Method for simultaneously determining contents of vitamin B1, vitamin B2 and vitamin B6

Abstract

The invention provides a method for simultaneously determining the contents of vitamin B1, vitamin B2 and vitamin B6, and belongs to the technical field of substance content determination. The method comprises the following steps: mixing a vitamin B1 standard substance with an acidic ethanol solution, mixing a vitamin B2 standard substance, glacial acetic acid and water, and mixing a vitamin B6 standard substance with a hydrochloric acid solution to respectively obtain standard stock solutions of vitamin B1, vitamin B2 and vitamin B6; respectively mixing the standard stock solutions of the vitamins B1, B2 and B6 with the mobile phase, the extracting solution and the sodium dihydrogen phosphate solution to respectively obtain standard working solutions of the vitamins B1, B2 and B6; mixing the premixed feed sample with the extracting solution to obtain a sample to-be-detected solution; and detecting the to-be-detected sample liquid by using a high performance liquid chromatograph to obtain the contents of the vitamins B1, B2 and B6 in the to-be-detected sample liquid. According to the determination method disclosed by the invention, the contents of the vitamins B1, B2 and B6 in the premix feed sample can be accurately determined at the same time, and the determination time is only one day.

Description

Technical Field

[0001] This invention relates to the field of substance content determination technology, and in particular to a method for simultaneously determining the content of vitamins B1, B2 and B6. Background Technology

[0002] Vitamins B1 (thiamine), B2 (riboflavin), and B6 (pyridoxine hydrochloride) are a large class of small-molecule organic compounds that are essential for maintaining normal life activities in animals.

[0003] Vitamin B1 is a coenzyme in energy metabolism and an essential substance for carbohydrate metabolism. It promotes appetite, aids reproduction, and has anti-polyneuritis, anti-beriberi, anti-gastrointestinal disorder, and anti-constipation effects. When animals are deficient in vitamin B1, they experience decreased appetite, weight loss, cardiovascular disorders, and decreased body temperature. Vitamin B2 promotes growth and functions as a component of certain enzyme systems in carbohydrate and amino acid metabolism. It has anti-angular cheilitis, glossitis, conjunctivitis, reproductive promotion, and anti-fatigue effects. When animals are deficient in vitamin B2, growth is stunted; piglets grow slowly and become anemic; adult pigs experience reproductive disorders; and poultry develop flexed claw paralysis. Vitamin B6 acts as a coenzyme in protein and nitrogen metabolism, is involved in red blood cell formation, and plays an important role in the endocrine system. When animals are deficient in vitamin B6, various livestock experience convulsions; pigs lose their appetite and grow poorly; chicks grow slowly; and hens produce fewer eggs.

[0004] Currently, there are three independent detection methods for vitamins B1, B2, and B6 in feed additives and premixes: GB / T 14700-2018 Determination of Vitamin B1 in Feed, GB / T 14701-2019 Determination of Vitamin B2 in Feed, and GB / T 14702-2018 Determination of Vitamin B6 in Premixed Feed. High-performance liquid chromatography (HPLC) suffers from cumbersome pretreatment methods and the inability to perform simultaneous determinations, making it unsuitable for large-scale testing.

[0005] Therefore, it is of great significance to explore an accurate, rapid and simultaneous method for determining the content of vitamins B1, B2 and B6 in additive premixed feed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for simultaneously determining the content of vitamins B1, B2, and B6, addressing the shortcomings of existing technologies. The method of this invention can simultaneously and accurately determine the content of vitamins B1, B2, and B6 in premixed feed samples. The detection results show no significant difference from international detection results, and the relative errors are all within the allowable error range of GB / T 18823. The operation is simple, and the error is very small. Furthermore, the method of this invention can rapidly and simultaneously determine the content of vitamins B1, B2, and B6 in premixed feed samples, requiring only one day for the determination.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for simultaneously determining the content of vitamins B1, B2, and B6, comprising the following steps: 1) Mix vitamin B1 standard and acidic ethanol solution to obtain vitamin B1 standard stock solution; Vitamin B2 standard, glacial acetic acid and water were mixed to obtain vitamin B2 standard stock solution; The vitamin B6 standard and hydrochloric acid solution were mixed to obtain the vitamin B6 standard stock solution; 2) Mix the vitamin B1 standard stock solution and the mobile phase to obtain the vitamin B1 standard working solution; The vitamin B2 standard stock solution and the extract were mixed to obtain the vitamin B2 standard working solution; The vitamin B6 standard stock solution and sodium dihydrogen phosphate solution were mixed to obtain the vitamin B6 standard working solution. 3) The standard working solutions of vitamin B1, B2 and B6 were detected by high performance liquid chromatography, and the standard curve equations of vitamin B1, B2 and B6 were obtained according to the external standard method. 4) Mix the premixed feed sample and the extract to obtain the sample solution to be tested; 5) The sample solution is tested using a high performance liquid chromatograph. The content of vitamins B1, B2 and B6 in the sample solution is obtained according to the standard curve equation in step 3) and the peak areas of vitamins B1, B2 and B6.

