Method for detecting B vitamins and application thereof

Abstract

The invention provides a method for detecting B vitamins and application thereof. The vitamin B is one or more of vitamin B1, vitamin B2, nicotinic acid, nicotinamide and vitamin B6; the method comprises the following steps: S1, pretreatment: heating a mixture to prepare a liquid sample to be detected; the mixture comprises a sample to be detected, an EDTA saturated solution, formic acid and water; s2, detection: respectively analyzing different standard samples and liquid samples to be detected by adopting a liquid chromatography ultraviolet detector to obtain analysis results of the standard samples and analysis results of the liquid samples to be detected; s3, analysis: comparing the analysis result of the to-be-detected liquid sample with the analysis result of each standard sample, and carrying out qualitative and quantitative analysis on the B vitamins in the to-be-detected sample. According to the method, the use of toxic reagents is reduced, the operation is simple, different B vitamins can be qualitatively and quantitatively detected at the same time, and the detection is rapid, simple, convenient and accurate.

Description

Technical Field

[0001] The present invention relates to the field of vitamin detection, and particularly relates to a method for detecting B vitamins and its application. Background Art

[0002] Infant formula milk powder is based on cow's milk or other animal milk or other animal and plant components as the basic components, and appropriate compound nutritional fortifiers and other auxiliary materials are added to make it as close to breast milk as possible, meeting the digestion, absorption and nutritional needs of infants; Food compound nutritional fortifiers refer to natural or synthetic food additives belonging to the scope of natural nutrients added to food to enhance nutritional components, and are additives used for the purpose of enhancing and supplementing the nutrition of food; The compound nutritional fortifiers used for nutritional fortification are mainly vitamins and minerals, and a series of improvements are made on this basis, such as adding dietary fiber, oligosaccharides, amino acids, unsaturated fatty acids, etc.

[0003] As an important part of compound nutritional fortifiers, whether the added amount of vitamins meets the standard has a great impact on the nutritional fortification effect of products and the growth and development of the human body. At present, there is no clear standard and detection method for compound nutritional fortifiers in the national standard. Selecting a suitable detection method to detect common vitamins in compound nutritional fortifiers can further accurately determine the dosage standard of vitamins and improve the detection accuracy and efficiency.

[0004] The known determination methods for vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6 include the following standards. The determination of vitamin B1 can be GB 5009.84-2016 "National Food Safety Standard Determination of Vitamin B1 in Foods", the determination of vitamin B2 can be GB 5009.85-2016 "National Food Safety Standard Determination of Vitamin B2 in Foods", the determination of niacin and nicotinamide can be GB 5009.89-2023 "National Food Safety Standard Determination of Niacin and Nicotinamide in Foods", and the determination of vitamin B6 can be GB 5009.154-2023 "National Food Safety Standard Determination of Vitamin B6 in Foods"; In addition, the following methods are also included: the high performance liquid chromatography detection method for the contents of vitamin B1, vitamin B2, vitamin B6, niacin and nicotinamide (publication number: CN117388398A), the detection method for the content of vitamin B in compound vitamin additives (publication number: CN104330484A). 12 Content detection method (publication number: CN104330484A).

[0005] The existing national standard methods have certain limitations. The detection process is rather cumbersome, requiring various enzymatic hydrolyses and pH value adjustments, etc. Different detectors are used for different B vitamins, and it is impossible to detect multiple B vitamins simultaneously. The reagent types used in the published patents are numerous and toxic. At the same time, there is no relevant detection method for vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6 in compound vitamin raw materials.

[0006] At present, the main methods for detecting B vitamins are microbiological analysis and chemical analysis. In recent years, the reported chemical analysis methods for B vitamins mainly include spectrophotometry, electrochemical methods, electrophoresis, and liquid chromatography.

[0007] (1) Microbiological analysis: Vitamins are essential factors for the growth of certain microorganisms. Vitamins are quantitatively detected based on the specificity between vitamins and certain microorganisms. As an international standard method for the analysis of some vitamins, compared with chemical analysis methods, the microbiological analysis method can reflect the total vitamin content in the matrix. Currently, vitamins B 12 , pantothenic acid, and folic acid are mostly detected by the microbiological method. The advantages of the microbiological detection method are high sensitivity and suitability for large-scale analytical operations. The limitations are low accuracy, poor repeatability, and long time consumption. In this method, the sample to be detected is compound vitamins with a high sample content and a large dilution factor; therefore, it is not suitable for the microbiological method.

[0008] (2) Spectrophotometry: Spectrophotometry is used to qualitatively and quantitatively analyze different B vitamins by utilizing the luminescence intensity and absorbance of different B vitamins within a specific wavelength range. Its advantages are low equipment price, simple operation, and wide application range. The limitation is low accuracy.

[0009] (3) Electrochemical analysis: Almost all vitamins have electrochemical activity. Electrochemical methods are used to perform qualitative and quantitative analysis by measuring the electrical signal value of a certain chemical system. Vitamins can undergo electron transfer in water based on the electrochemical oxidation or reduction reactions of vitamins in the electrolyte solution. The electrochemical sensor reports the vitamin concentration by measuring the current on the working electrode. Currently, the research focus in the electrochemical field is to develop new electrode materials that can be used to detect vitamins with improved sensitivity and selectivity, and the co-detection of multiple vitamins is also challenging in the electrochemical field.

[0010] (4) Capillary electrophoresis: Capillary electrophoresis (CE) is a liquid-phase separation technique that uses an electric field as the driving force and a capillary as the separation channel. Its advantages include low cost, low sample and reagent consumption, ease of automation, and time savings. When using capillary electrophoresis to detect B vitamins, the reproducibility is poor, and due to the small sample injection volume in capillary electrophoresis, the sensitivity is low when combined with individual detectors.

