Method for synchronously detecting water-soluble vitamins in compound feed based on HPLC (High Performance Liquid Chromatography) and application

Abstract

The invention discloses a method for synchronously detecting water-soluble vitamins in a compound feed based on HPLC (High Performance Liquid Chromatography) and application, and belongs to the technical field of vitamin detection. The detection method specifically comprises the following steps: uniformly mixing a to-be-detected compound feed, a standard substance and an extracting solution, performing ultrasonic treatment for 30 minutes, and passing through a 0.45 mu m needle type filter to obtain a to-be-detected solution of three vitamins and a standard substance at the same time; respectively detecting the to-be-detected solution and the standard solution by adopting a high performance liquid chromatography, and calculating the content of the water-soluble vitamins in the to-be-detected feed by using an external standard method according to an obtained high performance liquid chromatogram. According to the method, rapid separation and accurate quantification of vitamin B1, vitamin B2 and vitamin B6 are realized by optimizing chromatographic conditions and a pretreatment process. The method has the advantages of wide applicability, obviously reduced cost, high throughput and compatibility, strong specificity, high sensitivity, and safe and environment-friendly operation.

Description

Technical Field

[0001] The present invention belongs to the technical field of vitamin content detection, and in particular relates to a method and application of synchronous detection of water-soluble vitamins in compound feed based on HPLC. Background Art

[0002] Vitamins are essential nutrients for maintaining animal life. They cannot be synthesized in the animal body. Even if they are synthesized, it is difficult to meet the body's needs. Therefore, they are the most commonly used substances in feed additives. According to their solubility, vitamins are divided into fat-soluble vitamins and water-soluble vitamins. Water-soluble vitamins include vitamin B1, vitamin B2, vitamin B6, niacin, folic acid, pantothenic acid, vitamin B 12 and vitamin C. Vitamin B1 participates in nutrient metabolism, supports nervous system function and cellular energy production, vitamin B2 is widely involved in various redox reactions in the body, can promote the metabolism of sugar, fat and protein, and plays a certain role in maintaining the normal function of skin, mucous membranes and vision, vitamin B6 is closely related to amino acid metabolism, promotes glutamate decarboxylation, and is used clinically in animals to enhance immune function.

[0003] The current standard for the determination of vitamin B1 in compound feeds is GB / T 14700-2018, which requires extraction of compound feeds with an acidic ammonium chloride methanol solution. The filtered and centrifuged test solution is analyzed using a high-performance liquid chromatography-ultraviolet detector. High-performance liquid chromatography-fluorescence detection (HPLC-Fluorescence Detection) is used as an adjudication method, requiring acid hydrolysis, enzymatic hydrolysis, adsorption, separation, and purification of the compound feed before analysis. Vitamin B2 is primarily determined by reference to GB / T 14701-2019, Determination of Vitamin B2 in Feed. Compound feeds are extracted with heating in an acidic EDTA solution, and the filtered, centrifuged test solution is analyzed using a high-performance liquid chromatography-ultraviolet detector. High-performance liquid chromatography-fluorescence detection serves as an arbitration method, requiring the compound feed to be extracted with acid, oxidatively purified, and reduced with a reducing agent before analysis. Vitamin B6 is primarily determined by reference to GB / T 14702-2018, Determination of Vitamin B6 in Additive Premix Feeds - High-Performance Liquid Chromatography. Compound feeds are extracted with a phosphate solution, and the filtered, centrifuged test solution is analyzed using a high-performance liquid chromatography-ultraviolet detector or a fluorescence detector. In summary, the chromatographic conditions, pretreatment methods, and standard sample preparation methods used for the determination of the three vitamins differ, requiring multiple injections for analysis of different vitamins.

[0004] Patent CN109696504A discloses a method for simultaneously detecting multiple water-soluble vitamins in food. The method is summarized as follows: the sample is enzymatically hydrolyzed and purified by SPE. 18Although a reversed-phase column, gradient elution with phosphate buffer and methanol, coupled with ultraviolet (UV+) and fluorescence (FLD) detectors can simultaneously detect nine water-soluble vitamins, this method has cumbersome pretreatment steps and takes approximately two hours, making it unsuitable for testing multiple batches of products. Furthermore, the method does not adequately address the elution problem of water-soluble vitamins with large polarity differences, lacks sensitivity for trace levels of the vitamin cyanocobalamin, and cannot distinguish between structural analogs such as the three forms of vitamin B6: pyridoxine, pyridoxal, and pyridoxamine. Summary of the Invention

[0005] The present invention aims to solve the technical problems in the prior art and provides a method and application for synchronously detecting water-soluble vitamins in compound feed based on HPLC.

