Method for determining pyridoxal phosphate

Abstract

The invention discloses a method for determining pyridoxal phosphate, which comprises the following steps: preparing an extracting agent solution, preparing a derivative agent solution, preparing a phosphate buffer solution, preparing a sample solution, and preparing standard solutions with at least five concentrations, the standard solution is a solution with pyridoxal phosphate, and establishing a standard curve: performing liquid phase analysis on the standard solution with each concentration by using a high performance liquid chromatograph under a preset detection condition, measuring the peak area of each standard solution, and establishing the standard curve by taking the peak area of each standard solution as a vertical coordinate and the concentration as a horizontal coordinate; and content determination: carrying out liquid phase analysis on the sample solution by using a high performance liquid chromatograph under a preset detection condition, measuring the peak area, comparing the peak area with a standard curve to obtain the concentration, and calculating the content of pyridoxal phosphate in the sample by combining with the weighing of the sample. The method can accurately detect the content of pyridoxal phosphate in food or health food.

Description

Technical Field

[0001] The invention relates to the field of detection of effective components of health foods, foods or medicines, and in particular to a method for determining pyridoxal phosphate. Background Art

[0002] Pyridoxal phosphate, chemically known as 2-methyl-3-hydroxy-5-(phosphoryloxy)methyl-4-pyridinecarboxaldehyde, is an important vitamin B6 compound. It is not only a coenzyme of transaminase and decarboxylase in amino acid metabolism, but also promotes the decarboxylation of glutamate and enhances the production of γ-aminobutyric acid. Pyridoxal phosphate is also widely used in the synthesis of β-carbonylamide drugs and biological activity research. For example, it is mainly used in the treatment of Parkinson's syndrome in clinical practice to promote transaminase transamination and increase the content of dopamine in the body.

[0003] Due to the presence of aldehyde group, pyridoxal phosphate is active and easily undergoes condensation and nucleophilic addition reactions. It is unstable itself. Therefore, during the testing of food or health food, pyridoxal phosphate is easily degraded and it is difficult to accurately detect its content. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for determining pyridoxal phosphate, which can accurately detect the content of pyridoxal phosphate in food or health food.

[0005] The present invention is achieved through the following technical solutions:

[0006] A method for determining pyridoxal phosphate comprises the following steps:

[0007] Prepare extractant solution: Mix acetonitrile and water, add 0.1-1.0% phosphoric acid;

[0008] Prepare the derivatization agent solution: weigh 0.1-0.5g of the derivatization agent, add 20-30ml of acetonitrile and ultrasonically dissolve it, add 20-30ml of water and 0.1-1.0ml of hydrochloric acid, and mix well;

[0009] Prepare phosphate buffer: weigh a predetermined amount of potassium dihydrogen phosphate and add a certain amount of water to dissolve it, and adjust the pH to 2.5-4.0 with phosphoric acid;

[0010] Prepare sample solution: weigh 1.0-5.0g sample into a 50ml volumetric flask, add 5-25ml extractant solution, ultrasonically extract for 15-30min, add 1-10ml derivatization solution, derivatize in a constant temperature water bath for 10-30min, take out and cool to room temperature, add extractant solution to the mark, mix well;

[0011] Prepare at least five standard solutions of different concentrations: wherein the standard solution is a solution containing pyridoxal phosphate;

[0012] Establishing a standard curve: using a high performance liquid chromatograph under preset detection conditions, respectively performing liquid phase analysis on the standard solution corresponding to each concentration, measuring the peak area of ​​each standard solution, and establishing a standard curve with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa;

[0013] Content determination: Use a high performance liquid chromatograph to perform liquid phase analysis on the sample solution under preset detection conditions, measure its peak area, compare it with the standard curve to obtain its concentration, and combine the sample weighing to calculate the content of pyridoxal phosphate in the sample.

[0014] Furthermore, the preset detection conditions are:

[0015] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0016] Mobile phase: a mixture of acetonitrile and phosphate buffer;

[0017] Flow rate: 0.8~1.5ml / min;

[0018] Detection wavelength: 380~430nm;

[0019] Column temperature: 30-40°C;

[0020] Injection volume: 5~10ul.

