Detection method for measuring vitamin B12

Abstract

The invention relates to the technical field of analytical chemistry, in particular to a detection method for measuring vitamin B12. The invention discloses a detection method for measuring vitamin B12. The detection method comprises the following steps: preparing a sample solution and a standard solution; taking the sample solution and the standard solution, injecting the sample solution and the standard solution into a high performance liquid chromatograph for liquid chromatographic analysis, and determining the content of the vitamin B12; wherein the conditions of the liquid chromatographic analysis are as follows: a mobile phase A is 0.025% trifluoroacetic acid solution; a mobile phase B is acetonitrile; according to the method disclosed by the invention, an ion pair reagent is not added into a mobile phase, meanwhile, 546nm detection is selected according to the maximum absorption wavelength condition of the vitamin B12, the influence of most interference peaks is eliminated in the detection process of the method, and the B12 content detection can be accurately completed.

Description

Technical Field

[0001] The present application relates to the technical field of analytical chemistry, and in particular to a method for determining vitamin B12. Background Art

[0002] Most of the vitamin products currently available on the market are products with a mixture of multiple vitamins. The existing methods for detecting vitamin B12 generally use an absorption wavelength of around 361nm. During the experiment, it was found that due to the influence of various factors, there are many impurity peaks in the detection process, which can easily lead to the inability to completely separate vitamin B12 and cause inaccurate detection. At the same time, many detection methods use ion pair reagents as the mobile phase, which is more harmful to the chromatographic column.

[0003] Therefore, how to achieve accurate detection of vitamin B12 has become an urgent problem to be solved. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present application provides a method for determining vitamin B12.

[0005] The present application provides a method for determining vitamin B12, which adopts the following technical solution: A method for determining vitamin B12, the steps of the method are as follows: Prepare sample solution and standard solution; Take the sample solution and the standard solution, inject them into a high performance liquid chromatograph for liquid chromatography analysis, and determine the vitamin B12 content; preferably, the conditions for liquid chromatography analysis include: Mobile phase A: 0.025% trifluoroacetic acid solution; Mobile phase B: acetonitrile; The gradient elution method was used for elution. The gradient elution procedure was as follows: Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 5 95 5 22 2 98 27 2 98 27.5 95 5 32 95 5 .

[0006] Preferably, the conditions for the liquid chromatography analysis also include: the chromatographic column is an ODS column, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

[0007] Preferably, the conditions for the liquid chromatography analysis also include: a detection wavelength of 545-555 nm.

[0008] Further preferably, the detection wavelength is 545 nm, 546 nm, 547 nm, 548 nm, 549 nm, 550 nm, 551 nm, 552 nm, 553 nm, 554 nm or 555 nm.

[0009] More preferably, the detection wavelength is 546 nm.

[0010] Preferably, the injection volume is 50-150 μL.

[0011] Further preferably, the injection volume is 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL or 150 μL.

[0012] More preferably, the injection volume is 100 μL.

[0013] Preferably, the flow rate is 0.5-1.5 mL / min.

[0014] Further preferably, the flow rate is 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, 1 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, 1.4 mL / min, and 1.5 mL / min.

[0015] More preferably, the flow rate is 1 mL / min.

[0016] Preferably, the column temperature is 20-40°C.

[0017] Further preferably, the column temperature is 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C.

[0018] More preferably, the column temperature is 35°C.

[0019] Preferably, the detector is a diode array detector.

[0020] Preferably, the method for preparing the sample solution comprises the following steps: Weigh the test solution, add sodium acetate to dissolve it, ultrasonicate at 35-45℃ for 3-8min, cool to 20-25℃, add sodium acetate solution to the scale, shake well, centrifuge, filter, and prepare a sample solution with a mass concentration of 0.1-2.0ug / mL.

[0021] Preferably, the test sample in the test solution is a multivitamin; wherein the content of vitamin B12 in the multivitamin is 1.92-3.8 μg / g.

[0022] Preferably, the complex vitamin also includes at least one of calcium, magnesium, manganese, iron, zinc, selenium, copper, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B6, folic acid, biotin, vitamin C, vitamin K1, pantothenic acid, vitamin E and niacinamide.