[0006] In order to achieve the above purpose, the specific technical solutions of the present invention are as follows:

[0007] In one aspect, the present invention provides a method for synchronously detecting water-soluble vitamins in compound feed based on HPLC, comprising the following steps:

[0008] S1: Sample pretreatment: Mix the compound feed to be tested with the extract evenly, ultrasonicate for 30 min, and filter through a 0.45 μm syringe filter to obtain the water-soluble vitamin test solution.

[0009] S2: Standard sample pretreatment: Repeat S1 steps with the standard sample, mix and dilute to obtain a water-soluble vitamin mixed standard solution.

[0010] S3: HPLC detection: The test solution and the standard solution are detected respectively by high performance liquid chromatography, and the content of water-soluble vitamins in the test feed is calculated using the external standard method based on the obtained high performance liquid chromatography spectra.

[0011] Preferably, the water-soluble vitamins are vitamin B1, vitamin B2, and vitamin B6.

[0012] Preferably, the extract in S1 is a solution containing sodium heptanesulfonate and ammonium acetate, the pH of which is adjusted with phosphoric acid and then mixed with methanol in a certain proportion.

[0013] More preferably, the extract in S1 is a solution containing 0.05% sodium heptanesulfonate and 20 mmol / L ammonium acetate, the pH value of which is adjusted to 3.0 with phosphoric acid, and then mixed with methanol in a volume ratio of 80:20.

[0014] Preferably, when the compound feed to be tested is solid as described in S1, the compound feed sample needs to be treated in a water bath for 30 minutes and shaken every 10 minutes.

[0015] Preferably, the vitamin B2 standard is heated in a boiling water bath when dissolved.

[0016] The chromatographic conditions of the high performance liquid chromatography method described in S3 are:

[0017] High performance liquid chromatograph: Aglient 1260Ⅱ.

[0018] Chromatographic column: C 18 Reversed-phase chromatography column, length 150 mm, inner diameter 4.6 mm, particle size 4 μm; flow rate: 1.0 ml / min; temperature: 35°C; injection volume: 20 μL.

[0019] Mobile phase: Mobile phase A (aqueous phase) was a solution of 0.05% sodium heptanesulfonate and 20 mmol / L ammonium acetate, pH = 3.0; mobile phase B (organic phase) was methanol.

[0020] The detector of the high performance liquid chromatography used was UV, and the detection wavelength program was: 0-10 min 290 nm → 10-15 min 242 nm → 15-25 min 267 nm switching.

[0021] The gradient elution conditions are:

[0022] .

[0023] In a second aspect, the present invention provides an application of a method for simultaneous detection of water-soluble vitamins in compound feeds based on HPLC for rapid separation and accurate quantification of vitamin B1, vitamin B2, and vitamin B6.

[0024] The methodology of this invention was validated for the following: specificity, limit of detection and limit of quantification, standard curve, extraction recovery, precision and accuracy, and stability. The results were in compliance with the requirements of the "Guidelines for the Implementation of Validation and Validation of Chemical Analytical Methods for Conformity Assessment" (GB / T 27417-2017).

[0025] Beneficial effects:

[0026] 1. The combination of sodium heptanesulfonate and ammonium acetate as an extraction solvent demonstrates higher efficiency, selectivity, and stability than conventional extraction solvents for the extraction of charged water-soluble vitamins through a triple mechanism of "ion pairing, hydrophobicity, and buffering." This method is particularly suitable for the separation and analysis of trace polar compounds in complex matrices. Methanol, a highly polar organic reagent, precipitates proteins, improving extraction efficiency and reducing interference from excipients.

