample1、B2、B6Nicotinic acid and nicotinamide contents, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
Compared with the prior art, the invention has the following remarkable advantages:
1. vitamin B of the present invention1、B2、B6The high performance liquid chromatography detection method of the content of the nicotinic acid and the nicotinamide is suitable for any dosage form (soft capsules, tablets and the like);
2. the detection method of the invention can dissolve the sample by the extraction liquid, and can realize better vitamin B dissolution1、B2、B6Nicotinic acid, nicotinoylAmines, requiring only brief sonication to make vitamin B in the sample1、B2、B6The nicotinic acid and the nicotinamide are dissolved out thoroughly at one time, and the extract is used for fixing the volume to the scale, so that the sample treatment time is shortened;
3. the detection method of the invention can be used for detecting vitamin B with various structures1、B2、B6Nicotinic acid and nicotinamide are effectively separated, and the separation degree is more than 1.5 (shown in figure 1);
4. the detection method of the invention shortens the sample analysis time and improves the sample detection efficiency. The traditional method needs to run programs for multiple times, which takes about 240min, and the method can complete the operation of the programs once only in 30 min.
Drawings
FIG. 1 shows the detection method of the present invention for various structures of vitamin B1、B2、B6Performing a liquid chromatogram of high performance liquid chromatography detection on nicotinic acid and nicotinamide;
FIG. 2 is a liquid chromatogram of a sample assay according to the method described in example 1;
FIG. 3 is a liquid chromatogram of the sample detection of example 2;
FIG. 4 is a liquid chromatogram for the detection of a sample in example 3;
FIG. 5 is a liquid chromatogram for the detection of the sample of example 4.
Detailed Description
The technical solution of the present invention will be further specifically described below by way of specific examples. It is to be understood that the practice of the invention is not limited to the following examples, and that any variations and/or modifications may be made thereto without departing from the scope of the invention.
In the present invention, all parts and percentages are by weight, unless otherwise specified, and the equipment and materials used are commercially available or commonly used in the art. The methods in the following examples are conventional in the art unless otherwise specified.
The reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The following examples were tested on different samples.
Example 1
Vitamin B1、B2、B6The high performance liquid chromatography detection method of the content of the nicotinic acid and the nicotinamide comprises the following specific steps:
(1) preparing an extraction solution:
0.2% BHT dimethylsulfoxide solution: 2g BHT was weighed into a 1000ml beaker and dissolved in 1000ml with dimethyl sulfoxide.
Extraction solution: 10g of anhydrous citric acid is weighed, dissolved in 100ml of water, added with 0.2 percent BHT dimethyl sulfoxide solution to 1000ml and mixed evenly.
(2) Preparation of a mobile phase:
1.3g of sodium n-hexane sulfonate is weighed, dissolved in water and diluted to 1000 ml. 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly.
(3) Preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in a volumetric flask by using the extraction solution, heating in a water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, and fixing the volume to the scale by using the extraction liquid. Filtering part of the solution with 0.45um filter membrane to obtain the final product.
(5) Preparing a sample solution:
weighing 350mg of sample, placing the sample in a 50ml brown volumetric flask, dissolving the sample by using an extraction solution, heating the sample in a water bath at 70 ℃ to completely dissolve the sample, cooling the sample to room temperature, fixing the volume to the scale by using an extraction liquid, taking part of the solution to a 50ml centrifugal tube, placing the centrifugal tube in a centrifugal machine, centrifuging the centrifugal tube for about 15min, taking supernatant, and filtering the supernatant by using a 0.45um filter membrane to obtain the product.
(5) The conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80, the gradient is shown in table 1.