[0008] Preferably, the purity of the vitamin B1 standard in step 1) is ≥98%, and the concentration of the vitamin B1 standard in the vitamin B1 standard stock solution is 200~400μg / mL. The purity of the vitamin B2 standard is ≥95%, and the concentration of the vitamin B2 standard in the vitamin B2 standard stock solution is 100~200μg / mL; The purity of the vitamin B6 standard is ≥98%, and the concentration of the vitamin B6 standard in the vitamin B6 standard stock solution is 400~800μg / mL.

[0009] Preferably, the pH value of the acidic ethanol solution in step 1) is 3.5-4.3; the volume concentration of ethanol in the acidic ethanol solution is 15-25%; the mass-volume ratio of the vitamin B2 standard, glacial acetic acid and water is 0.006-0.008g: 0.5-1.5mL: 45-55mL; and the volume concentration of the hydrochloric acid solution is 0.8-0.9%.

[0010] Preferably, the concentrations of vitamin B1 standards in the vitamin B1 standard working solution in step 2) are 50~80μg / mL, 25~40μg / mL, 2.5~4μg / mL and 0.25~0.4μg / mL, respectively; The concentrations of vitamin B2 standard in the vitamin B2 standard working solution were 50~100μg / mL, 20~40μg / mL, 2~4μg / mL and 0.2~0.4μg / mL, respectively. The concentrations of vitamin B6 standard in the vitamin B6 standard working solution were 40~80μg / mL, 4~8μg / mL, 0.4~0.8μg / mL and 0.08~0.16μg / mL, respectively.

[0011] Preferably, the mobile phase in step 2) consists of mobile phase A and mobile phase B with a volume ratio of 7~9:1~3. Mobile phase A includes EDTA, sodium heptanesulfonate, glacial acetic acid, triethylamine and water, and mobile phase B is methanol. The extract consists of extract A and extract B in a volume ratio of 7~9:1~3. Extract A includes EDTA, glacial acetic acid, triethylamine and water, and extract B is methanol. The concentration of the sodium dihydrogen phosphate solution is 3.5~4.5 g / L.

[0012] Preferably, in the detection process described in steps 3) and 5), the chromatographic column is a C18 column; the particle size is 4~6μm; the flow rate is 1~1.5mL / min; the column temperature is 25~28℃; the injection volume is 10~20μL; and the detector is an ultraviolet or diode matrix detector.

[0013] Preferably, the standard curve equation for vitamin B1 in step 3) is y = 36771.9x - 972.652, R 2 =1.00000; The standard curve equation for vitamin B2 is y = 77294.8x + 14296.0, R0 2 =0.99996; the standard curve equation for vitamin B6 is y=38348.9x+1660.68, R 2 =1.00000; Where x represents the concentrations of vitamins B1, B2, and B6, and y represents the measured peak area.

[0014] Preferably, the premixed feed sample in step 4) is a vitamin premixed feed sample or a compound premixed feed sample.

[0015] Preferably, in step 4), the mass-to-volume ratio of the vitamin premixed feed sample to the extract is 0.05~0.15g:50mL; and the mass-to-volume ratio of the compound premixed feed sample to the extract is 1~2g:50mL. The extract consists of extract A and extract B in a volume ratio of 7~9:1~3. Extract A includes EDTA, glacial acetic acid, triethylamine and water, and extract B is methanol.

[0016] Preferably, in step 5), after obtaining the concentrations of vitamins B1, B2, and B6 in the sample solution based on the standard curve equation and the measured peak area, the content of vitamins B1, B2, and B6 is then obtained from the concentrations of vitamins B1, B2, and B6.

[0017] The beneficial effects of this invention include the following: 1) The determination method of the present invention can simultaneously and accurately determine the content of vitamins B1, B2 and B6 in premixed feed samples. The test results are not significantly different from international test results, and the relative errors are all within the allowable error range of GB / T 18823. The operation is simple and the error is very small.