[0023] Preferably, the method for preparing the test solution comprises the following steps: (1) grinding the vitamin complex, adding a sodium acetate solution, and mixing well to obtain a vitamin complex solution; (2) mixing diatomaceous earth and sodium carbonate to obtain a precipitating agent; (3) separating the precipitant into precipitant A and precipitant B; wherein the mass ratio of precipitant A to precipitant B is (0.4-0.6):(1-3); (4) Add precipitant A to the multivitamin solution and mix, then add precipitant B and mix, filter, and obtain a test solution.

[0024] Preferably, in step (1), the amount of the vitamin complex added is 1-4 parts by weight; the amount of the sodium acetate solution added is 20-30 mL; In the step (2), the amount of diatomaceous earth added is 2-5 parts by weight; the amount of sodium carbonate added is 4-7 parts by weight; The time for adding the precipitant A and mixing in step (4) is 2-5 minutes; the stirring speed is 50-100 rpm; In the step (4), the time for adding the precipitant B and mixing is 20-30 minutes; and the stirring speed is 200-300 rpm.

[0025] By adopting the above technical scheme, since the complex vitamin solution contains elements such as calcium, magnesium, manganese, iron, zinc, copper, etc., therefore, the elements such as calcium, magnesium, manganese, iron, zinc, copper, etc. will have a certain impact on the detection effect of vitamin B12 during the detection process. In this application, by adding a precipitant and adsorbing metal elements such as calcium, magnesium, manganese, iron, zinc, copper, etc. in the complex vitamin solution, the interference of metal elements such as calcium, magnesium, manganese, iron, zinc, copper, etc. on vitamin B12 during the detection process is effectively avoided, thereby effectively improving the detection efficiency of vitamin B12.

[0026] In the present application, the preparation method of the test solution is improved, the prepared precipitant is divided into precipitant A and precipitant B, which are added to the multivitamin solution in batches, and the mixing time and stirring speed are limited. The added precipitant A and precipitant B can adsorb metal elements such as calcium, magnesium, manganese, iron, zinc, and copper in the multivitamin solution, effectively improve the adsorption efficiency, and further avoid the interference of metal elements such as calcium, magnesium, manganese, iron, zinc, and copper on the detection results of vitamin B12.

[0027] Preferably, the preparation of the standard solution is: Precisely weigh vitamin B 12 24.2 mg of the standard substance was placed in a 10 mL volumetric flask, and 5% ethanol solution was added to dissolve it to the mark, and the solution was shaken to obtain the standard stock solution. Accurately pipette 1 mL of the standard mother solution, dilute it with sodium acetate solution, and make up to 100 mL as the standard intermediate solution; Pipette appropriate amount of standard intermediate solution respectively, and dilute with sodium acetate solution to prepare standard working curve solutions with concentrations of 0.0484μg / mL, 0.242μg / mL, 0.726μg / mL, 1.21μg / mL, 1.936μg / mL, and 2.42μg / mL.

[0028] The mobile phase of the method of the present application does not add ion pair reagents. At the same time, according to the maximum absorption wavelength of vitamin B12, 546nm is selected for detection. This method eliminates most of the interference peaks during the detection process and can accurately complete the B12 content test.

[0029] In summary, the present application includes at least one of the following beneficial technical effects: The present application provides a method for determining vitamin B12, which has good linearity, high precision, high recovery rate and high detection sensitivity; The present application provides a method for determining vitamin B12, which can separate vitamin B12 from complex vitamins. The method is simple, rapid, safe, reliable and easy to implement. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the HPLC spectrum of the sample solution in Example 1; Figure 2 is the HPLC spectrum of the blank solution; Figure 3 Vitamin B 12 HPLC spectrum of standard solution 1 (concentration 0.0484 μg / mL); Figure 4 Vitamin B 12 HPLC spectrum of standard solution 2 (concentration 0.242 μg / mL); Figure 5 Vitamin B 12 HPLC spectrum of standard solution 3 (concentration 0.726 μg / mL); Figure 6 Vitamin B 12 HPLC spectrum of standard solution 4 (concentration 1.21 μg / mL); Figure 7 Vitamin B12 HPLC spectrum of standard solution 5 (concentration 1.936 μg / mL); Figure 8 Vitamin B 12 HPLC spectrum of standard solution 6 (concentration 2.42 μg / mL); Fig. 9 It is the standard working curve diagram; Fig.10 The HPLC spectrum of the sample solution detected by the method of Comparative Example 1; DETAILED DESCRIPTION

[0031] The technical solution of the present application is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present application; some non-essential modifications and adjustments made by others based on the concept of the present application still fall within the protection scope of the present application.