[0021] Furthermore, the calculation formula in the content determination step is:

[0022]

[0023] Where:

[0024] C 样 —The concentration of the sample solution is calculated by checking the standard curve based on the peak area of ​​the sample solution, μg / mL;

[0025] W 样 — Sample weight, g;

[0026] V—dilution volume, ml.

[0027] Furthermore, the step of preparing at least five standard solutions of different concentrations specifically comprises:

[0028] Weigh 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, add 5-15 ml of water to dissolve, add acetonitrile to dilute to scale, and mix well. Pipette 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, add 5-25 ml of extractant solution and 1-10 ml of derivatization agent solution, and react in a constant temperature water bath of 40-60 ° C for 10-30 minutes, take out and cool to room temperature, add extractant solution to the scale and mix well to prepare standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug of pyridoxal phosphate per 1 ml, respectively.

[0029] Furthermore, the step of preparing phosphate buffered saline specifically comprises:

[0030] Weigh 3-7g potassium dihydrogen phosphate and add 1000ml water to dissolve it, and adjust the pH to 2.5-4.0 with phosphoric acid.

[0031] Furthermore, in the step of preparing the sample solution, the temperature of the constant temperature water bath is 40°C to 60°C.

[0032] Furthermore, the proportion of acetonitrile in the extractant is 30-70%.

[0033] Furthermore, the derivatizing agent is 2,4-dinitrophenylhydrazine, dansylhydrazine, or o-phenylenediamine.

[0034] Furthermore, in the mixed solution of acetonitrile and phosphate buffer, acetonitrile accounts for 10-50%.

[0035] Furthermore, the samples include tablets, capsules, powders, and soft candies in foods or health foods.

[0036] Compared with the prior art, the advantages of the present invention are:

[0037] 1. The present invention adopts a derivatization method to convert unstable pyridoxal phosphate into a stable derivative product, and then uses high performance liquid chromatography technology to accurately detect the content of the derivative product, thereby indirectly obtaining the content of pyridoxal phosphate.

[0038] 2. The derivatization method of the present invention has fast reaction and high efficiency, and is suitable for the determination of pyridoxal phosphate in foods such as tablets, capsules, powders, soft candies, or health foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is a chromatogram of the standard solution of pyridoxal phosphate in Example 1 of the present invention;

[0040] Figure 2 is a chromatogram of the sample solution in Example 1 of the present invention;

[0041] Figure 3is a chromatogram of the standard solution of pyridoxal phosphate in Example 2 of the present invention;

[0042] Figure 4 is a chromatogram of the sample solution in Example 2 of the present invention;

[0043] Figure 5 is a chromatogram of the standard solution of pyridoxal phosphate in Example 3 of the present invention;

[0044] Figure 6 It is the chromatogram of the sample solution in Example 3 of the present invention. DETAILED DESCRIPTION

[0045] The present invention provides a method for determining pyridoxal phosphate, which uses a derivatization method and can accurately detect the content of pyridoxal phosphate in food or health food. Specifically, it comprises the following steps:

[0046] 1) Prepare extractant solution: Mix acetonitrile and water, and add 0.1-1.0% phosphoric acid.

[0047] 2) Prepare the derivatization agent solution: weigh 0.1-0.5 g of the derivatization agent, add 20-30 ml of acetonitrile and ultrasonically dissolve it, add 20-30 ml of water and 0.1-1.0 ml of hydrochloric acid, and mix well.

[0048] 3) Preparation of phosphate buffer: Weigh a predetermined amount of potassium dihydrogen phosphate and add a certain amount of water to dissolve it, and adjust the pH to 2.5-4.0 with phosphoric acid.

[0049] 4) Prepare sample solution: weigh 1.0-5.0g sample into a 50ml volumetric flask, add 5-25ml extractant solution and ultrasonically extract for 15-30min, add 1-10ml derivatization solution, derivatize in a constant temperature water bath for 10-30min, take out and cool to room temperature, add extractant solution to the mark, and mix well.

[0050] 5) preparing standard solutions of at least five concentrations: wherein the standard solution is a solution containing pyridoxal phosphate.