[0018] 2) The determination method of the present invention can quickly and simultaneously determine the content of vitamins B1, B2 and B6 in premixed feed samples, and the determination time is only one day. Attached Figure Description

[0019] Figure 1 This is a standard curve diagram of the vitamin B1 standard working solution in Example 1; Figure 2 This is a chromatogram of the vitamin B1 standard working solution in Example 1; Figure 3 This is a standard curve diagram of the vitamin B2 standard working solution in Example 1; Figure 4 This is a chromatogram of the vitamin B2 standard working solution in Example 1; Figure 5 This is a standard curve diagram of the vitamin B6 standard working solution in Example 1; Figure 6 This is a chromatogram of the vitamin B6 standard working solution in Example 1; Figure 7 The chromatogram of vitamin B2 in Example 1 is shown below. Figure 8 This is a chromatogram of vitamin B2 from Example 2. Detailed Implementation

[0020] This invention provides a method for simultaneously determining the content of vitamins B1, B2, and B6, comprising the following steps: 1) Mix vitamin B1 standard and acidic ethanol solution to obtain vitamin B1 standard stock solution; Vitamin B2 standard, glacial acetic acid and water were mixed to obtain vitamin B2 standard stock solution; The vitamin B6 standard and hydrochloric acid solution were mixed to obtain the vitamin B6 standard stock solution; 2) Mix the vitamin B1 standard stock solution and the mobile phase to obtain the vitamin B1 standard working solution; The vitamin B2 standard stock solution and the extract were mixed to obtain the vitamin B2 standard working solution; The vitamin B6 standard stock solution and sodium dihydrogen phosphate solution were mixed to obtain the vitamin B6 standard working solution. 3) The standard working solutions of vitamin B1, B2 and B6 were detected by high performance liquid chromatography, and the standard curve equations of vitamin B1, B2 and B6 were obtained according to the external standard method. 4) Mix the premixed feed sample and the extract to obtain the sample solution to be tested; 5) The sample solution is tested using a high performance liquid chromatograph. The content of vitamins B1, B2 and B6 in the sample solution is obtained according to the standard curve equation in step 3) and the peak areas of vitamins B1, B2 and B6.

[0021] In step 1) of this invention, the purity of the vitamin B1 standard is preferably ≥98%, and the concentration of the vitamin B1 standard in the vitamin B1 standard stock solution is preferably 200~400 μg / mL, more preferably 250~350 μg / mL, and even more preferably 296.70 μg / mL; the vitamin B1 standard is preferably thiamine nitrate. The purity of the vitamin B2 standard is preferably ≥95%, and the concentration of the vitamin B2 standard in the vitamin B2 standard stock solution is preferably 100~200 μg / mL, more preferably 130~170 μg / mL, and even more preferably 135.52 μg / mL; The purity of the vitamin B6 standard is preferably ≥98%, and the concentration of the vitamin B6 standard in the vitamin B6 standard stock solution is preferably 400~800 μg / mL, more preferably 500~700 μg / mL, and even more preferably 624.86 μg / mL.

[0022] In step 1) of this invention, the pH value of the acidic ethanol solution is preferably 3.5-4.3, more preferably 4.0; the pH value is preferably adjusted using hydrochloric acid solution; the volume concentration of ethanol in the acidic ethanol solution is preferably 15-25%, more preferably 18-22%; the ethanol is preferably anhydrous ethanol; the vitamin B1 standard stock solution is preferably stored at 2-8°C in the dark and can be used for 3 months.

[0023] In step 1) of this invention, the preferred mass-to-volume ratio of vitamin B2 standard, glacial acetic acid, and water is 0.006-0.008 g: 0.5-1.5 mL: 45-55 mL, more preferably 0.007 g: 1 mL: 50 mL; the preferred glacial acetic acid is superior grade pure glacial acetic acid; the preferred water is deionized water; the preferred mixing method is to first boil the vitamin B2 standard and glacial acetic acid in a boiling water bath for 25-35 minutes, then cool and dilute to volume with deionized water; the preferred vitamin B2 standard stock solution is stored at 2-8°C protected from light and can be used for 6 months.

[0024] In step 1) of this invention, the volume concentration of the hydrochloric acid solution is preferably 0.8-0.9%; the mixed vitamin B6 standard is preferably dissolved in a portion of the hydrochloric acid by ultrasonication and then diluted to volume with the remaining hydrochloric acid; the ultrasonic dissolution time is preferably 4-6 minutes; the vitamin B6 standard stock solution is preferably stored at 2-8°C in the dark and can be used for 3 months.