[0027] 2. Optimized pre-treatment process with high-throughput compatibility: Only one extracting solution is needed for the extraction, followed by ultrasonic treatment and centrifugation to obtain three water-soluble vitamin sample test solutions and standard test solutions. No solid-phase extraction or derivatization treatment is required, making it suitable for centralized processing of batch samples and reducing the manpower consumption caused by repeated pre-treatment.

[0028] 3. Multi-component simultaneous detection overcomes the limitations of traditional methods: This invention utilizes an innovative HPLC mobile phase system and a multi-wavelength switching program for the UV detector to achieve baseline separation and simultaneous quantification of three vitamins under the same chromatographic conditions. The single injection analysis time is only 25 minutes, a 67% improvement in efficiency compared to traditional single-component determination methods (which require over 75 minutes). This method fills the gap in the ability to analyze three water-soluble vitamins in compound feeds using a single UV-based detector.

[0029] 4. The mobile phase utilizes sodium heptanesulfonate combined with ammonium acetate. Its core advantage lies in the synergistic regulation of analyte retention through the "ion-pair effect + buffer system," achieving both separation selectivity and method stability, making it particularly suitable for reversed-phase separations of polar charged compounds. Peak symmetry factors for vitamins B1, B2, and B6 are maintained above 1.5. Combined with column temperature control (35°C), baseline noise is maintained at ≤0.01 mAU and drift within 25 minutes is maintained at ≤0.05 mAU, meeting the requirements of long-term batch testing. This minimizes impurity interference, improving specificity, sensitivity, and separation performance. B vitamins are stable in acidic environments, and their strong phosphate buffering capacity reduces peak tailing. Optimizing pH can suppress vitamin ionization. Comparison of separation performance at pH 2.5, 3.0, and 3.5 revealed optimal separation at pH 3.0.

[0030] 5. The method is applicable to all types of feed samples, including premixes, concentrates, and compound feeds. By optimizing the mobile phase gradient elution procedure, it is compatible with the simultaneous detection of other water-soluble vitamins such as vitamin B3 and niacinamide, reserving a technical interface for subsequent standard upgrades.

[0031] 6. Low limit of detection and limit of quantification: Based on the signal-to-noise ratio (S / N), the limits of detection (LOD) of vitamins B1, B2, and B6 are 0.02 µg / ml, 0.007 µg / ml, and 0.006 µg / ml, respectively. The limits of quantification (LOQ) are 0.06 µg / ml, 0.06 µg / ml, and 0.02 µg / ml, respectively, meeting the detection requirements for trace vitamins in feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the chromatogram of the liquid compound feed in Example 1 (Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2);

[0033] Figure 2 This is the specific test chromatogram in Example 2 (Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2);

[0034] Figure 3 The standard curves of the three vitamins in Example 2 are shown below:

[0035] Figure 4 This is the chromatogram of the solid compound feed in Example 3 (Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2);

[0036] Figure 5 The chromatograms are compared under different experimental schemes in Example 4 (Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2);

[0037] Figure 6 The chromatograms are compared at different pH values of the mobile phase in Example 5 (Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2). DETAILED DESCRIPTION

[0038] The following will further explain the above content in detail through specific implementation methods in the form of examples. However, this should not be understood as limiting the scope of the above subject matter to the following examples. All technologies implemented based on the content of the present invention are within the scope.

[0039] Example 1

[0040] A method for detecting three water-soluble vitamins in a liquid compound feed is as follows:

[0041] Instruments and equipment: High performance liquid chromatograph with UV detector (diode matrix detector), pH meter with temperature control, accuracy 0.01, constant temperature water bath 0-100℃, analytical balance, syringe filter, 0.45μm organic filter membrane.

[0042] Reagents: ammonium acetate, phosphoric acid, sodium heptanesulfonate, methanol (chromatographic grade), vitamin B1, vitamin B2, and vitamin B6 standards were purchased from China National Institute for Food and Drug Control.

[0043] Preparation of the extract: Weigh appropriate amounts of sodium heptanesulfonate and ammonium acetate, dissolve them in water to prepare a solution containing 0.05% sodium heptanesulfonate and 20 mmol / L ammonium acetate. Adjust the pH to 3.0 with phosphoric acid. Take 800 ml of this solution and mix it with 200 ml of methanol.