[0051] 6) Establishing a standard curve: Using a high performance liquid chromatograph under preset detection conditions, perform liquid phase analysis on the standard solutions corresponding to each concentration, measure the peak area of ​​each standard solution, and establish a standard curve with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa.

[0052] 7) Content determination: Use high performance liquid chromatography to perform liquid phase analysis on the sample solution under preset detection conditions, measure its peak area, compare it with the standard curve to obtain its concentration, and combine with the sample weighing to calculate the content of pyridoxal phosphate in the sample.

[0053] Among them, the preset detection conditions are:

[0054] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0055] Mobile phase: a mixture of acetonitrile and phosphate buffer;

[0056] Flow rate: 0.8~1.5ml / min;

[0057] Detection wavelength: 380~430nm;

[0058] Column temperature: 30-40°C;

[0059] Injection volume: 5~10ul.

[0060] Among them, the calculation formula in the step content determination is:

[0061]

[0062] And in the formula:

[0063] C 样 —The concentration of the sample solution is calculated by checking the standard curve based on the peak area of ​​the sample solution, μg / mL;

[0064] W 样 — Sample weight, g;

[0065] V—dilution volume, ml.

[0066] The step of preparing at least five concentrations of standard solutions specifically includes: weighing 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, adding 5-15 ml of water to dissolve, adding acetonitrile to dilute to scale, and mixing. Respectively taking 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, adding 5-25 ml of extractant solution and 1-10 ml of derivatization agent solution, derivatizing in a constant temperature water bath of 40-60° C. for 10-30 minutes, taking out and cooling to room temperature, adding extractant solution to the scale and mixing, and preparing standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug of pyridoxal phosphate per 1 ml.

[0067] Steps: The preparation of phosphate buffer salt specifically includes: weighing 3-7g of potassium dihydrogen phosphate and adding 1000ml of water to dissolve it, and adjusting the pH to 2.5-4.0 with phosphoric acid.

[0068] Steps: When preparing the sample solution, the temperature of the constant temperature water bath is 40℃~60℃.

[0069] Optionally, the proportion of acetonitrile in the extractant is 30-70%.

[0070] Optionally, the derivatizing agent is 2,4-dinitrophenylhydrazine, dansylhydrazine, or o-phenylenediamine.

[0071] Optionally, the acetonitrile accounts for 10-50% of the mixed solution of acetonitrile and phosphate buffer.

[0072] Optionally, the sample can be a preparation such as tablets, capsules, powders, or soft candies in food or health food.

[0073] For ease of understanding, the following examples are provided:

[0074] Example 1

[0075] (1) Preparation of extractant solution

[0076] Measure 300 ml of acetonitrile and 700 ml of water, mix them, and add 0.1% phosphoric acid.

[0077] (2) Preparation of derivatization agent solution

[0078] Weigh 0.1 g of 2,4-dinitrophenylhydrazine, add 20 ml of acetonitrile and dissolve by ultrasonication, then add 30 ml of water and 0.1 ml of hydrochloric acid and mix well.

[0079] (3) Preparation of phosphate buffer

[0080] Weigh 3g of potassium dihydrogen phosphate and add 1000ml of water to dissolve it, and adjust the pH to 2.5 with phosphoric acid.

[0081] (4) Preparation of standard solution

[0082] Weigh 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, add 5 ml of water to dissolve, add acetonitrile to dilute to scale, and mix well. Pipette 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, add 5 ml of extractant solution and 1 ml of derivatization agent solution, derivatize in a 40 ° C constant temperature water bath for 10 minutes, take out and cool to room temperature, add extractant solution to dilute to scale and mix well, and make standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug per 1 ml.

[0083] (5) Prepare sample solution

[0084] Take 20 tablets and crush them (product name: Women's Multivitamin Tablets, batch number: GC2024190, manufacturer: Jiangsu Alande Nutrition Products Co., Ltd.), weigh 1.0g of the sample into a 50ml volumetric flask, add 5ml of the extractant solution and ultrasonically extract for 15min, add 1ml of the derivatization solution, derivatize in a 40℃ constant temperature water bath for 10min, take out and cool to room temperature, add the extractant solution to the scale, mix well, and use for high performance liquid chromatography determination.