[0025] In step 2) of this invention, the concentrations of vitamin B1 standards in the vitamin B1 standard working solution are preferably 50-80 μg / mL, 25-40 μg / mL, 2.5-4 μg / mL, and 0.25-0.4 μg / mL, and more preferably 59.34 μg / mL, 29.67 μg / mL, 2.967 μg / mL, and 0.2967 μg / mL, respectively. The concentrations of vitamin B2 standard in the vitamin B2 standard working solution are preferably 50-100 μg / mL, 20-40 μg / mL, 2-4 μg / mL and 0.2-0.4 μg / mL, and more preferably 67.76 μg / mL, 27.104 μg / mL, 2.71 μg / mL and 0.271 μg / mL; The concentrations of vitamin B6 standard in the vitamin B6 standard working solution are preferably 40-80 μg / mL, 4-8 μg / mL, 0.4-0.8 μg / mL, and 0.08-0.16 μg / mL, and more preferably 62.486 μg / mL, 6.249 μg / mL, 0.625 μg / mL, and 0.125 μg / mL.

[0026] In step 2) of this invention, the mobile phase preferably consists of mobile phase A and mobile phase B. Mobile phase A preferably includes EDTA, sodium heptanesulfonate, glacial acetic acid, triethylamine and water. The EDTA, sodium heptanesulfonate, glacial acetic acid and triethylamine are preferably of analytical grade. Mobile phase B is preferably methanol, and more preferably chromatographic grade methanol. The volume ratio of mobile phase A to mobile phase B is preferably 7~9:1~3, and more preferably 8:2.

[0027] In step 2) of this invention, the pH value of the mobile phase A is preferably 3.5-3.8, more preferably 3.6-3.7; the mass-volume ratio of EDTA, sodium heptanesulfonate, glacial acetic acid, triethylamine and water in the mobile phase A is preferably 45-55 mg: 0.8-1.3 g: 20-30 mL: 4-6 mL: 950-980 mL; more preferably 50 mg: 1.1 g: 25 mL: 5 mL: 970 mL; the mobile phase A is preferably obtained by ultrasonically dissolving EDTA and sodium heptanesulfonate in a portion of water, and then adding glacial acetic acid and triethylamine and making up to volume with water; the mobile phase A and mobile phase B are preferably obtained by ultrasonic degassing after mixing.

[0028] The glacial acetic acid and triethylamine described in this invention are used to adjust the pH value of mobile phase A.

[0029] In step 2) of the present invention, the extraction solution is preferably composed of extraction solution A and extraction solution B. Extraction solution A preferably includes EDTA, glacial acetic acid, triethylamine and water, and extraction solution B is preferably methanol. The volume ratio of extraction solution A to extraction solution B is preferably 7~9:1~3, and more preferably 8:2.

[0030] In step 2) of this invention, the preferred mass-to-volume ratio of EDTA, glacial acetic acid, triethylamine, and water in extract A is 45-55 mg: 20-30 mL: 4-6 mL: 950-980 mL; more preferably, it is 50 mg: 25 mL: 5 mL: 970 mL. Extract A is preferably prepared by ultrasonically dissolving EDTA in a portion of water, followed by adding glacial acetic acid and triethylamine, and then adjusting the volume with water.

[0031] The concentration of the sodium dihydrogen phosphate solution in step 2) of this invention is preferably 3.5~4.5 g / L, more preferably 3.9~4.2 g / L; the solvent of the sodium dihydrogen phosphate solution is preferably ultrapure water.

[0032] In the detection process described in steps 3) and 5) of this invention, the chromatographic column is preferably a C18 column; the length and inner diameter of the chromatographic column are preferably 150 mm and 4.6 mm, respectively; the particle size is preferably 4~6 μm, more preferably 5 μm; the flow rate is preferably 1~1.5 mL / min, more preferably 1.2 mL / min; the column temperature is preferably 25~28℃, more preferably 27℃; the injection volume is preferably 10~20 μL, more preferably 15~18 μL; the detector is preferably an ultraviolet or diode matrix detector; the detection wavelength of vitamin B1 is preferably 242 nm, the detection wavelength of vitamin B2 is preferably 267 nm, and the detection wavelength of vitamin B6 is preferably 290 nm.

[0033] The standard curve equation for vitamin B1 in step 3 of this invention is y = 36771.9x - 972.652, R 2=1.00000; The standard curve equation for vitamin B2 is y = 77294.8x + 14296.0, R0 2 =0.99996; the standard curve equation for vitamin B6 is y=38348.9x+1660.68, R 2 =1.00000; Where x represents the concentrations of vitamins B1, B2, and B6, and y represents the measured peak area.