TABLE 1
| Time (min) | Sodium n-hexane sulfonate glacial acetic acid (%) | Methanol (%) |
| 0 | 80 | 20 |
| 4.2 | 80 | 20 |
| 6.0 | 65 | 35 |
| 9.0 | 65 | 35 |
| 10.8 | 70 | 30 |
| 12.0 | 80 | 20 |
(6) And (3) calculating a detection result:
the peak sequence is vitamin B6Vitamin B2Vitamin B1The following formula is adopted to measure and calculate vitamin B in the sample1、B2、 B6The concentration of the content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)
In the formula: x: vitamin B in the sample1、B2、B6Content, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
The liquid chromatogram and the results of the sample detection in this example are shown in FIG. 2.
Example 2
Vitamin B1、B2、B6The high performance liquid chromatography detection method of the content of the nicotinic acid and the nicotinamide comprises the following specific steps:
(1) preparing an extraction solution:
0.2% BHT dimethylsulfoxide solution: 2g BHT was weighed into a 1000ml beaker and dissolved in 1000ml with dimethyl sulfoxide.
Extraction solution: 10g of anhydrous citric acid is weighed, dissolved in 100ml of water, added with 0.2 percent BHT dimethyl sulfoxide solution to 1000ml and mixed evenly.
(2) Preparation of a mobile phase:
1.3g of sodium n-hexane sulfonate is weighed, dissolved in water and diluted to 1000 ml. 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly.
(3) Preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in a volumetric flask by using the extraction solution, heating in a water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, and fixing the volume to the scale by using the extraction liquid. Filtering part of the solution with 0.45um filter membrane to obtain the final product.
(4) 2% octoxynol solution:
weighing 2ml of octoxynol into a 100ml volumetric flask, adding water to dissolve, fixing the volume to a scale, and uniformly mixing to obtain the octoxynol-containing beer.
(5) Preparing a sample solution:
weighing 500mg of sample, placing the sample in a 50ml brown volumetric flask, adding 5ml of 2% octoxynol solution, dissolving the sample by using an extraction solution, heating the mixture in a water bath at 70 ℃ to completely dissolve the sample, cooling the mixture to room temperature, and fixing the volume to the scale by using the extraction solution. Putting part of the solution into a 50ml centrifuge tube, centrifuging for about 15min in a centrifuge, and filtering the supernatant with a 0.45um filter membrane to obtain the final product.
(6) The conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80, the gradient is shown in table 1.
(7) And (3) calculating a detection result:
the peak appearance sequence is nicotinamide and vitamin B6Vitamin B2Vitamin B1The following formula is adopted to measure and calculate vitamin B in the sample1、B2、B6Concentration of nicotinamide content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)
In the formula: x: vitamin B in the sample1、B2、B6Nicotinamide content, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
The liquid chromatogram and the results of the sample detection in this example are shown in FIG. 3.
Example 3
Vitamin B1、B2、B6The high performance liquid chromatography detection method of the content of the nicotinic acid and the nicotinamide comprises the following specific steps:
(1) preparing an extraction solution:
0.2% BHT dimethylsulfoxide solution: 2g BHT was weighed into a 1000ml beaker and dissolved in 1000ml with dimethyl sulfoxide.
Extraction solution: 10g of anhydrous citric acid is weighed, dissolved in 100ml of water, added with 0.2 percent BHT dimethyl sulfoxide solution to 1000ml and mixed evenly.
(2) Preparation of a mobile phase:
1.3g of sodium n-hexane sulfonate is weighed, dissolved in water and diluted to 1000 ml. 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly.
(3) Preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in a volumetric flask by using the extraction solution, heating in a water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, and fixing the volume to the scale by using the extraction liquid. Filtering part of the solution with 0.45um filter membrane to obtain the final product.
(4) Preparing a sample solution:
weighing 1000mg of sample, placing the sample in a 50ml brown volumetric flask, dissolving the sample by using an extraction solution, heating the sample in a water bath at 70 ℃ to completely dissolve the sample, cooling the sample to room temperature, fixing the volume to the scale by using an extraction liquid, taking part of the solution to a 50ml centrifugal tube, placing the centrifugal tube in a centrifugal machine, centrifuging the centrifugal tube for about 15min, taking supernatant, and filtering the supernatant by using a 0.45um filter membrane to obtain the product.