[0011] (5) Liquid chromatography: As one of the main detection methods for B vitamins, currently, liquid chromatography is mainly divided into reverse-phase high-performance liquid chromatography (RP-HPLC), hydrophilic liquid chromatography (HILIC), and ultra-high-performance liquid chromatography (UPLC). By combining with detectors such as ultraviolet detector (UV), fluorescence detector (FLD), diode array detector (DAD), and mass spectrometry (MS), the separation and detection of various B vitamins can be achieved quickly and efficiently. RP-HPLC consists of a non-polar stationary phase and a mobile phase with a stronger polarity than the stationary phase, and it is the most commonly used technique for separating B vitamins. The stationary phase of the reverse liquid chromatography column is generally selected as a C18 chromatographic column. However, since water-soluble vitamins have poor retention ability in the reverse chromatographic column, it is necessary to change conditions such as the mobile phase to improve the peak shape and separation degree. The main measures include adding ion-pair reagents to the mobile phase, adjusting the pH value of the mobile phase, adding amine substances and electrolytes to the mobile phase. However, the accuracy of this method is insufficient, and it is difficult to give accurate qualitative and quantitative results. Summary of the Invention

[0012] In order to overcome the problems in the prior art that toxic reagents are used in the detection process and it is difficult to accurately qualitatively and quantitatively detect B vitamins, the present invention has established a new detection method by improving the pretreatment method, instrument conditions, etc., and provides a method for detecting B vitamins; this method reduces the use of toxic reagents, is easy to operate, and can simultaneously qualitatively and quantitatively detect different B vitamins, with rapid, simple, and accurate detection.

[0013] In the art, vitamins are divided into water-soluble vitamins and fat-soluble vitamins. The main targets detected by this method are water-soluble vitamins B1, B2, niacin, nicotinamide, and vitamin B6; among them, vitamins B1, B2, and B6 are usually also referred to as VB1, VB2, and VB6; vitamin B6 can also be referred to as pyridoxine.

[0014] The present invention provides a method for detecting B vitamins, wherein the B vitamins are one or more of vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6; the method comprises the following steps:

[0015] S1 Pretreatment: Heat the mixture to obtain a test liquid sample; the mixture includes a test sample, a saturated EDTA solution, formic acid, and water; the ratio of the mass of the test sample to the volume of the saturated EDTA solution is 0.3 g : (1.45 - 1.95) mL; the heating temperature is 55°C to 75°C; the heating time is 25 to 35 min;

[0016] S2 Detection: Use the ultraviolet detector of liquid chromatography to analyze different standard samples and the test liquid sample respectively, and obtain the analysis results of each standard sample and the analysis result of the test liquid sample; the standard sample is one or more of vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6; the detection wavelength of the ultraviolet detector is 270 - 280 nm; the chromatographic column of the liquid chromatography is a PFP chromatographic column; the mobile phase A of the liquid chromatography is a mixed aqueous solution of ammonium formate and formic acid; the elution method of the liquid chromatography adopts gradient elution;

[0017] S3 Analysis: Compare the analysis result of the test liquid sample with the analysis results of each standard sample to qualitatively and quantitatively analyze the B vitamins in the test sample.

[0018] In some embodiments, in the S1 pretreatment step, the preparation method of the mixture includes the following steps: sequentially add a saturated EDTA solution, the formic acid, and the water to a container containing the test sample.

[0019] In some embodiments, in the mixture, the ratio of the mass of the test sample, the volume of the saturated EDTA solution, and the volume of the formic acid is 0.3 g : (1.45 - 1.95) mL : (0.05 - 0.1) mL.

[0020] In some embodiments, in the mixture, the ratio of the mass of the test sample, the volume of the saturated EDTA solution, and the volume of the formic acid is 0.3 g : (1.5 - 1.8) mL : (0.08 - 0.1) mL.

[0021] In some embodiments, in the S1 pretreatment step, after the heating process, cool and make up the volume in sequence to obtain the test liquid sample; then filter the test liquid sample.

[0022] In some embodiments, in the S2 detection step, the column temperature of the PFP chromatographic column is 30 - 40°C.

[0023] In some embodiments, in the S2 detection step, the mobile phase B of the liquid chromatography is acetonitrile with a mass content of 100%.

[0024] In a specific embodiment, in the S2 detection step, the gradient elution method includes:

[0025] Within 0 - 20 min, the volume ratio of mobile phase A is 100% and the volume ratio of mobile phase B is 0%; within 20 - 25 min, the volume ratio of mobile phase A drops to ≤55% and the volume ratio of mobile phase B rises to ≥45%; within 25 - 32 min, the volume ratio of mobile phase A drops to 0% and the volume ratio of mobile phase B rises to 100%; within 32 - 39 min, the volume ratio of mobile phase A remains 0% and the volume ratio of mobile phase B remains 100%; within 39 - 39.1 min, the volume ratio of mobile phase A rises to 100% and the volume ratio of mobile phase B drops to 0%; within 39.1 - 50 min, the volume ratio of mobile phase A remains 100% and the volume ratio of mobile phase B remains 0%.

[0026] In some embodiments, in the S3 analysis step, the B vitamins in the sample to be tested are quantitatively analyzed by the external standard method.

[0027] The present invention also provides an application of a method for detecting B vitamins, which is used for detecting B vitamins in food raw materials, and the food raw materials include one or more of dairy raw materials, baking product raw materials, and meat product raw materials.

[0028] On the basis of conforming to common knowledge in the art, the above - mentioned preferred conditions can be arbitrarily combined to obtain various preferred examples of the present invention.

[0029] The reagents and raw materials used in the present invention are all commercially available.

[0030] The positive and progressive effects of the present invention are as follows:

[0031] The present invention has established a new detection method through improvements in pretreatment methods, instrument conditions, etc. This method reduces the use of toxic reagents, is simple to operate, and can simultaneously perform qualitative and quantitative detection of multiple B vitamins, with fast, simple, and accurate detection. Description of the Drawings

[0032] Figure 1 It is the chromatogram obtained by using the detection method of Example 1;

[0033] Figure 2 It is the chromatogram obtained by using the detection method of Example 3;

[0034] Figure 3 It is the chromatogram obtained by using the detection method of Comparative Example 1;

[0035] Figure 4 It is the chromatogram obtained by using the detection method of Comparative Example 2;

[0036] Figure 5It is the chromatogram obtained by using the detection method of Comparative Example 3;

[0037] Figure 6 It is the chromatogram obtained by using the detection method of Comparative Example 4;

[0038] Figure 7 It is the chromatogram obtained by using the detection method of Comparative Example 5;

[0039] Figure 8 It is the chromatogram obtained by using the detection method of Comparative Example 6;

[0040] Figure 9 It is the chromatogram obtained by using the detection method of Comparative Example 7;

[0041] Figure 10 It is the chromatogram obtained by using the detection method of Comparative Example 8;

[0042] Figure 11 It is the chromatogram obtained by using the detection method of Comparative Example 9;

[0043] Figure 12 It is the chromatogram obtained by using the detection method of Comparative Example 12;

[0044] Figure 13 It is the broken line graph of the deviation value between the results of detecting 21 raw materials by using the detection method of Example 1 in Application Example 3 and the standard range provided by the manufacturer. Detailed implementation mode