[0085] (6) High performance liquid chromatography conditions are:

[0086] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0087] Mobile phase: acetonitrile: phosphate buffer = 10:90;

[0088] Flow rate: 0.8ml / min;

[0089] Detection wavelength: 410nm;

[0090] Column temperature: 30°C;

[0091] Injection volume: 5ul;

[0092] (7) Establishing a standard curve

[0093] Using a high performance liquid chromatograph under preset detection conditions, i.e., the conditions of step (6), each concentration of the standard solution is subjected to liquid phase analysis to measure the peak area of ​​each standard solution, and a standard curve is established with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa, to obtain Figure 1 Chromatogram of a standard solution of pyridoxal phosphate.

[0094] (8) Content determination

[0095] The sample solution was analyzed by high performance liquid chromatography under the preset detection conditions, i.e., the conditions of step (6), to obtain Figure 2 The chromatogram of the sample solution was obtained. The peak area of ​​pyridoxal phosphate was obtained, and the concentration of pyridoxal phosphate in the sample solution was calculated according to the standard curve. Combined with the sample weighing and dilution volume, the content of pyridoxal phosphate in the sample was calculated according to the following formula to be 7.49 mg / g. The actual amount of pyridoxal phosphate added in the sample was 7.55 mg / g, and its recovery rate, that is, the ratio of the detected amount to the actual amount added, was 99.2%.

[0096] The calculation formula is:

[0097]

[0098] Where:

[0099] C 样 —The concentration of the sample solution is calculated by checking the standard curve based on the peak area of ​​the sample solution, μg / mL;

[0100] W 样 — Sample weight, g;

[0101] V—dilution volume, ml.

[0102] Example 2

[0103] (1) Preparation of extractant solution

[0104] Measure 400 ml of acetonitrile and 600 ml of water, mix them, and add 0.5% phosphoric acid.

[0105] (2) Preparation of derivatization agent solution

[0106] Weigh 0.3 g of dansyl hydrazide, add 30 ml of acetonitrile and dissolve by ultrasonication, then add 20 ml of water and 0.4 ml of hydrochloric acid and mix well.

[0107] (3) Preparation of phosphate buffer

[0108] Weigh 5 g of potassium dihydrogen phosphate and add 1000 ml of water to dissolve it, and adjust the pH to 3.0 with phosphoric acid.

[0109] (4) Preparation of standard solution

[0110] Weigh 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, add 5 ml of water to dissolve, add acetonitrile to dilute to scale, and mix well. Pipette 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, add 10 ml of extractant solution and 5 ml of derivatization agent solution, and derivatize in a 50 ° C constant temperature water bath for 20 minutes, take out and cool to room temperature, add extractant solution to dilute to scale and mix well, and make standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug per 1 ml.

[0111] (5) Prepare sample solution

[0112] Take out the contents of 20 soft capsules (product name: Resveratrol composite soft capsules, batch number: ZC2018692, manufacturer: Zhejiang Alande Biotechnology Co., Ltd.), weigh 2.0g of the sample into a 50ml volumetric flask, add 10ml of the extractant solution for ultrasonic extraction for 20min, add 5ml of the derivatization solution, derivatize in a 50℃ constant temperature water bath for 20min, take out and cool to room temperature, add the extractant solution to the scale, mix well, and use for high performance liquid chromatography determination.

[0113] (6) High performance liquid chromatography conditions are:

[0114] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0115] Mobile phase: acetonitrile: phosphate buffer = 20:80;

[0116] Flow rate: 1.0ml / min;

[0117] Detection wavelength: 420nm;

[0118] Column temperature: 35°C;

[0119] Injection volume: 8ul;

[0120] (7) Establishing a standard curve

[0121] Using a high performance liquid chromatograph under preset detection conditions, i.e., the conditions of step (6), each concentration of the standard solution is subjected to liquid phase analysis to measure the peak area of ​​each standard solution, and a standard curve is established with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa, to obtain Figure 3 Chromatogram of a standard solution of pyridoxal phosphate.