[0034] In step 3) of this invention, the external standard method preferably uses regression analysis based on the concentrations of vitamins B1, B2, and B6 and the measured peak areas to obtain the standard curve equation.

[0035] In step 4) of this invention, the premixed feed sample is preferably a vitamin premixed feed sample or a compound premixed feed sample; the vitamin premixed feed sample is preferably Anmei No. 1, Anmei No. 2 or Anmei No. 3; the compound premixed feed sample is preferably 114NB, 115NB or 116NB.

[0036] In step 4) of this invention, the preferred mass-to-volume ratio of the vitamin premixed feed sample to the extract is 0.05-0.15 g: 50 mL, more preferably 0.1 g: 50 mL; the preferred mass-to-volume ratio of the compound premixed feed sample to the extract is 1-2 g: 50 mL, more preferably 1.5 g: 50 mL.

[0037] In step 4) of the present invention, the extraction solution is preferably composed of extraction solution A and extraction solution B. Extraction solution A preferably includes EDTA, glacial acetic acid, triethylamine and water, and extraction solution B is preferably methanol. The volume ratio of extraction solution A to extraction solution B is preferably 7~9:1~3, and more preferably 8:2.

[0038] In step 4) of this invention, the preferred mass-to-volume ratio of EDTA, glacial acetic acid, triethylamine, and water in extract A is 45-55 mg: 20-30 mL: 4-6 mL: 950-980 mL; more preferably, it is 50 mg: 25 mL: 5 mL: 970 mL.

[0039] In step 4) of this invention, the mixing process preferably involves first dissolving the premixed feed sample in a portion of the extract, then cooling it and making up the volume with the extract; the dissolution temperature is 100°C and the time is 30-40 minutes, and the dissolution is preferably carried out in a water bath.

[0040] In step 5) of this invention, preferably, the concentrations of vitamins B1, B2, and B6 in the sample test solution are obtained according to the standard curve equation and the measured peak area, and then the content of vitamins B1, B2, and B6 is obtained from the concentrations of vitamins B1, B2, and B6; the formula for the concentration and content is preferably X=C×L / m, where X is the content of vitamins B1, B2, or B6 in mg / kg; C is the concentration of vitamins B1, B2, or B6 in μg / mL; L is the fixed volume of vitamins B1, B2, or B6 in mL; and m is the mass of vitamins B1, B2, or B6 in g.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Example 1

[0043] Weigh 0.0150 g of thiamine nitrate (vitamin B1) standard (purity 98.9%) into a 50 mL brown volumetric flask, dissolve and dilute to volume with acidic 20% ethanol solution to obtain a vitamin B1 standard stock solution with a concentration of 296.70 μg / mL. The acidic 20% ethanol solution was prepared by mixing 80 mL of hydrochloric acid solution (adjusted to pH 4.0 with 0.1 mol / L hydrochloric acid solution) and 20 mL of anhydrous ethanol. Store the vitamin B1 standard stock solution at 5°C protected from light.

[0044] Weigh 0.0070 g of vitamin B2 standard (purity 96.8%) into a 50 mL brown volumetric flask, add 1 mL of analytical grade glacial acetic acid, and boil in a boiling water bath for 30 min. After complete dissolution, remove from heat, cool, and dilute to volume with deionized water to obtain a vitamin B2 standard stock solution with a concentration of 135.52 μg / mL. Store the vitamin B2 standard stock solution at 5 °C protected from light.

[0045] Weigh 0.0157 g of vitamin B6 standard (purity 99.5%) into a 25 mL brown volumetric flask, add 18 mL of hydrochloric acid solution, sonicate for 5 min, and after complete dissolution, dilute to volume with hydrochloric acid solution to obtain a vitamin B6 standard stock solution with a concentration of 624.86 μg / mL. The hydrochloric acid solution was prepared by diluting 8.5 mL of 0.1 mol / L hydrochloric acid with water to a final volume of 1000 mL. Store the vitamin B6 standard stock solution at 5°C protected from light.