(5) The conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80, the gradient is shown in table 1.
(6) And (3) calculating a detection result:
the peak appearance sequence is nicotinamide and vitamin B6Vitamin B2Vitamin B1The following formula is adopted to measure and calculate vitamin B in the sample1、B2、B6Concentration of nicotinamide content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)
In the formula: x: vitamin B in the sample1、B2、B6Nicotinamide content, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
The liquid chromatogram and the results of the sample detection in this example are shown in FIG. 4.
Example 4
Vitamin B1、B2、B6The high performance liquid chromatography detection method for the content of nicotinic acid and nicotinamide comprises the following steps:
(1) preparing an extraction solution:
0.2% BHT dimethylsulfoxide solution: 2g BHT was weighed into a 1000ml beaker and dissolved in 1000ml with dimethyl sulfoxide.
Extraction solution: 10g of anhydrous citric acid is weighed, dissolved in 100ml of water, and then 0.2 percent of BHT dimethyl sulfoxide solution is added to the mixture until the mixture is scaled and mixed evenly.
(2) Preparation of a mobile phase:
1.3g of sodium n-hexane sulfonate is weighed, dissolved in water and diluted to 1000 ml. 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly.
(3) Preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in volumetric flasks by using the extraction solution, heating in water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, fixing the volume to a scale by using the extraction liquid, and filtering a part of solution by using a 0.45um filter membrane to obtain the nicotinamide.
(4) Preparing a sample solution:
weighing 600mg of sample, placing the sample in a 50ml brown volumetric flask, dissolving the sample by using an extraction solution, heating the sample in a water bath at 70 ℃ to completely dissolve the sample, cooling the sample to room temperature, fixing the volume to the scale by using an extraction liquid, taking part of the solution to a 50ml centrifugal tube, placing the centrifugal tube in a centrifugal machine, centrifuging the centrifugal tube for about 15min, taking supernatant, and filtering the supernatant by using a 0.45um filter membrane to obtain the product.
(5) The conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80, the gradient is shown in table 1.
(6) And (3) calculating a detection result:
the peak appearance sequence is nicotinamide and vitamin B6Vitamin B2Vitamin B1The following formula is adopted to measure and calculate vitamin B in the sample1、B2、B6Concentration of nicotinamide content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)
In the formula: x: vitamin B in the sample1、B2、B6Nicotinamide content, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
The liquid chromatogram and the results of the sample detection in this example are shown in FIG. 5.
The following results were obtained by comparing the HPLC detection methods of examples 1 to 4 with those of GB5009.248-2016, and are shown in Table 2.
TABLE 2
| Whether or not enzymatic hydrolysis is required | Operating time min | |
| Examples 1 to 4 | Whether or not | 20-30min or so |
| GB5009.89-2016 Nicotinamide nicotinate | Whether or not | About 30-40min |
| GB5009.84-2016 test for vitamin B1 | Is that | About 70-80min |
| GB5009.85-2016 test for vitamin B2 | Is that | About 70-80min |
| GB5009.154-2016 test for vitamin B6 | Whether or not | About 30-40min |
The following results were obtained from a comparison of the data in table 1: the vitamin B is measured by adopting the high performance liquid chromatography detection method1、 B2、B6The content of nicotinic acid and nicotinamide can not only be used for one-time mixing vitamin B1、B2、B6Nicotinic acid and nicotinamide are detected, and the operation time is obviously shortened.
The embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same or similar parts among the embodiments are referred to each other. The device disclosed by the embodiment corresponds to the method disclosed by the embodiment, so that the description is simple, and the relevant points can be referred to the method part for description.