[0032] The raw materials involved in the present application are all commercially available products, wherein the present application is further described in detail below in conjunction with the embodiments.

[0033] Preparation Example 1: Preparation of test solution: (1) Grind 2 g of multivitamin C into fine powder, add 25 ml of sodium acetate solution, and mix well to obtain a multivitamin solution; (2) 3.5 g of diatomaceous earth and 5.5 g of sodium carbonate were mixed to obtain a precipitant; (3) dividing the precipitant into precipitant A and precipitant B; wherein the mass ratio of precipitant A to precipitant B is 0.5:2.5; (4) Add precipitant A to the multivitamin solution and mix for 3 minutes; stir at 80 rpm, add precipitant B and mix for 25 minutes; stir at 250 rpm, filter, and obtain a test solution.

[0034] Preparation Example 2: Preparation of test solution: (1) Grind 2 g of multivitamin C into fine powder, add 25 ml of sodium acetate solution, and mix well to obtain a multivitamin solution; (2) Mix 2 g of diatomaceous earth and 4 g of sodium carbonate to obtain a precipitant; (3) dividing the precipitant into precipitant A and precipitant B; wherein the mass ratio of precipitant A to precipitant B is 0.4:1; (4) Add precipitant A to the multivitamin solution and mix for 2 minutes; the stirring speed is 50 rpm, add precipitant B and mix for 20 minutes; the stirring speed is 200 rpm, filter, and obtain the test solution.

[0035] Preparation Example 3: Preparation of test solution: (1) Grind 2 g of multivitamin C into fine powder, add 25 ml of sodium acetate solution, and mix well to obtain a multivitamin solution; (2) 5 g of diatomaceous earth and 7 g of sodium carbonate were mixed to obtain a precipitant; (3) dividing the precipitant into precipitant A and precipitant B; wherein the mass ratio of precipitant A to precipitant B is 0.6:3; (4) Add precipitant A to the multivitamin solution and mix for 5 minutes; the stirring speed is 100 rpm, add precipitant B and mix for 30 minutes; the stirring speed is 300 rpm, filter, and obtain the test solution.

[0036] Example 1: Multivitamin Sample Detection A method for determining vitamin B12, the steps are as follows: 1. Instrument: Electronic balance, ultrasonic cleaning machine, high performance liquid chromatography, centrifuge.

[0037] 2. Reagents: Glacial acetic acid (AR), trifluoroacetic acid (AR), anhydrous ethanol (AR), acetonitrile (chromatographic grade), anhydrous sodium acetate (AR); Vitamin B 12 Standard product (source: China Food and Drug Inspection Institute, content 90.5%, batch number: 100248-201705, CAS: 68-19-9).

[0038] Multivitamin sample: purchased from Jiangsu Tianmeijian Nature Bioengineering Co., Ltd., Siweiyi multivitamin mineral chewable tablets, tablet weight is 1g, batch number 20240911; among them, each 1.5g of multivitamin contains vitamin B12 1.92-3.8μg / g, calcium 112.5-187.5mg, magnesium 33.75-56.25mg, manganese 0.51-0.84mg, iron 6-10mg, zinc 4.5-7.5mg, selenium 15-25μg, copper 0.15-0.25mg, vitamin A192-400μg, vitamin D3 4-7.5μg, vitamin B1 4.8-10mg, vitamin B2 2.8-6.3mg, vitamin B6 2.4-5mg, folic acid 120-250μg, biotin 20-45μg, vitamin C 48-108mg, vitamin K1 24-50μg, pantothenic acid 3.2-7.2mg, vitamin E 6-13.5mg, niacinamide 10-22.5mg.

[0039] 3. Preparation of solution: Sodium acetate solution: weigh 10.2g sodium acetate, add 40mL glacial acetic acid, and add water to dissolve to 500mL.

[0040] 0.025% trifluoroacetic acid solution (mobile phase A): Take 0.25 mL of trifluoroacetic acid, add water to make up to 1000 mL, mix well, filter, and degas to obtain.