[0044] Sample pretreatment: Weigh 4.0 g (accurate to 0.0001 g) of liquid compound feed into a 25 ml brown conical flask, add about 15 ml of the extract, ultrasonically treat for 30 minutes, and then dilute to the mark with the extract.

[0045] Standard preparation:

[0046] Preparation of vitamin B1 standard stock solution: Weigh 10 mg of vitamin B1 standard (accurate to 0.0001g), add the extract to dissolve by ultrasonication and dilute to 100 ml. Store in a brown bottle and store in a refrigerator at 2℃-8℃. It can be used for 3 months. The concentration of the solution is 100 μg / ml.

[0047] Preparation of vitamin B2 standard stock solution: Weigh 10 mg (accurate to 0.0001 g) of vitamin B2 standard, add the extract and boil in a boiling water bath for 30 min. After the solid particles are completely dissolved, dilute the volume to 100 ml and store in a brown bottle in a refrigerator at 2°C-8°C. It can be used for 3 months. The concentration of the solution is 100 µg / ml.

[0048] Preparation of vitamin B6 standard stock solution: Weigh 10 mg of vitamin B6 standard (accurate to 0.0001g), add the extract to dissolve by ultrasonication and dilute to 100 ml. Store in a brown bottle and store in a refrigerator at 2℃-8℃. It can be used for 3 months. The concentration of the solution is 100 μg / ml.

[0049] The working solution of the vitamin mixed standard was prepared as follows: 1 ml of the three vitamin standard stock solutions were accurately measured and transferred to a 10 ml volumetric flask. The vitamin concentration of the solution was 10 µg / ml and the solution was prepared immediately before use.

[0050] HPLC detection:

[0051] HPLC condition design:

[0052] High performance liquid chromatography: Aglient 1260Ⅱ; chromatographic column: C 18 Reversed-phase column, 150 mm long, 4.6 mm inner diameter, 4 µm particle size; detector: UV detector; flow rate: 1.0 ml / min; temperature: 35°C; injection volume: 20 µl; wavelength: 290 nm for 0-10 min, 242 nm for 10-15 min, and 267 nm for 15-25 min.

[0053] Mobile phase preparation: Mobile phase A (aqueous phase): Weigh appropriate amounts of sodium heptane sulfonate and ammonium acetate, dissolve in water to prepare a solution containing 0.05% sodium heptane sulfonate and 20 mmol / L ammonium acetate, adjust the pH to 3.0 with phosphoric acid, and filter through a 0.45 μm filter membrane; Mobile phase B (organic phase): methanol.

[0054] Gradient elution was used and the elution conditions were as follows:

[0055] .

[0056] The detection spectrum of three water-soluble vitamins in liquid compound feed is as follows Figure 1 shown.

[0057] Example 2 Methodology Verification

[0058] 1. Methodological validation: The specificity, limit of detection and limit of quantification, standard curve, spiked recovery, precision and accuracy, and stability of this method were validated with reference to the Implementation Guide for Validation and Validation of Chemical Analysis Methods for Conformity Assessment (GB / T 27417-2017).

[0059] 1.1 Specificity

[0060] Blank solvent test: The extract was directly passed through a 0.22µm syringe filter and injected into the HPLC instrument for 6 consecutive injections.

[0061] Excipient Interference Test: 18 ml of a blank feed matrix containing no target compound was added with 2 ml of a 100 µg / ml mixed vitamin standard working solution to prepare a composite feed sample with a vitamin concentration of 10 µg / ml. Six replicates of each of the above samples were assayed under the assay conditions selected in Example 1.

[0062] 1.2 Limit of detection and limit of quantification

[0063] 100 µl of blank feed matrix without the target compound was spiked with vitamin standard substances to prepare assays containing the three vitamins at concentrations of 0.1, 1.0, 10.0, and 20.0 µg / ml. Each concentration was replicated five times. Determinations were performed according to the conditions described in Example 1. A signal-to-noise ratio (S / N) ≥ 3 was defined as the limit of detection (LOD), and an S / N ≥ 10 was defined as the limit of quantification (LOQ).