[0045] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions noted in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0046] The present invention provides a method for detecting B vitamins, where the B vitamins are one or more of vitamin B1, vitamin B2, nicotinic acid, nicotinamide and vitamin B6; the method includes the following steps:

[0047] S1 Pretreatment: Heating the mixture to obtain a sample solution to be detected;

[0048] The mixture includes a sample to be tested, a saturated EDTA solution, formic acid and water; the ratio of the mass of the sample to be tested to the volume of the saturated EDTA solution is 0.3 g : (1.45 - 1.95) mL; the method for preparing the mixture includes the following steps: sequentially adding the saturated EDTA solution, the formic acid and the water into the container containing the sample to be tested; the ratio of the mass of the sample to be tested to the fixed volume of the sample solution to be tested is 0.3 g : 50 mL; in the mixture, the water includes the water for dissolving the sample to be tested and the water for volume fixation, and the ratio of the volume of the water for dissolving the sample to be tested to the fixed volume of the sample solution to be tested is 1 : 2;

[0049] The heating temperature is 55°C to 75°C; the heating time is 25 to 35 min; after the heating process, it is sequentially cooled and volume-fixed to obtain the sample solution to be tested; then the sample solution to be tested is subjected to a filtration treatment;

[0050] S2 Detection: Using an ultraviolet detector of a liquid chromatograph, different standard samples and the sample solution to be tested are analyzed respectively to obtain the analysis results of each standard sample and the analysis result of the sample solution to be tested; the standard samples are one or more of vitamin B1, vitamin B2, nicotinic acid, nicotinamide and vitamin B6;

[0051] The detection wavelength of the ultraviolet detector is 270 - 280 nm;

[0052] The chromatographic column of the liquid chromatograph is a PFP chromatographic column; the specification of the PFP chromatographic column is 4.6×250 mm, 5 μm; the column temperature of the PFP chromatographic column is 30 - 40°C;

[0053] The mobile phase A of the liquid chromatograph is a mixed aqueous solution of ammonium formate and formic acid; in the mobile phase A, the mass content of ammonium formate is 0.75 g / L, and the volume content of formic acid is 0.2%, where % is the volume percentage of formic acid in the mixed aqueous solution of ammonium formate and formic acid; the mobile phase B of the liquid chromatograph is acetonitrile with a mass content of 100%;

[0054] The elution method of the liquid chromatograph uses a gradient elution method; the gradient elution method includes:

[0055] Within 0 - 20 min, the volume percentage of mobile phase A is 100%, and the volume percentage of mobile phase B is 0%; within 20 - 25 min, the volume percentage of mobile phase A drops to ≤55%, and the volume percentage of mobile phase B rises to ≥45%; within 25 - 32 min, the volume percentage of mobile phase A drops to 0%, and the volume percentage of mobile phase B rises to 100%; within 32 - 39 min, the volume percentage of mobile phase A remains 0%, and the volume percentage of mobile phase B remains 100%; within 39 - 39.1 min, the volume percentage of mobile phase A rises to 100%, and the volume percentage of mobile phase B drops to 0%; within 39.1 - 50 min, the volume percentage of mobile phase A remains 100%, and the volume percentage of mobile phase B remains 0%.

[0056] S3 Analysis: Compare the analysis results of the sample to be tested with the analysis results of each standard sample to qualitatively and quantitatively analyze the B vitamins in the sample to be tested; quantitatively analyze the B vitamins in the sample to be tested by the external standard method.

[0057] In the present invention, the PFP chromatographic column refers to a polyaryl fluoride chromatographic column commonly used in the art.

[0058] In the S1 pretreatment step of the present invention, the saturated EDTA solution refers to a saturated aqueous solution of EDTA; the solute of the saturated EDTA solution is ethylenediaminetetraacetic acid (EDTA); the preparation process of the saturated EDTA solution can be carried out as follows: Weigh 12 g of EDTA disodium salt into a 250 mL beaker, add 90 mL of water, heat and stir until completely dissolved, and after standing overnight, the supernatant is the saturated EDTA solution. Those skilled in the art know that under different room temperature conditions, the content of the solute EDTA in the saturated EDTA solution will vary slightly.

[0059] Among them, the saturated aqueous solution of EDTA can provide more coordination sites, so as to more effectively combine with metal ions in B vitamins to form stable chelates. This high-concentration environment also helps to reduce the interference of metal ions and improve the selectivity and efficiency of the chelation reaction. In addition, EDTA can quickly react with metal ions, reducing the reaction time and improving the efficiency and accuracy of the experiment.

[0060] In the S1 pretreatment step of the present invention, generally, when the volume of the saturated EDTA solution is 1.45 mL - 1.95 mL, the content of solid EDTA is preferably 0.1462 g - 0.1967 g.

[0061] In the present invention, first adding an EDTA saturated solution to the sample to be tested can enable the metal ions in the sample to be better complexed; adding a certain amount of formic acid to adjust the pH value of the mixture to 2.5 - 3.5, so as to improve the ionization efficiency, enhance the separation effect, and improve the extraction efficiency of vitamin B2; adding water can make the extraction of the sample to be tested more sufficient during the shaking process.

[0062] In the S1 pretreatment step of the present invention, the formic acid refers to formic acid with a content of 100%.

[0063] In the present invention, those skilled in the art should know that in the mixture, the water refers to the additionally added water, rather than the water in the EDTA saturated solution.

[0064] In a certain embodiment, in the mixture, the water is primary water.

[0065] In the present invention, the primary water can be understood according to the routine in the art, generally referring to water with a conductivity not higher than 0.01 mS / m.

[0066] In a certain embodiment, after successively adding the EDTA saturated solution and the formic acid to 0.3 g of the sample to be tested, 25 mL of water is added to fully dissolve the sample to be tested; after complete dissolution, water is added to make the volume of the sample solution to be tested up to 50 mL.

[0067] In a certain embodiment, after successively adding the EDTA saturated solution and the formic acid to 0.6 g of the sample to be tested, 50 mL of water is added to fully dissolve the sample to be tested; after complete dissolution, water is added to make the volume of the sample solution to be tested up to 100 mL.

[0068] In the present invention, those skilled in the art should know that the sample solution to be tested is a solution containing the sample to be tested.

[0069] In a certain embodiment, in the mixture, the mass ratio of the sample to be tested, the volume of the EDTA saturated solution, and the volume of the formic acid is 0.3 g : (1.5 - 1.8) mL : (0.08 - 0.1) mL.