[0041] Acetonitrile (mobile phase B): Take 500 mL of acetonitrile and ultrasonicate for 20 minutes.

[0042] 5% ethanol solution: Take 5 mL of ethanol and add water to make up to 100 mL, mix well.

[0043] 4. Analysis methods 4.1 Instrument conditions and parameters Chromatographic conditions: Chromatographic column: ODS column, 250mm*4.6mm, 5μm; Wavelength: 546nm; Injection volume: 100 μL; Flow rate: 1.0 mL / min; Column temperature: 35 °C; Detector: diode array detector; Mobile phase A: 0.025% trifluoroacetic acid solution; Mobile phase B: acetonitrile; Mobile phase: Gradient elution method is used, see the table below: Table 1 Mobile phase gradient elution table Time (min) Mobile phase A (%) Mobile phase B (%) 0 95 5 5 95 5 22 2 98 27 2 98 27.5 95 5 32 95 5 4.2 Preparation of sample solution: Preparation of sample solution: Take the test solution (obtained from Preparation Example 1) and place it in a 50mL volumetric flask, add 6ml of sodium acetate solution, ultrasonicate at 40°C for 5min, cool to 25°C, then slowly add sodium acetate solution to the scale, shake well, centrifuge at 3500r / min for 5min, pass the supernatant through a 0.45μm filter membrane, and prepare a sample solution with a mass concentration of 0.1ug / mL.

[0044] 4.3 Preparation of standard solution Precisely weigh vitamin B 12 24.2 mg of the standard substance was placed in a 10 mL volumetric flask, and 5% ethanol solution was added to dissolve it to the mark, and the solution was shaken to obtain the standard stock solution. Accurately pipette 1 mL of the standard mother solution, dilute it with sodium acetate solution, and make up to 100 mL as the standard intermediate solution; Pipette appropriate amount of standard intermediate solution respectively, and dilute with sodium acetate solution to prepare standard working curve solutions with concentrations of 0.0484μg / mL, 0.242μg / mL, 0.726μg / mL, 1.21μg / mL, 1.936μg / mL, and 2.42μg / mL.

[0045] 4.4. Sample determination Accurately pipette 100 μL of sample solution and standard working curve solution respectively, inject into liquid chromatograph, and measure to obtain; the HPLC spectrum of sample solution is shown in Figure 1 .

[0046] 4.5 Calculation The standard curve is used to obtain a regression equation. The peak area corresponding to the sample is substituted into the regression equation to calculate the sample concentration. The content is then calculated using the following formula: Where: X—Vitamin B in the sample 12 Content, μg / tablet; C—Vitamin B in the sample under the regression equation 12 Concentration, μg / mL; V—dilution volume of the product, mL; m—product specification, g / piece; W—sample mass, g.

[0047] Example 2: Methodological Validation Methodological verification of the determination method of Example 1: 1. Method blank test 1.1 Test methods Preparation of blank solution: Sodium acetate solution: the preparation method is the same as in Example 1; Vitamin B 12 Preparation of standard solution: Precisely weigh vitamin B 12 24.2 mg of the standard substance was placed in a 10 mL volumetric flask, and 5% ethanol solution was added to dissolve it to the mark, and the solution was shaken to obtain the standard stock solution. Accurately pipette 1 mL of the standard mother solution, dilute it with sodium acetate solution, and make up to 100 mL as the standard intermediate solution; Take appropriate amount of standard intermediate solution and dilute it with sodium acetate solution to the concentration of 0.0484μg / mL, 0.242μg / mL, 0.726μg / mL, 1.21μg / mL, 1.936μg / mL, 2.42μg / mL vitamin B 12 Standard solution.

[0048] Precisely pipette 100 μL of blank solution and vitamin B 12 100 μL of the standard solution was injected into the liquid chromatograph for determination, and the blank solution and vitamin B 12 Comparison of peak retention times of standard solutions.

[0049] 1.2 Test results The HPLC spectrum of the blank solution is as follows Figure 2 As shown; Vitamin B 12 The HPLC spectrum of the standard solution is as follows Figure 3-Figure 8 shown.

[0050] 1.3 Test conclusion according to Figure 3-Figure 8 It can be seen that the blank solution contains vitamin B 12 There is no absorption peak at the peak retention time, indicating that the blank solution has no interference with the determination results.