[0122] (8) Content determination

[0123] The sample solution was analyzed by high performance liquid chromatography under the preset detection conditions, i.e., the conditions of step (6), to obtain Figure 4 The peak area of ​​pyridoxal phosphate was obtained, and the concentration of pyridoxal phosphate in the sample solution was calculated according to the standard curve. The content of pyridoxal phosphate in the sample was calculated to be 9.80 mg / g according to the calculation formula in combination with the sample weighing and dilution volume. The actual amount of pyridoxal phosphate added in the sample was 9.85 mg / g, and the recovery rate was 99.5%.

[0124] Example 3

[0125] (1) Preparation of extractant solution

[0126] Measure 500 ml of acetonitrile and 500 ml of water, mix them, add 0.3% phosphoric acid

[0127] (2) Preparation of derivatization agent solution

[0128] Weigh 0.5g o-phenylenediamine and add 25ml acetonitrile to dissolve by ultrasonic, add 30ml water and 0.5ml hydrochloric acid, mix well

[0129] (3) Preparation of phosphate buffer

[0130] Weigh 6g potassium dihydrogen phosphate and dissolve it in 1000ml water. Adjust the pH to 3.5 with phosphoric acid.

[0131] (4) Preparation of standard solution

[0132] Weigh 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, add 5 ml of water to dissolve, add acetonitrile to dilute to scale, and mix well. Pipette 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, add 15 ml of extractant solution and 10 ml of derivatization agent solution, and react in a 60 ° C constant temperature water bath for 30 minutes. Take out and cool to room temperature, add extractant solution to dilute to scale and mix well to make standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug per 1 ml.

[0133] (5) Prepare sample solution

[0134] Crush 20 soft candies (product name: Children's multivitamin soft candy, batch number: F19034, manufacturer: Taizhou Alande Nutrition Products Co., Ltd.), weigh 3.0g of the sample into a 50ml volumetric flask, add 15ml of the extractant solution and ultrasonically extract for 30min, add 5ml of the derivatization solution, and derivatize in a 60℃ constant temperature water bath for 30min, take out and cool to room temperature, add the extractant solution to the scale, mix well, and use for high performance liquid chromatography determination.

[0135] (6) High performance liquid chromatography conditions are:

[0136] Chromatographic column: octadecyl bonded silica gel chromatographic column;

[0137] Mobile phase: acetonitrile: phosphate buffer = 30:70;

[0138] Flow rate: 1.1 ml / min;

[0139] Detection wavelength: 390nm;

[0140] Column temperature: 40°C;

[0141] Injection volume: 10ul.

[0142] (7) Establishing a standard curve

[0143] Using a high performance liquid chromatograph under preset detection conditions, i.e., the conditions of step (6), each concentration of the standard solution is subjected to liquid phase analysis to measure the peak area of ​​each standard solution, and a standard curve is established with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa, to obtain Figure 5 Chromatogram of a standard solution of pyridoxal phosphate.

[0144] (8) Content determination

[0145] The sample solution was analyzed by high performance liquid chromatography under the preset detection conditions, i.e., the conditions of step (6), to obtain Figure 6 The peak area of ​​pyridoxal phosphate was obtained, and the concentration of pyridoxal phosphate in the sample solution was calculated according to the standard curve. The content of pyridoxal phosphate in the sample was 5.32 mg / g according to the calculation formula, combined with the sample weighing and dilution volume. The actual amount of pyridoxal phosphate added in the sample was 5.35 mg / g, and the recovery rate was 99.4%.

[0146] The invention adopts a derivatization method to first convert unstable pyridoxal phosphate in food or health products into a stable derivative product, and then uses a high performance liquid chromatography technology to accurately detect the content of the derivative product, so that the content of pyridoxal phosphate can be indirectly obtained. The invention has fast reaction and high efficiency, and is suitable for the determination of pyridoxal phosphate in food or health foods such as tablets, capsules, powders, and soft candies.

[0147] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.