The methods for detecting the contents of vitamin B1, vitamin B2, vitamin B6, nicotinic acid and nicotinamide by high performance liquid chromatography are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (6)
1. Vitamin B1、B2、B6The high performance liquid chromatography detection method for the content of the nicotinic acid and the nicotinamide is characterized by comprising the following steps:
s1, preparation of an extraction solution:
preparing a BHT dimethyl sulfoxide solution with the mass concentration of 0.2%; weighing 10g of anhydrous citric acid, adding 100ml of water for dissolving, then adding 0.2% BHT dimethyl sulfoxide solution to 1000ml, and uniformly mixing;
s2, preparation of mobile phase sodium n-hexane sulfonate glacial acetic acid:
weighing 1.3g of sodium n-hexane sulfonate, adding water to dissolve and diluting to 1000 ml; 986.5ml of the mixture is taken and added with 13.5ml of glacial acetic acid to be mixed evenly to obtain sodium n-hexane sulfonate glacial acetic acid;
s3, preparation of a 2% octoxynol solution:
weighing 2ml of octoxynol into a 100ml volumetric flask, adding water to dissolve, fixing the volume to a scale, and uniformly mixing to obtain the octoxynol-containing beer.
S4, preparing a standard solution:
precisely weighing vitamin B1、B2、B6Dissolving 10mg of each, 100mg of nicotinamide and 20mg to 100ml of nicotinic acid in a volumetric flask by using an extraction solution, heating in a water bath at 70 ℃ to completely dissolve the nicotinamide and the nicotinic acid, cooling to room temperature, and fixing the volume to the scale by using an extraction liquid;
s5, sample solution preparation:
weighing a proper amount of sample, placing the sample in a 50ml brown volumetric flask, dissolving the sample by using an extraction solution, heating the solution in a water bath at the temperature of 70 +/-2 ℃ to completely dissolve the sample, cooling the solution to room temperature, metering the volume to a scale by using an extraction liquid, centrifuging and filtering;
s6, detecting by liquid chromatography, and calculating vitamin B in the sample according to the detection result1、B2、B6And the concentration of nicotinamide content.
2. Vitamin B as claimed in claim 11、B2、B6The high performance liquid chromatography detection method for the content of the nicotinic acid and the nicotinamide is characterized in that the centrifugal treatment comprises the following steps: the rotation speed of 2500-.
3. Vitamin B as claimed in claim 11、B2、B6The high performance liquid chromatography detection method for the content of the nicotinic acid and the nicotinamide is characterized in that in the standard solution and the sample treatment step, the obtained solutions are filtered by adopting filter membranes, and the filter membranes are 0.45um filter membranes.
4. Vitamin B as claimed in claim 11、B2、B6The high performance liquid chromatography detection method for the content of the nicotinic acid and the nicotinamide is characterized in that in the liquid chromatography detection, the conditions of the liquid chromatography detection are as follows: a chromatographic column: c18Column, 150X 4.6mm, 5 μm; flow rate: 1.0mL/min, detection wavelength: 280nm, sample size ((A)): 3 μ L, column temperature: at 30 ℃, mobile phase: methanol: sodium n-hexane sulfonate glacial acetic acid 20: 80.
5. vitamin B as claimed in claim 11、B2、B6The high performance liquid chromatography detection method for the content of nicotinic acid and nicotinamide is characterized in that gradient elution is adopted in liquid chromatography detection, and the concentration of methanol and sodium n-hexane sulfonate glacial acetic acid is changed along with time:
6. vitamin B as claimed in claim 11、B2、B6The high performance liquid chromatography detection method for the content of nicotinic acid and nicotinamide is characterized in that in the calculation process of the detection result, the following formula is adopted to measure and calculate the vitamin B in the sample1、B2、B6Concentration of niacin, niacinamide content:
X＝Asample (A)*V*CSign board/ASign board/WSample (A)，
In the formula: x: vitamin B in the sample1、B2、B6Nicotinic acid and nicotinamide contents, mg/g;
and C, marking: the concentration of each vitamin in the standard solution is mg/mL;
v: the volume of a sample is determined to be mL;
w: weighing g of the sample;
marking A: peak area of the standard solution;
a sample A: peak area of sample solution.
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