[0064] 1.3 Standard curve

[0065] The extracts were used to prepare three vitamin mixed standard solutions at concentrations of 1.0 µg / ml, 5 µg / ml, 10 µg / ml, 20 µg / ml, 50 µg / ml, and 100 µg / ml, respectively. The assays were performed according to the assay conditions described in Example 1. Linear regression was performed using the weighted least squares method, with concentration as the abscissa and chromatographic peak area as the ordinate, to calculate the regression equation and correlation coefficient.

[0066] 1.4 Spike recovery

[0067] Three vitamin standards were added to blank feed matrix samples that did not contain the target vitamins to prepare three vitamins with different concentrations. After extraction and treatment according to the sample pretreatment method, the samples were detected by high performance liquid chromatography, and their concentrations and recoveries were calculated.

[0068] 1.5 Precision and Accuracy

[0069] 1.5.1 Intra-assay precision: Weigh an appropriate amount of vitamin reference substance to prepare a vitamin B1 concentration of 88 µg / ml, a vitamin B2 concentration of 130 µg / ml, and a vitamin B6 concentration of 200 µg / ml. Repeat five times for each concentration and calculate the intra-assay coefficient of variation (CV).

[0070] 1.5.2 Inter-batch precision: Prepare samples according to the intra-batch precision. Prepare and test three batches of samples on different days and calculate the inter-batch coefficient of variation (CV).

[0071] 1.6 Stability: After pretreatment, place the sample in an automatic sampler for 12 hours and 24 hours, record the content changes, and evaluate its stability.

[0072] 2. Results Analysis

[0073] 2.1 Specificity

[0074] No interference peaks appeared at the retention time of the target compound in the blank solvent and blank matrix, indicating that the solvent and matrix had no interference. When the excipients were present in the blank matrix, the retention time and peak shape of the main component peak were consistent with those of the single injection, and the separation degree between the excipient peak and the main component peak was 1.5, indicating that the excipients did not interfere with the main component detection. Figure 2 .

[0075] 2.2 Limit of detection and limit of quantification

[0076] The signal-to-noise ratio was used to calculate the LOD and LOQ values in this validation. For vitamin B1, the LOD was 0.02 µg / ml and the LOQ was 0.06 µg / ml; for vitamin B2, the LOD was 0.007 µg / ml and the LOQ was 0.06 µg / ml; and for vitamin B6, the LOD was 0.006 µg / ml and the LOQ was 0.02 µg / ml.

[0077] 2.3 Standard curve

[0078] The results show that the three vitamins have a good linear relationship with the chromatographic peak area. The correlation coefficients are all 3 9 or above. Figure 3 Vitamin standard curve equation.

[0079] 2.4 Spike recovery

[0080] The vitamin standards were prepared at low, medium, and high concentrations of 1.5, 15.0, and 30.0 µg / g, respectively, with five replicates for each concentration. The recoveries of vitamin B1, vitamin B2, and vitamin B1 in the compound feed ranged from 96% to 110%, 92% to 107%, and 90% to 103%, all within the 90% to 110% range (Table 1).

[0081] Table 1 Spiked recovery

[0082] .

[0083] 2.5 Precision

[0084] 2.5.1 Intra-batch precision

[0085] In the intra-batch precision validation, the same batch of compound feed samples was selected and the vitamin B1 content was determined with five replicate injections within the limit of quantification (LOQ) of each sample. The relative standard deviation (RSD) was 2%. Similarly, the RSDs for vitamins B2 and B6 were all within 5%, demonstrating the excellent reproducibility of this method within the same batch of samples.

[0086] 2.5.2 Inter-batch precision

[0087] During inter-batch precision verification, three different batches of compound feed samples were selected and the contents of vitamins B1, B2, and B6 were determined using the aforementioned method. The results for vitamin B1 across the different batches were 79.7, 79.0, and 78.0 µg / ml, respectively, with a calculated relative standard deviation (RSD) of 1%. The inter-batch RSDs for the other two vitamins were all below 5%, demonstrating the method's excellent stability across different batches and minimal impact from batch variability.

[0088] Table 2 Precision

[0089] .

