CN118010861A - Method for rapidly detecting multiple water-soluble vitamins in vitamin premix - Google Patents
Method for rapidly detecting multiple water-soluble vitamins in vitamin premix Download PDFInfo
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- CN118010861A CN118010861A CN202311873134.XA CN202311873134A CN118010861A CN 118010861 A CN118010861 A CN 118010861A CN 202311873134 A CN202311873134 A CN 202311873134A CN 118010861 A CN118010861 A CN 118010861A
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- 229940088594 vitamin Drugs 0.000 title claims abstract description 39
- 229930003231 vitamin Natural products 0.000 title claims abstract description 39
- 235000013343 vitamin Nutrition 0.000 title claims abstract description 39
- 239000011782 vitamin Substances 0.000 title claims abstract description 39
- 235000019742 Vitamins premix Nutrition 0.000 title claims abstract description 29
- 238000000034 method Methods 0.000 title claims abstract description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims abstract description 21
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 14
- 239000011259 mixed solution Substances 0.000 claims abstract description 10
- 239000012528 membrane Substances 0.000 claims abstract description 9
- 238000002347 injection Methods 0.000 claims abstract description 7
- 239000007924 injection Substances 0.000 claims abstract description 7
- 239000000523 sample Substances 0.000 claims abstract description 7
- 239000012488 sample solution Substances 0.000 claims abstract description 6
- 238000002137 ultrasound extraction Methods 0.000 claims abstract description 6
- 239000006228 supernatant Substances 0.000 claims abstract description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 6
- 238000005119 centrifugation Methods 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 150000003722 vitamin derivatives Chemical class 0.000 abstract description 10
- 238000005259 measurement Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- GHOKWGTUZJEAQD-ZETCQYMHSA-N (D)-(+)-Pantothenic acid Chemical compound OCC(C)(C)[C@@H](O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-ZETCQYMHSA-N 0.000 description 18
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 description 16
- 235000019161 pantothenic acid Nutrition 0.000 description 9
- 239000011713 pantothenic acid Substances 0.000 description 9
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 description 8
- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 8
- 229960000304 folic acid Drugs 0.000 description 8
- 235000019152 folic acid Nutrition 0.000 description 8
- 239000011724 folic acid Substances 0.000 description 8
- 229940055726 pantothenic acid Drugs 0.000 description 8
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 8
- RBCOYOYDYNXAFA-UHFFFAOYSA-L (5-hydroxy-4,6-dimethylpyridin-3-yl)methyl phosphate Chemical compound CC1=NC=C(COP([O-])([O-])=O)C(C)=C1O RBCOYOYDYNXAFA-UHFFFAOYSA-L 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 7
- 238000012417 linear regression Methods 0.000 description 7
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 6
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229960003966 nicotinamide Drugs 0.000 description 2
- 235000005152 nicotinamide Nutrition 0.000 description 2
- 239000011570 nicotinamide Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000013582 standard series solution Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 101710146995 Acyl carrier protein Proteins 0.000 description 1
- RGJOEKWQDUBAIZ-IBOSZNHHSA-N CoASH Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCS)O[C@H]1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-IBOSZNHHSA-N 0.000 description 1
- RGJOEKWQDUBAIZ-UHFFFAOYSA-N coenzime A Natural products OC1C(OP(O)(O)=O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 RGJOEKWQDUBAIZ-UHFFFAOYSA-N 0.000 description 1
- 239000005516 coenzyme A Substances 0.000 description 1
- 229940093530 coenzyme a Drugs 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229940014662 pantothenate Drugs 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
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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)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a rapid detection method of multiple water-soluble vitamins in vitamin premix, which relates to the technical field of detection and comprises the following steps: adding hydrochloric acid solution into vitamin premix for special medical formula food for dissolving and ultrasonic extraction, fixing volume with hydrochloric acid solution, and shaking to obtain mixed solution; centrifuging the mixed solution, taking supernatant, and passing through a membrane to obtain a sample injection solution; and taking the sample solution to detect in high performance liquid chromatography. The method has the advantages of good separation effect, high accuracy and the like, can meet the measurement of the content of the water-soluble vitamins in the vitamin premix for the formula food with special medical application, and optimizes the detection method for only detecting one vitamin at a time by adopting the national food safety standard at present.