[0070] In a certain embodiment, in the mixture, the mass ratio of the sample to be tested, the volume of the EDTA saturated solution, and the volume of the formic acid is 0.3 g : 1.5 mL : 0.1 mL.

[0071] By adding an appropriate amount of formic acid, the extraction rate of the vitamin to be tested can be improved, while ensuring consistency with the initial mobile phase, avoiding the influence of the solvent peak on the determination, and reducing the solvent effect.

[0072] In the present invention, the process of heating the mixture can include shaking the mixture under constant temperature conditions.

[0073] In the S1 pretreatment step of the present invention, hydrolysis occurs by heating the mixture; based on this, the heating temperature can be referred to as the "hydrolysis temperature", and the heating time can be referred to as the "hydrolysis time".

[0074] In one embodiment, the temperature of the heating is 55°C, 60°C, 65°C, 70°C or 75°C.

[0075] In one embodiment, the time of the heating is 25 min, 28 min, 30 min or 35 min.

[0076] In one embodiment, in the S2 detection step, the detection wavelength of the ultraviolet detector is 270 nm or 280 nm; when the detection wavelength of the ultraviolet detector is 280 nm, the obtained chromatogram baseline is smoother.

[0077] In one embodiment, in the S2 detection step, the gradient elution method is as follows:

[0078]

[0079] In the present invention, preferably, the mobile phase A of the liquid chromatography is a mixed aqueous solution of ammonium formate and formic acid; after adding the ion pair reagent, a longer equilibration time is required, and there are also problems such as poor reproducibility of retention values, unstable baseline and some other separation problems. Therefore, generally, the combination of the ion pair reagent and the gradient elution method is not recommended; in this application, by using the above ion pair reagent as the mobile phase A, the accuracy of analysis can be improved.

[0080] In the present invention, preferably, the mobile phase B is acetonitrile with a mass content of 100%. Acetonitrile has a strong elution ability, especially in the case of a low mixing ratio; in addition, the polarity index of acetonitrile is also relatively high, which means that in liquid chromatography, the elution range of the solution prepared by mixing acetonitrile and water is relatively wide, and its elution ability gradually decreases as the proportion of the organic phase increases. This difference is particularly important for the separation of certain specific compounds, especially in multi-component analysis, and acetonitrile can provide better separation effects.

[0081] In one embodiment, in the S2 detection step, the column temperature of the PFP chromatographic column is 40°C.

[0082] In one embodiment, in the S2 detection step, the external standard method is used to quantify the sample to be measured.

[0083] In one embodiment, in the S2 detection step, preferably, the standard sample is prepared as follows: Weigh 50 mg of vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6 respectively into a 50 mL volumetric flask, dissolve with primary water and make up to the scale; shake well for standby; among them, the solubility of vitamin B2 in water is relatively low, and its standard sample needs to be shaken at 70 °C for 1 h.

[0084] In one embodiment, in the S2 detection step, in the standard curve to be plotted by the external standard method, the concentrations of each standard sample are as follows:

[0085] Table 1

[0086]

[0087] In one embodiment, the method for detecting B vitamins includes the following steps:

[0088] A. Select a sample to be tested, that is, a sample of B vitamins to be detected;

[0089] B. Weigh 0.3 g of the sample to be tested into a 50 mL volumetric flask;

[0090] C. Add 1.5 mL of saturated EDTA solution to the volumetric flask in step B;

[0091] D. Add 0.1 mL of formic acid to the volumetric flask in step C;

[0092] E. Add 25 mL of primary water to the volumetric flask in step D to form a mixture;

[0093] F. Shake the mixture in the volumetric flask well and place it in a constant temperature of 70 °C and shake for 30 min;

[0094] G. Take out the volumetric flask in step F and cool it to room temperature, then make up to the fixed volume scale of the volumetric flask with primary water and shake well to obtain a sample solution to be tested;

[0095] H. Filter the sample solution to be tested in step G through a water-based filter membrane;

[0096] I. Separate the sample solution to be tested after filtration in step H through a chromatographic column and detect it with a liquid chromatography ultraviolet detector;

[0097] J. Compare the retention time of the target chromatographic peak in step H with the retention time of the chromatographic peak of the standard substance to check whether there is a target substance in the sample to be tested; if there is a target substance, then use the external standard method for quantification.

[0098] In one embodiment, the method for detecting B vitamins includes the following steps:

[0099] A. Select a sample to be tested, that is, a sample of B vitamins to be detected;

[0100] B. Weigh 0.6 g of the sample to be tested into a 100 mL volumetric flask;

[0101] C. Add 3.0 mL of saturated EDTA solution to the volumetric flask in step B;

[0102] D. Add 0.2 mL of formic acid to the volumetric flask in step C;

[0103] E. Add 50 mL of primary water to the volumetric flask in step D to form a mixture;

[0104] F. Shake the mixture in the volumetric flask well, place it in a constant temperature bath at 70 °C and shake for 30 min;

[0105] G. Take out the volumetric flask in step F and cool it to room temperature, then make up the volume to the calibration mark of the volumetric flask with primary water and shake well to obtain the sample solution to be tested;

[0106] H. Filter the sample solution to be tested in step G through a water-based filter membrane;

[0107] I. Separate the sample solution to be tested after filtration in step H through a chromatographic column and detect it using a liquid chromatography ultraviolet detector;

[0108] J. Compare the retention time of the chromatographic peak of the target substance obtained in step H with the retention time of the chromatographic peak of the reference substance to check whether the target substance exists in the sample to be tested; if the target substance exists, then use the external standard method for quantification.

[0109] In the present invention, the room temperature is the conventional understanding in the art, specifically 15 - 30 °C.

[0110] The present invention also provides an application of a method for detecting B vitamins, which is used for detecting B vitamins in food raw materials, and the food raw materials include one or more of dairy raw materials, baking product raw materials and meat product raw materials.

[0111] In some embodiments of the present invention, the raw materials of the B vitamins in the test samples in the comparative examples and application examples are sourced from three manufacturers, namely A, B, and C. Among them, manufacturer A is Beijing Jinkangpu Food Technology Co., Ltd., and its raw material batch numbers are: 6030305236424, 603211222H053, 603181122G189, 603160223A887, 603110523C690, 603010323B215, 603020323B252; manufacturer B is DSM Vitamins (Shanghai) Co., Ltd., and its raw material batch numbers are: VR22101397, VR23041289, VR23041261, VR23071083, VR23061212, VR23121470, VR23121438, VR2401123, VR22121011, VR23041364, VR23121404; manufacturer C is Glanbia Nutritionals (Suzhou) Co., Ltd., and its raw material batch numbers are: 0001173026, 0001173028, 0001182783.