[0051] 2. Confirmation of linear range 2.1 Test data 100 μL of each of 6 standard working curve solutions of different concentrations (prepared by the same method as in Example 1) were accurately aspirated and injected into the liquid chromatograph. The measurement results are shown in Table 2.

[0052] Table 2 Standard working curve solution data table 2.2 Standard working curve With the concentration in Table 2 as the horizontal axis and the peak area as the vertical axis, the standard working curve is as follows Fig. 9 shown.

[0053] 2.3. Linearity test conclusion Linearity evaluation: The correlation is 0.9999, so this method is used to determine vitamin B 12 It shows good linearity between the concentrations of 0.04832μg / mL and 2.416μg / mL, which meets the requirements of GB / T27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment": GB / T27417-2017 requires a correlation of ≥0.99.

[0054] 3. Limit of detection and limit of quantification The detection limit DL and quantification limit QL of the analytical method were calculated from the signal-to-noise ratio (S / N). DL was defined as the concentration of the analyte corresponding to S / N=3, and QL was defined as the concentration of the analyte corresponding to S / N=10.

[0055] 3.1 Detection Limit When the signal-to-noise ratio (S / N) is 3, vitamin B 12 The detection limit was 0.012 μg / mL.

[0056] 3.2 Limit of Quantitation When the signal-to-noise ratio (S / N) is 10, vitamin B 12 The limit of quantification was 0.036 μg / mL.

[0057] 4. Precision test 4.1 Test methods Weigh 6 portions of multivitamin samples, treat the samples according to the sample solution preparation method of Example 1 to prepare sample solutions, detect the sample contents, and calculate the RSD (%) thereof.

[0058] 4.2 Experimental data Table 2 Test data table 4.3 Test conclusion 6 samples of vitamin B 12 The content RSD is 1.5%, indicating that the method has good precision and meets the requirements of GB / T27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment": GB / T27417-2017 requires RSD (%) ≤ 7.5%.

[0059] 5. Recovery rate test 5.1 Test methods Vitamin B12 at a concentration of 2.42 μg / mL 12 Preparation of standard solution: Precisely weigh vitamin B 12 24.2 mg of the standard substance was placed in a 10 mL volumetric flask, and 5% ethanol solution was added to dissolve it to the mark, and the solution was shaken to obtain the standard stock solution. Accurately pipette 1 mL of the standard mother solution, dilute it with sodium acetate solution, and make up to 100 mL as the standard intermediate solution; Take an appropriate amount of the standard intermediate solution and dilute it with sodium acetate solution to a concentration of 2.42 μg / mL of vitamin B 12 Standard solution.

[0060] Spiking: 9 samples of multivitamin were accurately weighed and divided into three groups, each with three samples. Vitamin B12 with a concentration of 2.42 μg / mL was accurately added to each group. 12 The standard solution 2.0mL, 4.0mL, 6.0mL, and the sample solution were prepared according to the method of Example 1.

[0061] 5.2. Test data are as follows Table 3 Recovery test data The measured amount added = the measured amount of the standard sample - the measured amount of the sample; Recovery rate (%) = measured amount added / theoretical amount added × 100%; 5.3 Test conclusion The average recovery rate is 96.9%, and the relative standard deviation (RSD) is 3.1%, which shows that this method has good accuracy and the recovery rate meets the requirements of GB / T27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment": the measured component is 1-100 mg / kg, and the recovery rate is 90-110%.

[0062] 6. Conclusion Through vitamin B 12 The linearity, precision, blank, detection limit, quantification limit and recovery rate tests of the content determination method were all in line with the requirements of GB / T27417-2017 "Guidelines for Confirmation and Validation of Chemical Analysis Methods for Conformity Assessment", proving that the content determination method is scientific and effective and can be used to determine the content of vitamin B 12 The multivitamins serve the purpose of quality control.

[0063] Comparative Example: The difference between this comparative example and Example 1 is that the detection wavelength is 361 nm.

[0064] The test results of the comparative example are Fig.10 As shown, according to Fig.10 It can be seen that when the detection wavelength is 361nm, the interference of the miscellaneous peaks is serious and accurate measurement cannot be performed.

[0065] Embodiment 3: The difference from Example 1 is that the test solution is obtained from Preparation Example 2.