Description
Technical Field
The invention relates to the field of detection, in particular to a rapid detection method for multiple water-soluble vitamins in vitamin premix.
Background
The special medical formula food is specially processed and prepared to meet the special requirements of people with limited eating or specific disease states on nutrition or diet. Wherein the raw material vitamin premix provides a plurality of water-soluble vitamins for the product, for example pantothenic acid is a B group water-soluble vitamin, and the vitamin B 5 and the throughout polyacid are alike. Pantothenic acid is a necessary nutrient substance for maintaining normal physiological functions of human body, is a necessary precursor of coenzyme A and acyl carrier protein, also comprises water-soluble vitamins such as folic acid, nicotinamide, vitamin B 1 and the like, and is an important nutrition enhancer added into formula food with special medical application.
At present, the detection method in the national food safety standard generally can only detect one vitamin at a time, and has the advantages of high requirements on experimental staff when developed, complicated pretreatment operation steps, large workload, low efficiency and long time consumption, and greatly reduces the efficiency of vitamin detection. Often, multiple vitamins are added into the formula food with special medical application, and the vitamins are not added singly but are compounded by specially designed vitamin premix. The related regulations of the formula food for special medical use have strict requirements on the vitamin content, so that the accurate quantification of various vitamin components in the vitamin premix is a key for effectively controlling the vitamin content in the final product. Therefore, aiming at the vitamin premix, it is important to develop a high performance liquid chromatography detection method capable of simultaneously detecting the contents of multiple vitamins.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to overcome the defect that the detection method in the prior art can only detect one vitamin at a time, thereby providing a rapid detection method for a plurality of water-soluble vitamins in vitamin premix, and a high performance liquid chromatography detection method capable of detecting a plurality of vitamin contents simultaneously.
Therefore, the invention provides a rapid detection method of a plurality of water-soluble vitamins in a vitamin premix, which comprises the following steps:
s1: adding hydrochloric acid solution into vitamin premix for special medical formula food for dissolving and ultrasonic extraction, fixing volume with hydrochloric acid solution, and shaking to obtain mixed solution;
S2: centrifuging the mixed solution, taking supernatant, and passing through a membrane to obtain a sample injection solution;
S3: and taking the sample solution to detect in high performance liquid chromatography.
Preferably, the concentration of the hydrochloric acid solution is 0.1mol/L.
Preferably, the dissolution temperature is 40-60 ℃.
Preferably, the ultrasonic time is 10-40 min
Preferably, the centrifugation speed is 4000r/min and the centrifugation time is 2min.
Preferably, the membrane is a 0.22 μm aqueous membrane.
Preferably, the chromatographic column specification of the high performance liquid chromatography is a C18 column with the size of 250mm multiplied by 4.6mm and the particle size of 5 μm.
Preferably, the column temperature of the chromatographic column of the high performance liquid chromatography is 30 ℃, the sample injection amount is 10 mu L, the detection wavelength is 210nm and 270nm, and the flow rate is 1.0mL/min.
Preferably, in the high performance liquid chromatography, mobile phase a is phosphoric acid solution (ph=2.0), mobile phase B is acetonitrile, and gradient elution conditions are:
| Time (min) | Mobile phase (A) | Mobile phase (B) |
| 0 | 100 | 0 |
| 12 | 100 | 0 |
| 14 | 85 | 15 |
| 20 | 85 | 15 |
| 22 | 100 | 0 |
| 35 | 100 | 0 |
。
The technical scheme of the invention has the following advantages:
1. The rapid detection method of multiple water-soluble vitamins in vitamin premix provided by the invention comprises the steps of extracting six different water-soluble vitamins in vitamin premix for formula food with special medical application through hydrochloric acid solution, then directly measuring by a high performance liquid chromatograph (chromatographic column is C18:250X4.6 mm, particle size is 5 μm), separating and obtaining a target object, and synchronously measuring vitamin B 1, nicotinamide, vitamin B 6, vitamin B 2, folic acid and pantothenic acid in samples containing multiple water-soluble vitamins and fat-soluble vitamins through optimizing mobile phase proportion and using a C18 chromatographic column, so that accurate quantification is realized.