[0046] Add 50 mg of analytical grade EDTA, 1.1 g of analytical grade sodium heptanesulfonate, and 700 mL of deionized water to a 1000 mL volumetric flask. Dissolve the solutions by sonication until homogeneous. Then add 25 mL of analytical grade glacial acetic acid and 5 mL of analytical grade triethylamine. Dilute to the mark with deionized water, mix well, and filter through a 0.45 μm aqueous filter membrane. Mix 800 mL of this solution with 200 mL of chromatographic grade methanol and degas by sonication to obtain the mobile phase. Mix 0.2 mL of vitamin B1 stock solution with 0.8 mL of mobile phase to obtain vitamin B1 working solution 1 with a vitamin B1 standard concentration of 59.34 μg / mL. Mix 0.1 mL of vitamin B1 stock solution with 0.9 mL of mobile phase to obtain vitamin B1 working solution 2 with a concentration of 29.67 μg / mL. Mix 0.1 mL of working solution 2 with 0.9 mL of mobile phase to obtain vitamin B1 working solution 3 with a concentration of 2.967 μg / mL. Mix 0.1 mL of working solution 3 with 0.9 mL of mobile phase to obtain vitamin B1 working solution 4 with a standard concentration of 0.2967 μg / mL. The peak areas of vitamin B1 standard working solutions were determined using high-performance liquid chromatography (HPLC). The peak areas of vitamin B1 standard working solutions 1-4 were 2181963, 1088211, 108629, and 10384, respectively. Regression analysis was performed based on the concentration of vitamin B1 and the measured peak areas, yielding the standard curve equation y = 36771.9x - 972.652, R0. 2 =1.00000, where x is the concentration of vitamin B1 and y is the peak area corresponding to that concentration. Standard curves for vitamin B1 at different concentrations are shown below. Figure 1 As shown, the chromatogram of a vitamin B1 standard working solution with a concentration of 59.34 μg / mL is as follows. Figure 2 As shown.

[0047] Add 50 mg of analytical grade EDTA and 700 mL of deionized water to a 1000 mL volumetric flask, and sonicate until the EDTA is completely dissolved. Then add 25 mL of analytical grade glacial acetic acid and 5 mL of analytical grade triethylamine, and dilute to the mark with deionized water and shake well. Mix 800 mL of this solution with 200 mL of methanol to obtain the extract. Mix 0.5 mL of vitamin B2 stock solution and 0.5 mL of extract to obtain vitamin B2 standard working solution 1 with a vitamin B2 standard concentration of 67.76 μg / mL. Mix 0.2 mL of vitamin B2 stock solution and 0.8 mL of extract to obtain vitamin B2 standard working solution 2 with a concentration of 27.104 μg / mL. Mix 0.1 mL of standard working solution 2 and 0.9 mL of extract to obtain vitamin B2 standard working solution 3 with a concentration of 2.71 μg / mL. Mix 0.1 mL of standard working solution 3 and 0.9 mL of extract to obtain vitamin B2 standard working solution 4 with a standard concentration of 0.271 μg / mL. The peak areas of vitamin B2 standard working solutions were determined using high-performance liquid chromatography (HPLC). The peak areas of vitamin B2 standard working solutions 1-4 were 5238722, 2142801, 216806, and 21766, respectively. Regression analysis was performed based on the concentration of vitamin B2 and the measured peak areas, yielding the standard curve equation y = 77294.8x + 14296.0, R0. 2 =0.99996, where x is the concentration of vitamin B2 and y is the peak area corresponding to that concentration. The standard working curves for vitamin B2 at different concentrations are shown below. Figure 3 As shown, the chromatogram of the vitamin B2 standard working solution with a concentration of 67.76 μg / mL is as follows. Figure 4 As shown.

[0048] Dissolve 3.9 g of sodium dihydrogen phosphate in 1000 mL of ultrapure water and filter through a 0.45 μm aqueous filter membrane to obtain a sodium dihydrogen phosphate solution. Mix 0.1 mL of vitamin B6 stock solution and 0.9 mL of sodium dihydrogen phosphate solution to obtain vitamin B6 working solution 1 with a standard concentration of 62.486 μg / mL. Mix 0.1 mL of vitamin B6 working solution 1 and 0.9 mL of sodium dihydrogen phosphate solution to obtain vitamin B6 working solution 2 with a concentration of 6.249 μg / mL. Mix 0.1 mL of working solution 2 and 0.9 mL of sodium dihydrogen phosphate solution to obtain vitamin B6 working solution 3 with a concentration of 0.625 μg / mL. Mix 0.2 mL of working solution 3 and 0.8 mL of extract to obtain vitamin B6 working solution 4 with a standard concentration of 0.125 μg / mL. The peak areas of vitamin B6 standard working solutions were determined using high-performance liquid chromatography (HPLC). The peak areas corresponding to vitamin B6 standard working solutions 1-4 were 2397644, 244337, 24525, and 4814, respectively. Regression analysis was performed based on the concentration of vitamin B6 and the measured peak areas, yielding the standard curve equation y = 38348.9x + 1660.68, with R² = 1.00000, where x is the concentration of vitamin B6 and y is the peak area corresponding to that concentration. The standard working curves for different concentrations of vitamin B6 are shown below. Figure 5 As shown, the chromatogram of the vitamin B6 standard working solution with a concentration of 62.486 μg / mL is as follows. Figure 6 As shown in the figure. During the high-performance liquid chromatography (HPLC) detection process, the chromatographic column was a C18 column, 150 mm long, 4.6 mm inner diameter, and 5 μm particle size; the mobile phase was the same as described above, the flow rate was 1.2 mL / min, the column temperature was 26 ℃, the injection volume was 15 μL, and the detector was an ultraviolet matrix detector. The detection wavelengths for vitamin B1 were 242 nm, vitamin B2, and vitamin B6, respectively.