[0112] Example 1

[0113] This example provides a method for detecting B vitamins. The method includes the following steps:

[0114] S1 Pretreatment: Heat the mixture to obtain a test liquid sample; the preparation method of the mixture includes the following steps:

[0115] A. Select a compound raw material containing vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6 as the test sample;

[0116] B. Weigh 0.3 g of the test sample into a 50 mL volumetric flask;

[0117] C. Add 1.5 mL of saturated EDTA solution to the volumetric flask in step B; the preparation method of the saturated EDTA solution is as follows: Add more than 12 g of EDTA solid to every 100 mL of water, heat and stir to dissolve, cool to room temperature, and then take the volume required for mixing;

[0118] D. Add 0.1 mL of formic acid to the volumetric flask in step C;

[0119] E. Add 25 mL of primary water to the volumetric flask in step D to form a mixture;

[0120] F. Shake the mixture in the volumetric flask well and place it in a constant temperature at 70 °C and shake for 30 min;

[0121] G. Take out the volumetric flask in step F and cool it to room temperature, then make up the volume to the volumetric scale of the volumetric flask with primary water and shake well to obtain a test liquid sample;

[0122] H. Filter the test liquid sample in step G through a 0.22 μm aqueous filter membrane;

[0123] The heating temperature of the mixture is 70 °C;

[0124] The heating time of the mixture is 30 min.

[0125] S2 Detection: Use the ultraviolet detector of liquid chromatography to analyze different standard samples and the test liquid sample respectively to obtain the analysis results of each standard sample and the test liquid sample; The standard samples are vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6;

[0126] The detection conditions of liquid chromatography are as follows:

[0127] a) The chromatographic column is: a 4.6×250 mm, 5 μm PFP chromatographic column;

[0128] b) The mobile phase consists of mobile phase A and mobile phase B; Among them,

[0129] Mobile phase A is a mixed aqueous solution of ammonium formate and formic acid, and its preparation method is as follows: Weigh 0.75 g of ammonium formate, dissolve it in 500 mL of water, add 2 mL of anhydrous formic acid, and then make up the volume to 1000 mL with primary water;

[0130] Mobile phase B: The content is 100% acetonitrile;

[0131] c) The conditions for gradient elution are shown in Table 2, and Table 2 is the elution program of the mobile phase.

[0132] Table 2

[0133]

[0134] d) Column temperature: 40 °C;

[0135] e) Injection volume: 10 μL;

[0136] f) The detection wavelength of the ultraviolet detector is 280 nm;

[0137] The preparation method of the standard sample is: Weigh 50 mg of vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6 respectively in a 50 mL volumetric flask, dissolve them with primary water and make up the volume to the scale; Shake well and set aside; Among them, the solubility of vitamin B2 in water is relatively low, and its standard sample needs to be shaken at 70 °C for 1 h.

[0138] S3 Analysis: Compare the analysis results of the test liquid sample with those of each standard sample to qualitatively and quantitatively analyze the B vitamins in the test sample. Specifically, quantitatively analyze the B vitamins in the test sample by the external standard method; among them, for the preparation of the standard curve: dilute to the concentrations in Table 1 with 2% formic acid solution.

[0139] Figure 1 is the chromatogram obtained by the detection method of Example 1.

[0140] Example 2

[0141] This example provides a method for detecting B vitamins. In the S1 pretreatment step, the heating temperature is 60°C. Except for this, other conditions are the same as those in Example 1.

[0142] Example 3

[0143] This example provides a method for detecting B vitamins. In the S2 detection step, the detection wavelength of the ultraviolet detector is 270 nm. Except for this, other conditions are the same as those in Example 1.

[0144] Figure 2 is the chromatogram obtained by the detection method of Example 3.

[0145] Comparative Examples 1 - 6

[0146] This group of comparative examples discloses a method for detecting B vitamins. The detection wavelengths of the ultraviolet detectors in this group of comparative examples are different from those in Example 1. Specifically, in the S2 detection step, the detection wavelength of the ultraviolet detector in Comparative Example 1 is 210 nm; the detection wavelength of the ultraviolet detector in Comparative Example 2 is 220 nm; the detection wavelength of the ultraviolet detector in Comparative Example 3 is 230 nm; the detection wavelength of the ultraviolet detector in Comparative Example 4 is 240 nm; the detection wavelength of the ultraviolet detector in Comparative Example 5 is 250 nm; the detection wavelength of the ultraviolet detector in Comparative Example 6 is 260 nm. Other conditions are the same as those in Example 1.

[0147] Figures 3-8 are the chromatograms obtained by the detection methods of Comparative Examples 1 - 6 respectively.

[0148] From Figures 3-8 it can be seen that at low wavelengths of 210 - 260 nm, the baseline drifts; thus affecting the detection results of vitamin B1, vitamin B2, nicotinic acid, nicotinamide, and vitamin B6.

[0149] However, from Figures 1-2 it can be seen that when the detection wavelength of the ultraviolet detector is 270 nm (the detection method of Example 3), 280 nm (the detection method of Example 1), the resolution and baseline of the chromatogram are both good.

[0150] Therefore, the detection wavelength of the ultraviolet detector is 270 nm or 280 nm, which can take into account the detection sensitivities of five substances, namely vitamin B1, vitamin B2, niacin, nicotinamide, and vitamin B6.

[0151] Comparative Example 7

[0152] This comparative example discloses a method for detecting B vitamins. The chromatographic column of the liquid chromatography in this comparative example is different from that in Example 1; specifically, in the S2 detection step of Comparative Example 7, the chromatographic column is a C18 chromatographic column. Other conditions are the same as those in Example 1.

[0153] Figure 9 is the chromatogram obtained by using the detection method of Comparative Example 7. As Figure 9 can be seen, when using a C18 chromatographic column, the ionic strength is relatively low. In addition, formate is relatively easy to absorb moisture, making it difficult to weigh, and it may also volatilize in the mobile phase; there are impurity peaks before and after the peaks of nicotinamide and vitamin B2, and the separation effect of the C18 chromatographic column is lower than that of the PFP chromatographic column in Example 1.