2. The rapid detection method of the multiple water-soluble vitamins in the vitamin premix provided by the invention adopts the high performance liquid chromatography C18 chromatographic column to measure the content of six different water-soluble vitamins in the vitamin premix for the formula food with special medical application, samples are subjected to ultrasonic extraction, centrifugal precipitation and impurity passing through a membrane feeding machine, are separated by the C18 chromatographic column with the particle size of 5 mu m with the particle size of 250mm multiplied by 4.6mm, and are detected by an ultraviolet detector in double wavelength, and the external standard method is quantitative.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a high performance liquid chromatogram of a sample solution of the present invention;
FIG. 2 is a high performance liquid chromatogram of a standard series of solutions of the present invention.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The specific experimental procedures or conditions are not noted in the examples and may be followed by the operations or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.
Example 1
S1: 1g of vitamin premix for the formula food with the special medical application to be measured is weighed into a 100mL conical flask, 50mL of 0.1mol/L hydrochloric acid solution with the temperature of 40-60 ℃ is added for dissolution, and ultrasonic extraction is carried out for 30min after uniform mixing. Shaking for 3-5 times during ultrasonic extraction, taking out, cooling to room temperature, fixing volume with 0.1mol/L hydrochloric acid solution, and shaking to obtain mixed solution. The obtained mixed solution is transferred into a 100mL volumetric flask, and is uniformly mixed after the volume is fixed to the scale by using 0.1mol/L hydrochloric acid solution.
S2: transferring the mixed solution obtained in the step S1 into a centrifuge tube, and completely separating solid and liquid in the mixed solution at the centrifugation rate of 4000r/min for 2min, and taking supernatant to pass through a 0.22 mu m water-based filter membrane to obtain a sample injection solution.
S3: 1.5mL of sample solution is taken and detected in high performance liquid chromatography to obtain the content of vitamin B 1, nicotinamide, vitamin B 6, vitamin B 2, folic acid and pantothenic acid which are six different water-soluble vitamins in the sample solution. The column temperature of the chromatographic column is 30 ℃, the sample injection amount is 10 mu L, the detection wavelength is 210nm and 270nm, and the flow rate is 1.0mL/min. In the high performance liquid chromatography, mobile phase a was phosphoric acid solution (ph=2.0), mobile phase B was acetonitrile, and gradient elution conditions were as shown in table 1 below.
TABLE 1 gradient elution procedure
| Time (min) | Mobile phase (A) | Mobile phase (B) |
| 0 | 100 | 0 |
| 12 | 100 | 0 |
| 14 | 85 | 15 |
| 20 | 85 | 15 |
| 22 | 100 | 0 |
| 35 | 100 | 0 |
The map obtained by the method is shown in figure 1, and can be seen from the figure, six different water-soluble vitamins of vitamin B 1, nicotinamide, vitamin B 6, vitamin B 2, folic acid and pantothenic acid in figure 1 are well separated, and the peak shape is neat and clear and easy to read. Six water-soluble vitamin standard series solutions are tested and analyzed, and the obtained standard series solution chromatograms are shown in figure 2.