Claims

1. A method for determining pyridoxal phosphate, characterized in that: The steps include: Prepare extractant solution: Mix acetonitrile and water, add 0.1-1.0% phosphoric acid; Prepare the derivatization agent solution: weigh 0.1-0.5g of the derivatization agent, add 20-30ml of acetonitrile and ultrasonically dissolve it, add 20-30ml of water and 0.1-1.0ml of hydrochloric acid, and mix well; Prepare phosphate buffer: weigh a predetermined amount of potassium dihydrogen phosphate and add a certain amount of water to dissolve it, and adjust the pH to 2.5-4.0 with phosphoric acid; Prepare sample solution: weigh 1.0-5.0g sample into a 50ml volumetric flask, add 5-25ml extractant solution, ultrasonically extract for 15-30min, add 1-10ml derivatization solution, derivatize in a constant temperature water bath for 10-30min, take out and cool to room temperature, add extractant solution to the mark, mix well; Prepare at least five standard solutions of different concentrations: wherein the standard solution is a solution containing pyridoxal phosphate; Establishing a standard curve: using a high performance liquid chromatograph under preset detection conditions, respectively performing liquid phase analysis on the standard solution corresponding to each concentration, measuring the peak area of ​​each standard solution, and establishing a standard curve with the peak area of ​​each standard solution as the ordinate and the concentration as the abscissa; Content determination: Use a high performance liquid chromatograph to perform liquid phase analysis on the sample solution under preset detection conditions, measure its peak area, compare it with the standard curve to obtain its concentration, and combine the sample weighing to calculate the content of pyridoxal phosphate in the sample.

2. The method for determining pyridoxal phosphate according to claim 1, wherein The preset detection conditions are: Chromatographic column: octadecyl bonded silica gel chromatographic column; Mobile phase: a mixture of acetonitrile and phosphate buffer; Flow rate: 0.8~1.5ml / min; Detection wavelength: 380~430nm; Column temperature: 30-40°C; Injection volume: 5~10ul.

3. The method for determining pyridoxal phosphate according to claim 1, wherein The calculation formula in the content determination step is: Where: C 样 —The concentration of the sample solution is calculated by checking the standard curve based on the peak area of ​​the sample solution, μg / mL; W 样 — Sample weight, g; V—dilution volume, ml.

4. The method for determining pyridoxal phosphate according to claim 1, wherein The step of preparing at least five standard solutions of different concentrations specifically comprises: Weigh 5 mg of pyridoxal phosphate reference substance into a 20 ml volumetric flask, add 5-15 ml of water to dissolve, add acetonitrile to dilute to scale, and mix well. Pipette 0.5 ml, 1 ml, 2 ml, 4 ml, and 5 ml of the above solution into a 50 ml volumetric flask, add 5-25 ml of extractant solution and 1-10 ml of derivatization agent solution, and derivatize in a 40-60 ° C constant temperature water bath for 10-30 minutes, take out and cool to room temperature, add extractant solution to the scale and mix well to make standard solutions containing 2.5 ug, 5 ug, 10 ug, 20 g, and 25 ug of pyridoxal phosphate per 1 ml.

5. The method for determining pyridoxal phosphate according to claim 1, wherein The step of preparing phosphate buffered saline specifically comprises: Weigh 3-7g potassium dihydrogen phosphate and add 1000ml water to dissolve it, and adjust the pH to 2.5-4.0 with phosphoric acid.

6. The method for determining pyridoxal phosphate according to claim 1, wherein The step: in preparing the sample solution, the temperature of the constant temperature water bath is 40°C to 60°C.

7. The method for determining pyridoxal phosphate according to claim 1, wherein The acetonitrile accounts for 30-70% of the extractant.

8. The method for determining pyridoxal phosphate according to claim 1, wherein The derivatizing agent is 2,4-dinitrophenylhydrazine, dansylhydrazine and o-phenylenediamine.

9. The method for determining pyridoxal phosphate according to claim 2, wherein In the mixed solution of acetonitrile and phosphate buffer, acetonitrile accounts for 10-50%.

10. The method for determining pyridoxal phosphate according to claim 1, characterized in that: The samples include tablets, capsules, powders and soft candies in food or health food.

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