[0066] After testing, the content of vitamin B12 in the sample was 3.52μg / g.

[0067] Embodiment 4: The difference from Example 1 is that the test solution is obtained from Preparation Example 3.

[0068] After testing, the content of vitamin B12 in the sample was 3.6μg / g.

Claims

1. A method for determining vitamin B12, characterized in that: The detection method steps are as follows: Prepare sample solution and standard solution; Take the sample solution and the standard solution, inject them into a high performance liquid chromatograph for liquid chromatography analysis, and determine the vitamin B12 content; wherein the conditions of the liquid chromatography analysis include: Mobile phase A: 0.025% trifluoroacetic acid solution; Mobile phase B: acetonitrile; The gradient elution method was used for elution. The gradient elution procedure was as follows: 。 2. A method for determining vitamin B12 according to claim 1, characterized in that: The conditions of the liquid chromatography analysis also include: the chromatographic column is an ODS column, the length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the filler particle size is 5 μm.

3. A method for determining vitamin B12 according to claim 1, characterized in that: The conditions of the liquid chromatography analysis also include: detection wavelength of 545-555 nm; injection volume of 50-150 μL; flow rate of 0.5-1.5 mL / min; column temperature of 20-40° C.; and the detector is a diode array detector.

4. A method for determining vitamin B12 according to claim 3, characterized in that: The detection wavelength was 546 nm; the injection volume was 100 μL; the flow rate was 1 mL / min; and the column temperature was 35° C.

5. A method for determining vitamin B12 according to claim 1, characterized in that: The sample solution was prepared in the following steps: Weigh the test solution, add sodium acetate to dissolve it, ultrasonicate at 35-45℃ for 3-8min, cool to 20-25℃, add sodium acetate solution to the scale, centrifuge, filter, and prepare a sample solution with a mass concentration of 0.1-2.0ug / mL.

6. A method for determining vitamin B12 according to claim 5, characterized in that: The test sample in the test solution is a multivitamin; wherein the content of vitamin B12 in the multivitamin is 1.92-3.8 μg / g.

7. A method for determining vitamin B12 according to claim 6, characterized in that: The complex vitamin also includes at least one of calcium, magnesium, manganese, iron, zinc, selenium, copper, vitamin A, vitamin D3, vitamin B1, vitamin B2, vitamin B6, folic acid, biotin, vitamin C, vitamin K1, pantothenic acid, vitamin E and niacinamide.

8. A method for determining vitamin B12 according to claim 6, characterized in that: The preparation method of the test solution comprises the following steps: (1) grinding the vitamin complex, adding a sodium acetate solution, and mixing well to obtain a vitamin complex solution; (2) mixing diatomaceous earth and sodium carbonate to obtain a precipitating agent; (3) separating the precipitant into precipitant A and precipitant B; wherein the mass ratio of precipitant A to precipitant B is (0.4-0.6):(1-3); (4) Add precipitant A to the multivitamin solution and mix, then add precipitant B and mix, filter, and obtain a test solution.

9. A method for determining vitamin B12 according to claim 8, characterized in that: In the step (1), the amount of the vitamin complex added is 1-4 parts by weight; the amount of the sodium acetate solution added is 20-30 mL; In the step (2), the amount of diatomaceous earth added is 2-5 parts by weight; the amount of sodium carbonate added is 4-7 parts by weight; The time for adding the precipitant A and mixing in step (4) is 2-5 minutes; the stirring speed is 50-100 rpm; In the step (4), the time for adding the precipitant B and mixing is 20-30 minutes; and the stirring speed is 200-300 rpm.

10. A method for determining vitamin B12 according to claim 1, characterized in that: Preparation of the standard solution: Precisely weigh vitamin B 12 24.2 mg of the standard substance was diluted with 5% ethanol solution and made up to 10 mL. The solution was shaken well and used as the standard stock solution. Accurately pipette 1 mL of the standard mother solution, dilute it with sodium acetate solution, and make up to 100 mL as the standard intermediate solution; Pipette appropriate amount of standard intermediate solution respectively, and dilute with sodium acetate solution to prepare standard working curve solutions with concentrations of 0.0484μg / mL, 0.242μg / mL, 0.726μg / mL, 1.21μg / mL, 1.936μg / mL, and 2.42μg / mL.

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