[0090] 2.6 Stability

[0091] When the sample solution of compound feed pretreatment is stored at room temperature for ≤72 hours, the target water-soluble vitamin content is stable (change ≤5%). When the sample solution is refrigerated at 4°C for one week, the content of each vitamin changes ≤5%, indicating good stability. If extended storage is required, the sample solution can be sealed and refrigerated at 4°C. Reliable results are obtained within ≤72 hours. Pretreated samples should be analyzed as soon as possible. If storage is necessary, refrigeration is preferred to ensure stability.

[0092] The stability results are shown in Table 3 below:

[0093] Table 3 Stability results

[0094] .

[0095] The test results of the liquid compound feed in Example 1 are as follows: the vitamin B1 content is 81 mg / L; the vitamin B2 content is 127 mg / L; and the vitamin B6 content is 200 mg / L, which are consistent with the theoretical values of the sample.

[0096] Example 3 Solid compound feed detection

[0097] Weigh 0.25 g (accurate to 0.0001 g) of solid compound feed into a 25 ml brown conical flask, add about 15 ml of the extract and heat in a boiling water bath for 30 min. Shake once every 10 min. After the solid particles are completely dissolved, adjust the volume to the scale and calculate the content on a high performance liquid chromatograph according to the treatment method and chromatographic conditions in Example 1.

[0098] The test results are as follows:

[0099] The content of vitamin B1 is 1511 mg / kg, the content of vitamin B2 is 4204 mg / kg, and the content of vitamin B6 is 1200 mg / kg, which are consistent with the theoretical values of the sample. The detection spectrum is as follows Figure 4 .

[0100] Example 4 Comparison of different test methods

[0101] The standard pretreatment, sample pretreatment, mobile phase and chromatographic conditions in Comparative Example 1 are different from those in Example 1. Figure 5 : Among them, A and C in the figure are chromatograms of the reference substance and sample solution under the technical scheme of Example 1; B and D are chromatograms of the reference substance and sample solution under the technical scheme of the comparative example; among them, Peak 1 is vitamin B6; Peak 2 is vitamin B1; Peak 3 is vitamin B2.

[0102] Comparative Example 1 detection method is as follows:

[0103] Standard Preparation: Accurately weigh 10 mg of each vitamin B1, vitamin B2, and vitamin B6 reference solution into a 100 mL volumetric flask. Add an appropriate amount of water and shake in a 65°C waterbath for 15 minutes. Cool to room temperature, dilute to the mark, and shake well to prepare the reference solution. Accurately measure 1 mL of each standard stock solution into a 10 mL volumetric flask. The vitamin concentration of each solution is 10 µg / mL. Prepare immediately before use.

[0104] Sample pretreatment: Accurately weigh about 2.00 g, place in a 50 mL volumetric flask, add appropriate amount of water, shake in a 65°C water bath for 15 min, cool to room temperature, dilute to the scale, shake well, and use as the test solution.

[0105] The mobile phase was sodium heptanesulfonate solution (containing 0.5% glacial acetic acid and 0.05% triethylamine)-methanol (65:35, v / v).

[0106] Chromatographic conditions: flow rate: 1.0 mL / min; detection wavelength: 280 nm; injection volume: 20 μL, column temperature: 25°C.

[0107] Analysis of results: The traditional isocratic elution of sodium heptane sulfonate-methanol (65:35) as a mobile phase cannot take into account the different retention characteristics of multiple water-soluble vitamins. Vitamins exhibit leading peaks with asymmetric peak shapes, sample baseline noise greater than 5 mAU, and a significant decrease in sensitivity. Excessive sample impurities affect the separation effect. Compared with the sodium heptane sulfonate-ammonium acetate-methanol gradient elution system of the present invention, problems such as asymmetric peaks and excessive impurity peaks exist. Therefore, the addition of 0.5 mmol / L sodium heptane sulfonate and 10 mmol / L ammonium acetate to the mobile phase, through an optimized gradient elution program, can accurately match the elution requirements of each vitamin at different time periods. The peak shape is sharp and symmetrical, the tailing factor T is equal to 1.02, and the resolution (Rs) is greater than 2.0, achieving efficient separation and improving detection sensitivity and accuracy.