[0154] Comparative Examples 8 - 9

[0155] This group of comparative examples discloses a method for detecting B vitamins. The mobile phase of the chromatographic column and its elution method in this comparative example are different from those in Example 1. Specifically, in the S2 detection step of Comparative Examples 8 - 9, sodium octanesulfonate (Comparative Example 8) and sodium hexanesulfonate (Comparative Example 9) are respectively selected as the mobile phase A, and the mobile phase B is acetonitrile. The isocratic elution method is adopted, and elution is carried out according to the volume ratio of aqueous phase: organic phase (mobile phase A: mobile phase B) of 85:15. Other conditions are the same as those in Example 1.

[0156] Figures 10-11 are the chromatograms obtained by using the detection methods of Comparative Examples 8 and 9 respectively. As Figure 10 can be seen, when sodium octanesulfonate is selected as the ion pair reagent in Comparative Example 8, vitamin B2 and vitamin B6 do not peak under isocratic elution, that is, vitamin B2 and vitamin B6 cannot be detected; as Figure 11 can be seen, when sodium hexanesulfonate is selected as the ion pair reagent in Comparative Example 9, the elution time of vitamin B2 under isocratic elution is 13 min, but there are impurity peaks near the target peak, interfering with the detection result.

[0157] While in Example 1, ammonium formate is selected as the ion pair reagent and a certain amount of formic acid is added, and gradient elution can well achieve the separation of chromatographic peaks and at the same time ensure that the elution time of vitamin B2 is advanced.

[0158] Comparative Examples 10 - 12

[0159] This group of comparative examples discloses a method for detecting B vitamins. The elution method in this comparative example is different from that in Example 1; specifically, the elution method in the S2 detection step of Comparative Examples 10-12 is isocratic elution, and the isocratic elution of this group of comparative examples is carried out according to the volume ratio of aqueous phase: organic phase (mobile phase A: mobile phase B) of 95:5 (Comparative Example 10), 90:10 (Comparative Example 11), and 85:15 (Comparative Example 12).

[0160] Among them, mobile phase A is a mixed aqueous solution of ammonium formate and formic acid, and its preparation method is as follows: Weigh 0.75 g of ammonium formate, dissolve it in 500 mL of water, add 2 mL of anhydrous formic acid, and then make up the volume to 1000 mL with primary water; mobile phase B: acetonitrile with a mass content of 100%.

[0161] Figure 12 Figure 7 is the chromatogram obtained by using the detection method of Comparative Example 12. When the volume ratio of mobile phase A to mobile phase B in isocratic elution is 85:15, the chromatographic peaks can be effectively separated, but the peak time of vitamin B2 is about 40 min. In Example 1, gradient elution is used, and vitamin B2 elutes at 28 min. There will be impurity peaks from the elution of vitamin B2 to 45 min, which proves that gradient elution has a better effect than isocratic elution.

[0162] Comparative Examples 13-17

[0163] This group of comparative examples discloses a method for detecting B vitamins. The dosage of the extraction reagent in this comparative example is different from that in Example 1; specifically, in the mixture preparation method of the S1 pretreatment step of Comparative Examples 13-17, the addition amounts of the EDTA saturated solution are 1.0 mL (Comparative Example 13), 2.0 mL (Comparative Example 14), 2.5 mL (Comparative Example 15), 5.0 mL (Comparative Example 16), and 10.0 mL (Comparative Example 17) respectively, and other conditions are the same as those in Example 1.

[0164] The test sample of this detection method selects a B vitamin raw material from manufacturer A, in which the niacinamide content ranges from 15575 to 20139 mg / kg, the vitamin B2 content ranges from 1566 to 2034 mg / kg, the vitamin B6 content ranges from 1700 to 2300 mg / kg, and the vitamin B1 content ranges from 3000 to 3800 mg / kg.

[0165] Table 3 shows the detection results with different addition amounts of the extraction reagent.

[0166] Table 3

[0167]

[0168] When the volume of the EDTA saturated solution added is 1.5 mL, it is the result of detecting a B vitamin raw material from Manufacturer A using the detection method of Example 1.

[0169] As can be seen from the above table, when the addition amount of the extracting agent is 1.0 mL (Comparative Example 13), 2.0 mL (Comparative Example 14), 2.5 mL (Comparative Example 15), 5.0 mL (Comparative Example 16), or 10.0 mL (Comparative Example 17), some of the detection results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will fall within the range of their standard contents, but they still do not meet the standards, and it is impossible to accurately measure niacinamide, vitamin B2, vitamin B6, and vitamin B1 simultaneously. When the volume of the EDTA saturated solution added in Example 1 is used (that is, the volume of the added EDTA saturated solution is 1.5 mL), the detection results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will all fall within the range of their standard contents.

[0170] Comparative Example 18

[0171] This comparative example discloses a method for detecting B vitamins. The type of the extracting reagent in this comparative example is different from that in Example 1, and only primary water is used as the extracting agent; specifically, the method for preparing the mixture in the S1 pretreatment step of Comparative Example 18 includes the following steps:

[0172] A. Using the B vitamin raw materials from the above-mentioned Manufacturers A, B, and C as the test samples;

[0173] B. Weighing 0.3 g of the test sample into a 50 mL volumetric flask;

[0174] C. Adding 25 mL of primary water to the volumetric flask in step B to form a mixture;

[0175] D. Shaking the mixture in the volumetric flask well and placing it in a constant temperature of 70 °C and shaking for 30 min;

[0176] E. Taking out the volumetric flask in step D and cooling it to room temperature, then making up the volume to the volumetric scale of the volumetric flask with primary water and shaking well to obtain the test liquid sample;

[0177] F. Passing the test liquid sample in step E through a 0.22 μm water-based filter membrane.

[0178] Other conditions are the same as those in Example 1.

[0179] Table 4 shows the detection results of different types of extracting reagents for B vitamin raw materials from different manufacturers and the standard ranges provided by the manufacturers.

[0180] Table 4

[0181]

[0182] Among them, when the extraction reagent is "primary water + EDTA + formic acid", the results of raw materials from three manufacturers A, B, and C are detected respectively by the detection method of Example 1.

[0183] In the above table, the preparation method of the mixture in Comparative Example 18 corresponding to "primary water" is as follows: Weigh 0.3 g of the sample to be tested, add 25 mL of primary water, hydrolyze at 70 °C for 30 min, cool to room temperature after taking out, and make up the volume with primary water. The preparation method of the mixture in Example 1 corresponding to "primary water + EDTA + formic acid" is as follows: Add 1.5 mL of saturated EDTA solution, 0.1 mL of formic acid solution and 25 mL of primary water to a 50 mL volumetric flask containing 0.3 g of the sample to be tested, place it in a water bath thermostatic shaker at 70 °C for 30 min, take it out and let it stand at room temperature, and make up the volume to the scale with primary water.