Example 2
The standard curve was drawn and linear regression was performed with the peak area y of the map of example 1 as ordinate and the concentrations x (ug/mL) of vitamin B 1, nicotinamide, vitamin B 6, vitamin B 2, folic acid and pantothenic acid standard solutions as abscissa. Vitamin B 1 is in the concentration range of 0.1-1.0 ug/mL, vitamin B 2 is in the concentration range of 0.05-1.0 ug/mL, vitamin B 6 is in the concentration range of 0.1-1.0 ug/mL, nicotinamide is in the concentration range of 0.5-10 ug/mL, folic acid is in the concentration range of 1-20 ug/mL, pantothenic acid is in the concentration range of 0.8-4 ug/mL, and the obtained peak area has good linear relation with the concentration. Vitamin B 1 linear regression equation y= 32.37372x-0.33771, correlation coefficient 0.99945; vitamin B 2 linear regression equation y=123.38219x+1.71038, correlation coefficient 0.99927; vitamin B 6 linear regression equation is y=27.12917x+0.16689, correlation coefficient is 0.99966; the linear regression equation of nicotinamide is y= 37.21464x-0.13787, and the correlation coefficient is 0.99994; the folic acid linear regression equation is y=79.16001x+7.42900, and the correlation coefficient is 0.99979; the linear regression equation for pantothenate was y=8.22770x+0.37133 and the correlation coefficient was 0.99953.
Vitamin B 1, nicotinamide, vitamin B 6, vitamin B 2, folic acid and pantothenic acid standard solutions were repeatedly injected 6 times by the method of example 1, peak areas were measured, relative Standard Deviation (RSD) was calculated as shown in the following table, RSD was less than 5%, and the results revealed that the precision was good and the instrument stability was good.
The vitamin premix for the same formula food with special medical application is analyzed and detected by the method in the example 1, 6 parallel tests are carried out, and the average value and RSD of the 6 tests are shown in the table below.
Therefore, the results obtained by the measurement method of the invention have excellent precision and stability.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.
Claims (9)
1. A rapid detection method for a plurality of water-soluble vitamins in a vitamin premix is characterized by comprising the following steps:
s1: adding hydrochloric acid solution into vitamin premix for special medical formula food for dissolving and ultrasonic extraction, fixing volume with hydrochloric acid solution, and shaking to obtain mixed solution;
S2: centrifuging the mixed solution, taking supernatant, and passing through a membrane to obtain a sample injection solution;
S3: and taking the sample solution to detect in high performance liquid chromatography.
2. The method for rapid detection of multiple water-soluble vitamins in a vitamin premix according to claim 1, wherein the concentration of the hydrochloric acid solution is 0.1mol/L.
3. The method for rapid detection of multiple water-soluble vitamins in a vitamin premix according to claim 1, wherein the dissolution temperature is 40-60 ℃.
4. The method for rapidly detecting a plurality of water-soluble vitamins in a vitamin premix according to claim 1, wherein the ultrasonic time is 10-40 min.
5. The method for rapid detection of multiple water-soluble vitamins in a vitamin premix according to claim 1, wherein the centrifugation rate is 4000r/min and the centrifugation time is 2min.
6. The method for rapid detection of multiple water-soluble vitamins in a vitamin premix according to claim 1, wherein the membrane is a 0.22 μm aqueous membrane.
7. The method for rapidly detecting a plurality of water-soluble vitamins in a vitamin premix according to claim 1, wherein the high performance liquid chromatography is characterized in that the specification of a chromatographic column is 250mm×4.6mm, and the size of a C18 column with a particle size of 5 μm.
8. The method for rapidly detecting a plurality of water-soluble vitamins in a vitamin premix according to claim 7, wherein the column temperature of the chromatographic column of the high performance liquid chromatography is 30 ℃, the sample injection amount is 10 μl, the detection wavelength is 210nm and 270nm, and the flow rate is 1.0mL/min.
9. The method for rapid detection of multiple water-soluble vitamins in vitamin premix according to claim 8, wherein mobile phase a is phosphoric acid solution (ph=2.0) in high performance liquid chromatography, mobile phase B is acetonitrile, and gradient elution conditions are:
。
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|---|---|---|---|
| CN202311873134.XA CN118010861A (en) | 2023-12-29 | 2023-12-29 | Method for rapidly detecting multiple water-soluble vitamins in vitamin premix |
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|---|---|---|---|
| CN202311873134.XA CN118010861A (en) | 2023-12-29 | 2023-12-29 | Method for rapidly detecting multiple water-soluble vitamins in vitamin premix |
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| CN118010861A true CN118010861A (en) | 2024-05-10 |
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