[0108] Example 5 Comparison of mobile phase pH tests

[0109] To evaluate the effect of different mobile phase pH values on vitamin peak shape and its impact on analysis time, we aimed to reduce peak tailing (tailing factor T between 0.95 and 1.05) and peak broadening, thereby improving detection sensitivity and quantitative accuracy. While maintaining separation effectiveness, we also sought to minimize analysis cycles and improve detection efficiency. The specific implementation plan is as follows:

[0110] The mobile phase system was sodium heptanesulfonate-ammonium acetate solution. The pH of the mobile phase was adjusted to 2.5, 3.0, and 3.5 by adding phosphoric acid solution. The mobile phase at each pH value was filtered through a 0.45 μm microporous filter membrane and ultrasonically degassed for 30 minutes. The rest of the conditions remained the same as in Example 1. The analytical spectrum is shown in FIG. Figure 6 ; ABC in the figure are chromatograms of standard solutions when the mobile phase pH is 3.5, 3.0, and 2.5 respectively.

[0111] Analysis of results: At pH 3.5, the retention time of vitamins was prolonged, the vitamin B6 peak was asymmetrical, overlapping peaks appeared, and the resolution was less than 1.0. Vitamin B1 and vitamin B2 were not completely separated, resulting in double peaks. At pH 2.5, the retention time of vitamins was advanced, overlapping peaks appeared for vitamin B6, and the resolution was less than 1.0. Vitamin B1 had double peaks and could not be completely separated. At pH 3.0, the target peak and adjacent peaks were well separated by baseline, with a resolution (Rs) greater than 1.5. The peak shape was sharp and symmetrical, and the tailing factor was between 0.95 and 1.05, enabling accurate peak area integration and quantitative analysis.

Claims

1. A method for simultaneous detection of water-soluble vitamins in compound feed based on HPLC, comprising the following steps: S1. Sample pretreatment: Mix the compound feed to be tested and the extract evenly, ultrasonicate for 30 min, and filter through a 0.45 μm syringe filter to obtain the water-soluble vitamin test solution; S2. Standard sample pretreatment: Repeat step S1 with the standard sample, mix and dilute to obtain a water-soluble vitamin mixed standard solution; S3. HPLC detection: The test solution and the standard solution are detected respectively by high performance liquid chromatography, and the content of water-soluble vitamins in the test feed is calculated using the external standard method according to the obtained high performance liquid chromatography spectra; The water-soluble vitamins are vitamin B1, vitamin B2, vitamin B 6； The extract in S1 is prepared by adjusting the pH of a solution containing sodium heptanesulfonate and ammonium acetate with phosphoric acid and then mixing it with methanol in a certain proportion; The chromatographic conditions of the high performance liquid chromatography described in S3 are as follows: mobile phase A is a solution of 0.05% sodium heptanesulfonate and 20 mmol / L ammonium acetate, pH=3.0; mobile phase B is methanol.

2. The detection method according to claim 1, wherein The extract in S1 is a solution containing 0.05% sodium heptanesulfonate and 20 mmol / L ammonium acetate, the pH value of which is adjusted to 3.0 with phosphoric acid, and then mixed with methanol at a ratio of 80:

20.

3. The detection method according to claim 1, wherein The chromatographic conditions of the high performance liquid chromatography are as follows: the high performance liquid chromatograph is Aglient 1260Ⅱ; C 18 The reversed-phase column was 150 mm long, 4.6 mm inner diameter, and 4 µm particle size. The flow rate was 1.0 ml / min, the temperature was 35°C, and the injection volume was 20 µL.

4. The detection method according to claim 3, characterized in that The high performance liquid chromatography adopts gradient elution, and the elution conditions are as follows: .

5. The detection method according to claim 4, characterized in that The detector of the high performance liquid chromatography was UV, and the detection wavelength program was: 0-10 min 290 nm → 10-15 min 242 nm → 15-25 min 267 nm switching.

6. The detection method according to claim 1, characterized in that When the compound feed to be tested is solid, the sample needs to be treated in a water bath for 30 minutes and shaken every 10 minutes.

7. The detection method according to claim 1, characterized in that The vitamin B2 standard was dissolved in a boiling water bath.

8. Use of the HPLC-based simultaneous detection method for water-soluble vitamins in compound feeds as described in any one of claims 1 to 7 in the rapid separation and accurate quantification of vitamin B1, vitamin B2, and vitamin B6.

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