[0049] 0.1173 g of Anmei No. 1 was placed in a 50 mL brown volumetric flask, and 35 mL of the above extraction solution was added. The flask was boiled in a 100℃ water bath for 35 min, shaking it for the first few minutes to prevent solid clumping. After cooling, the volume was adjusted to the mark with the extraction solution, mixed, and filtered. The filtrate was filtered through a 0.45 μm aqueous membrane and injected into a liquid chromatography vial to obtain the sample to be tested. The sample was analyzed using the above high-performance liquid chromatography (HPLC). The concentrations of vitamins B1, B2, and B6 in the sample were found to be 28.131 μg / mL, 39.731 μg / mL, and 34.489 μg / mL, respectively, based on the peak area and the standard curve equation. Using the formula X = C × L / m, the contents of vitamins B1, B2, and B6 in the sample were found to be 11991.05 mg / kg, 16935.64 mg / kg, and 14701.19 mg / kg, respectively. The chromatogram of vitamin B2 is as follows Figure 7 As shown.

[0050] Example 2

[0051] The mass of Anmei No. 1 was 0.1169 g, and all other conditions were the same as in Example 1. The sample was analyzed using the above-mentioned high-performance liquid chromatography (HPLC). The concentrations of vitamins B1, B2, and B6 in the sample were obtained as 27.836 μg / mL, 40.557 μg / mL, and 34.572 μg / mL, respectively, based on peak area and standard curve equation. Using the formula X = C × L / m, the contents of vitamins B1, B2, and B6 in the sample solution were found to be 11905.90 mg / kg, 17346.88 mg / kg, and 14787.00 mg / kg, respectively. The chromatogram of vitamin B2 is shown below. Figure 8 As shown.

[0052] As shown in Examples 1 and 2, the average contents of vitamins B1, B2, and B6 in the test samples were 11948.5 mg / kg, 17141.3 mg / kg, and 14744.1 mg / kg, respectively, and the absolute differences in the contents of vitamins B1, B2, and B6 in the test samples were 85.15 mg / kg, 411.24 mg / kg, and 85.80 mg / kg, respectively. Calculated using the "precision" or "repeatability" criteria in the national standard method, the permissible errors for vitamins B1, B2, and B6 in the test samples were 597.42 mg / kg, 857.06 mg / kg, and 737.20 mg / kg, respectively. Therefore, the errors in vitamins B1, B2, and B6 in Examples 1 and 2 of this invention are all within the permissible error range.

[0053] Example 3

[0054] The contents of vitamins B1, B2 and B6 in Anmei No. 2, Anmei No. 3, 114NB, 115NB and 116NB were determined using the method described in Example 2. The test results were compared with the results of the national standard method, as shown in Table 1.

[0055] Table 1. Comparison of the test results of Example 3 with the test results of the national standard method.

[0056] As shown in Table 1, the results of simultaneously determining vitamins B1, B2, and B6 in premixed feed samples using the method of the present invention are not significantly different from the results of the national standard test. The relative errors are all within the allowable error range of GB / T 18823, indicating that the test method is accurate. The results that require 3 days to be obtained using the national standard method can be obtained in one day using the method of the present invention.

[0057] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for simultaneous determination of vitamin Bl, B2 and B6 content, characterized in that, The method comprises the following steps: 1) mixing a vitamin B1 standard and an acidic ethanol solution to obtain a vitamin B1 standard stock solution; mixing a vitamin B2 standard, glacial acetic acid and water to obtain a vitamin B2 standard stock solution; mixing a vitamin B6 standard and a hydrochloric acid solution to obtain a vitamin B6 standard stock solution; 2) mixing the vitamin B1 standard stock solution and a mobile phase to obtain a vitamin B1 standard working solution; mixing the vitamin B2 standard stock solution and an extraction solution to obtain a vitamin B2 standard working solution; mixing the vitamin B6 standard stock solution and a sodium dihydrogen phosphate solution to obtain a vitamin B6 standard working solution; 3) detecting the vitamin B1, B2 and B6 standard working solutions by using a high performance liquid chromatograph, and obtaining standard curve equations of the vitamin B1, B2 and B6 according to an external standard method; 4) mixing a premixed feed sample and an extraction solution to obtain a sample to-be-tested solution; 5) detecting the sample to-be-tested solution by using a high performance liquid chromatograph, and obtaining the content of the vitamin B1, B2 and B6 in the sample to-be-tested solution according to the standard curve equations in step 3) and the peak areas of the vitamin B1, B2 and B6.