[0184] As can be seen from the above table, the content of the target substance measured by the detection method of Comparative Example 18 is partially not within the range of the standard content, while the content of the target substance measured by the detection method of Example 1 (i.e., the extraction reagent is "primary water + EDTA + formic acid") is within the range of the standard content; this proves the reliability of the method of this application.

[0185] Comparative Examples 19 - 22

[0186] This group of comparative examples discloses a method for detecting B vitamins. The heating temperature (hydrolysis temperature) in this comparative example is different from that in Example 1; specifically, the temperature for heating the mixture in the S1 pretreatment step of Comparative Examples 19 - 22 is 30 °C (Comparative Example 19), 40 °C (Comparative Example 20), 50 °C (Comparative Example 21), and 80 °C (Comparative Example 22) respectively. Other conditions are the same as those in Example 1.

[0187] The sample to be tested for this detection method is a B vitamin raw material from manufacturer A, where the range of nicotinamide is 15575 - 20139 mg / kg, the content range of vitamin B2 is 1566 - 2034 mg / kg, the content range of vitamin B6 is 1700 - 2300 mg / kg, and the content range of vitamin B1 is 3000 - 3800 mg / kg.

[0188] Table 5 shows the detection results at different heating temperatures.

[0189] Table 5

[0190]

[0191] Among them, when the heating temperature is 60 °C, it is the result of detecting a B vitamin raw material from manufacturer A by the detection method of Example 2; when the heating temperature is 70 °C, it is the result of detecting a B vitamin raw material from manufacturer A by the detection method of Example 1.

[0192] As can be seen from the above table, when the heating temperature is 30°C (Comparative Example 19), 40°C (Comparative Example 20), 50°C (Comparative Example 21), or 80°C (Comparative Example 22), some of the test results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will fall within the range of their standard contents, but they still do not meet the standards, and it is impossible to accurately measure niacinamide, vitamin B2, vitamin B6, and vitamin B1 simultaneously. When the heating temperature of Example 1 or Example 2 is adopted (i.e., the heating temperature is 60°C or 70°C), the test results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will all fall within the range of their standard contents.

[0193] Comparative Examples 23 - 25

[0194] This group of comparative examples discloses a method for detecting B vitamins. The heating time (hydrolysis time) in this group of comparative examples is different from that of Example 1; specifically, in the S1 pretreatment step of Comparative Examples 23 - 25, the heating time of the heating mixture is 10 min (Comparative Example 23), 20 min (Comparative Example 24), and 40 min (Comparative Example 25) respectively. Other conditions are the same as those of Example 1.

[0195] The test sample of this detection method selects a B vitamin raw material from Manufacturer A, in which the range of niacinamide is 15575 - 20139 mg / kg, the content range of vitamin B2 is 1566 - 2034 mg / kg, the content range of vitamin B6 is 1700 - 2300 mg / kg, and the content range of vitamin B1 is 3000 - 3800 mg / kg.

[0196] Table 6 shows the test results for different heating times.

[0197] Table 6

[0198]

[0199] Among them, when the heating time is 30 min, it is the result of detecting a B vitamin raw material from Manufacturer A using the detection method of Example 1.

[0200] As can be seen from the above table, when the heating time is 10 min (Comparative Example 23), 20 min (Comparative Example 24), or 40 min (Comparative Example 25), some of the test results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will fall within the range of their standard contents, but they still do not meet the standards, and it is impossible to accurately measure niacinamide, vitamin B2, vitamin B6, and vitamin B1 simultaneously. When the heating time of Example 1 is adopted (i.e., the heating time is 30 min), the test results of niacinamide, vitamin B2, vitamin B6, and vitamin B1 will all fall within the range of their standard contents.

[0201] Application Example 1 - Accuracy Test 1

[0202] In this application example, the contents of vitamin B1, vitamin B2, niacinamide, and vitamin B6 in one B vitamin raw material from each of the three manufacturers A, B, and C were detected to verify the accuracy of the detection method in Example 1. Table 7 shows the detection results of the B vitamin raw materials from different manufacturers and the standard ranges provided by the manufacturers.

[0203] Table 7

[0204]

[0205] As can be seen from this table, the detection results of the contents of niacinamide, vitamin B1, vitamin B2, and vitamin B6 in the above three raw materials all fall within the standard ranges provided by the manufacturers; the detection method in Example 1 has good accuracy.

[0206] In addition, a blank test was conducted on a mixture without the sample to be measured. The preparation method of this mixture without the sample to be measured is as follows: 1.5 mL of the EDTA saturated solution in Example 1, 0.1 mL of formic acid, and 25 mL of primary water were successively added to a 50 mL volumetric flask; the materials in the volumetric flask were shaken well and placed in a constant temperature shaker at 70 °C for 30 min; the volumetric flask was taken out and cooled, and then made up to the calibration mark of the volumetric flask with primary water and shaken well to obtain the test liquid sample. Other operations were carried out using exactly the same test steps, reagents, and dosages as those for the analysis of the sample to be measured.

[0207] No B vitamins were detected in this blank experiment, proving the effectiveness of the test results.

[0208] Application Example 2 - Stability Test

[0209] This application example verified the stability of the detection method in Example 1. Repetitive verification experiments were respectively carried out on one B vitamin raw material from each of the three manufacturers A, B, and C. Among them, 7 groups of parallel experiments were carried out on the raw material from manufacturer A, and 6 groups of parallel experiments were carried out on the raw materials from manufacturers B and C, obtaining the detection results in Table 8. Table 8 shows the multiple detection results of the same raw material.

[0210] Table 8

[0211]

[0212] As can be seen from the above table, the precisions of the detection method in Example 1 for niacinamide, vitamin B2, vitamin B6, and vitamin B1 in the raw materials of Manufacturer A are 1.24%, 0.18%, 0.97%, and 0.55% respectively; the precisions of the detection method in Example 1 for niacinamide, vitamin B2, vitamin B6, and vitamin B1 in the raw materials of Manufacturer B are 1.04%, 0.17%, 0.80%, and 0.53% respectively; the precisions of the detection method in Example 1 for niacinamide, vitamin B2, vitamin B6, and vitamin B1 in the raw materials of Manufacturer C are 0.48%, 0.20%, 0.79%, and 0.33% respectively.