2. The method of claim 1, wherein, The purity of the vitamin B1 standard in step 1) is greater than or equal to 98%, and the concentration of the vitamin B1 standard in the vitamin B1 standard stock solution is 200-400 μg / mL; The purity of the vitamin B2 standard is greater than or equal to 95%, and the concentration of the vitamin B2 standard in the vitamin B2 standard stock solution is 100-200 μg / mL; The purity of the vitamin B6 standard is greater than or equal to 98%, and the concentration of the vitamin B6 standard in the vitamin B6 standard stock solution is 400-800 μg / mL.

3. The method according to claim 1 or 2, characterized in that, The pH value of the acidic ethanol solution in step 1) is 3.5-4.3, the volume concentration of ethanol in the acidic ethanol solution is 15-25%, the mass-volume ratio of the vitamin B2 standard, glacial acetic acid and water is 0.006-0.008 g: 0.5-1.5 mL: 45-55 mL, and the volume concentration of the hydrochloric acid solution is 0.8-0.9%.

4. The method of claim 3, wherein, The concentration of the vitamin B1 standard in the vitamin B1 standard working solution in step 2) is 50-80 μg / mL, 25-40 μg / mL, 2.5-4 μg / mL and 0.25-0.4 μg / mL, respectively; The concentration of the vitamin B2 standard in the vitamin B2 standard working solution is 50-100 μg / mL, 20-40 μg / mL, 2-4 μg / mL and 0.2-0.4 μg / mL, respectively; The concentration of the vitamin B6 standard in the vitamin B6 standard working solution is 40-80 μg / mL, 4-8 μg / mL, 0.4-0.8 μg / mL and 0.08-0.16 μg / mL, respectively.

5. The method of claim 4, wherein, The mobile phase in step 2) is composed of a mobile phase A and a mobile phase B in a volume ratio of 7-9: 1-3, the mobile phase A comprises EDTA, heptanesulfonic acid sodium, glacial acetic acid, triethylamine and water, and the mobile phase B is methanol. The extraction solution is composed of extraction solution A and extraction solution B in a volume ratio of 7-9:1-3, the extraction solution A comprises EDTA, glacial acetic acid, triethylamine and water, and the extraction solution B is methanol. The concentration of the sodium dihydrogen phosphate solution is 3.5-4.5 g / L.

6. The method of claim 4, wherein, In the detection of steps 3) and 5), the chromatographic column is a C18 column, the particle size is 4-6 mu m, the flow rate is 1-1.5 mL / min, the column temperature is 25-28 DEG C, the injection volume is 10-20 mu L, and the detector is an ultraviolet or diode matrix detector.

7. The method according to claim 5 or 6, characterized in that, Step 3) The standard curve equation of vitamin B1 is y = 36771.9x - 972.652, R 2 = 1.00000; the standard curve equation of vitamin B2 is y = 77294.8x + 14296.0, R 2 = 0.99996; the standard curve equation of vitamin B6 is y = 38348.9x + 1660.68, R 2 = 1.00000; In the formula, x is the concentration of vitamins B1, B2 and B6, and y is the measured peak area.

8. The method of claim 7, wherein, The premix feed sample in step 4) is a vitamin premix feed sample or a compound premix feed sample.

9. The method of claim 8, wherein, The mass-volume ratio of the vitamin premix feed sample and the extraction solution is 0.05-0.15 g:50 mL, and the mass-volume ratio of the compound premix feed sample and the extraction solution is 1-2 g:50 mL. The extraction solution is composed of extraction solution A and extraction solution B in a volume ratio of 7-9:1-3, the extraction solution A comprises EDTA, glacial acetic acid, triethylamine and water, and the extraction solution B is methanol.

10. The assay method according to claim 9, characterized in that, In step 5), the concentration of vitamins B1, B2 and B6 in the sample to be tested is obtained according to the standard curve equation and the measured peak area, and then the content of vitamins B1, B2 and B6 is obtained from the concentration of vitamins B1, B2 and B6.

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