[0213] Among them, the calculation formula for precision is as follows:

[0214]

[0215] It can be seen that the precision of the detection method in Example 1 < 2%, which can meet the experimental requirements.

[0216] Application Example 3 Accuracy Test 2

[0217] In this application example, different raw materials from different manufacturers were further detected; specifically, the detection method in Example 1 was used to verify 21 raw materials from 3 manufacturers, namely A, B, and C, as the samples to be tested; among them, the number of raw materials from Manufacturer A is 7, specifically including Manufacturer A Raw Materials 1 - 7; the number of raw materials from Manufacturer B is 11, specifically including Manufacturer B Raw Materials 1 - 11; the number of raw materials from Manufacturer C is 3, specifically including Manufacturer C Raw Materials 1 - 3.

[0218] Table 9 shows the detection results of B - group vitamin raw materials from different manufacturers.

[0219] Table 9

[0220]

[0221] As can be seen from the above table, the detection results of the contents of niacinamide, vitamin B1, vitamin B2, and vitamin B6 are all within the standard ranges provided by the manufacturers.

[0222] Figure 13This is a line graph showing the deviation values between the test results of 21 raw materials as test samples using the detection method of Example 1 in this application example and the standard range provided by the manufacturer. The test results of 21 raw materials from 3 manufacturers were compared with the standard range provided by the manufacturer. The average deviation of niacinamide in the raw materials of each manufacturer from the manufacturer's standard was 0.19%, the average deviation of vitamin B2 from the manufacturer's standard was 1.4%, the average deviation of vitamin B6 from the manufacturer's standard was 3.0%, the average deviation of vitamin B1 from the manufacturer's standard was 0.32%, and the maximum deviation of individual raw materials from the manufacturer's standard was -9.36%. It can be seen that the deviation of the detection method in Example 1 from the standard value provided by the manufacturer is <10%, which can meet the detection requirements.

[0223] Application Example 4 Accuracy Test 3

[0224] In this application example, the contents of niacinamide, vitamin B1, vitamin B2, and vitamin B6 in one of the B-vitamin raw materials of Manufacturer A were detected to verify the accuracy of the detection method in Example 2; Table 10 shows the test results of the B-vitamin raw materials of Manufacturer A and the standard range provided by the manufacturer.

[0225] Table 10

[0226]

[0227] It can be seen from this table that the test results of the contents of niacinamide, vitamin B1, vitamin B2, and vitamin B6 in the B-vitamin raw materials of Manufacturer A all fall within the standard range provided by the manufacturer; the detection method in Example 2 has good accuracy.

Claims

1. A method for detecting B vitamins, characterized in that, The B vitamins are one or more of vitamin B1, vitamin B2, niacin, niacinamide and vitamin B6; the method comprises the following steps: S1 pretreatment: heating the mixture to prepare the liquid sample to be tested; The mixture includes a sample to be tested, a saturated EDTA solution, formic acid and water; the ratio of the mass of the sample to be tested to the volume of the saturated EDTA solution is 0.3 g: (1.45-1.95) mL; The heating temperature is 55°C to 75°C; The heating time is 25 to 35 minutes; S2 detection: using a liquid chromatograph ultraviolet detector to analyze different standard samples and the liquid sample to be tested, respectively, to obtain the analysis results of each of the standard samples and the analysis results of the liquid sample to be tested; The standard sample is one or more of vitamin B1, vitamin B2, niacin, niacinamide and vitamin B6; The detection wavelength of the ultraviolet detector is 270-280nm; The chromatographic column of the liquid chromatography is a PFP chromatographic column; The mobile phase A of the liquid chromatography is a mixed aqueous solution of ammonium formate and formic acid; The elution method of the liquid chromatography adopts a gradient elution method; S3 Analysis: Compare the analysis results of the liquid sample to be tested with the analysis results of each of the standard samples, and conduct qualitative and quantitative analysis on the B vitamins in the sample to be tested.

2. The method for detecting B vitamins according to claim 1, characterized in that In the S1 pre-treatment step, the method for preparing the mixture comprises the following steps: sequentially adding the EDTA saturated solution, the formic acid and the water into a container containing the sample to be tested.

3. The method for detecting B vitamins according to claim 1 or 2, characterized in that, In the mixture, the ratio of the mass of the sample to be tested, the volume of the saturated EDTA solution and the volume of the formic acid is 0.3 g: (1.45-1.95) mL: (0.05-0.1) mL.

4. The method for detecting B vitamins according to any one of claims 1-3, characterized in that, In the mixture, the ratio of the mass of the sample to be tested, the volume of the saturated EDTA solution and the volume of the formic acid is 0.3 g: (1.5-1.8) mL: (0.08-0.1) mL.

5. The method for detecting B vitamins according to any one of claims 1-4, characterized in that, In the pre-treatment step S1, after the heating process, the liquid sample to be tested is obtained by cooling and constant volume in sequence; The liquid sample to be tested is then filtered.

6. The method for detecting B vitamins according to any one of claims 1-5, characterized in that, In the S2 detection step, the column temperature of the PFP chromatographic column is 30-40°C.

7. The method for detecting B vitamins according to any one of claims 1-6, characterized in that, In the detection step S2, the mobile phase B of the liquid chromatography is acetonitrile with a mass content of 100%.

8. The method for detecting B vitamins according to claim 7, wherein In the S2 detection step, the gradient elution method comprises: From 0 to 20 min, the volume proportion of mobile phase A is 100%, and the volume proportion of mobile phase B is 0%; 20-25 min, the volume proportion of mobile phase A decreased to ≤55%, and the volume proportion of mobile phase B increased to ≥45%; 25-32 min, the volume proportion of mobile phase A decreased to 0%, and the volume proportion of mobile phase B increased to 100%; 32-39 min, the volume proportion of mobile phase A was maintained at 0%, and the volume proportion of mobile phase B was maintained at 100%; 39-39.1min, the volume proportion of mobile phase A increased to 100%, and the volume proportion of mobile phase B decreased to 0%; From 39.1 to 50 min, the volume percentage of mobile phase A is kept at 100%, and the volume percentage of mobile phase B is kept at 0%.

9. The method for detecting B vitamins according to any one of claims 1-8, characterized in that, In the S3 analysis step, the B vitamins in the sample to be tested are quantitatively analyzed by the external standard method.

10. Use of a method for detecting B vitamins, characterized in that, It is used for the detection of B vitamins in food raw materials, and the food raw materials include one or more of dairy product raw materials, baking product raw materials, and meat product raw materials.

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