Method for detecting racemic ketoisoleucine calcium impurity in compound alpha-keto acid tablet

Abstract

The invention discloses a method for detecting racemic ketoisoleucine calcium impurities in a compound alpha-keto acid tablet, which comprises the following steps: dissolving a racemic ketoisoleucine calcium reference substance in a solvent to obtain a reference substance solution, taking the solvent as a blank solution, injecting the blank solution into a liquid chromatograph until a base line is stable, and measuring the content of the racemic ketoisoleucine calcium impurities in the compound alpha-keto acid tablet. Injecting the reference substance working solution into a liquid chromatograph for detection; the method comprises the following steps: dissolving a sample in a solvent to obtain a test solution, injecting the test solution into a liquid chromatograph, recording a chromatogram, calculating the single impurity of the racemic ketoisoleucine calcium by an external standard method, and calculating the total amount of the impurities. The invention belongs to the technical field of racemic ketoisoleucine calcium impurity determination, and particularly relates to a method which can accurately detect some specific racemic ketoisoleucine calcium degradation impurities, is not interfered by other components in compound amino acid, meets the requirement that the degree of separation among the impurities meets the applicability of a chromatographic system, and is high in detection accuracy. The sample treatment is simple and convenient, the sample detection result is accurate and reliable, and the sensitivity is high.

Description

Technical Field

[0001] The invention belongs to the technical field of racemic ketoisoleucine calcium impurity determination, and specifically refers to a method for detecting racemic ketoisoleucine calcium impurities in a compound alpha-keto acid tablet. Background Art

[0002] In order to more objectively reflect the racemic ketoisoleucine calcium impurity in the present invention, an attempt is made to establish a new HPLC method.

[0003] This method detects several currently recognized specific impurities of racemic ketoisoleucine calcium based on the HPLC method for detecting all impurities of racemic ketoisoleucine calcium. Each impurity can be detected and is not affected by other ingredients in the compound α-ketoacid tablets. The separation between each impurity is good (greater than 1.5), meeting the requirements for the suitability of the chromatographic system. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a method for detecting the impurity of racemic ketoisoleucine calcium in compound α-ketoacid tablets, and establishes a more comprehensive, objective and efficient detection method for controlling the total impurity of racemic ketoisoleucine calcium in compound α-ketoacid tablets.

[0005] The technical solution adopted by the present invention is as follows: The present invention provides a method for detecting racemic ketoisoleucine calcium impurities in compound α-ketoacid tablets, which comprises the following steps:

[0006] Step 1: dissolving the racemic ketoisoleucine calcium impurity reference substance in a solvent to obtain a racemic ketoisoleucine calcium impurity reference substance solution, taking the solvent as a blank solution, injecting the blank solvent into a liquid chromatograph until the baseline is stable, and then injecting the racemic ketoisoleucine calcium impurity reference substance solution into the liquid chromatograph for detection;

[0007] Step 2: dissolve the sample in a solvent to obtain a test solution, inject the test solution into a liquid chromatograph, record the chromatogram, calculate the single impurity of racemic ketoisoleucine calcium by an external standard method, and calculate the total amount of impurities.

[0008] Preferably, the mobile phase flow rate is 1.0-1.5 ml / min, the injection volume is 10-20 μl, the column temperature is 25° C.-35° C., mobile phase A and mobile phase B are used for gradient elution, and a UV detector is used for detection at a detection wavelength of 205 nm.

[0009] Preferably, the mobile phase A is potassium hexafluorophosphate-phosphoric acid aqueous solution (0.2 mol / L to 0.5 mol / L potassium hexafluorophosphate, 0.1% to 0.15% phosphoric acid), and the mobile phase B is acetonitrile. The chromatographic column is C18-amide, 5 μm 150×4.6 mm.

[0010] Preferably, the gradient elution process is as follows:

[0011] Time / min Mobile phase A / % Mobile phase B / % 0 95 5 5 95 5 15 70 30 30 70 30 31 95 5 38 95 5

[0012] Preferably, the concentration of the racemic ketoisoleucine calcium impurity reference solution is 0.3-0.6 μg / ml.

[0013] Furthermore, the racemic ketoisoleucine calcium impurity reference solution is prepared as follows:

[0014] (1) Preparation of racemic ketoisoleucine calcium reference stock solution (0.5 mg / ml): weigh about 50 mg of racemic ketoisoleucine calcium reference into a 100 ml volumetric flask, add acetonitrile: 0.01 mol / L hydrochloric acid solution in a ratio of 3:7, sonicate for 10 min, dilute to scale, and shake well;

[0015] (2) Preparation of racemic ketoisoleucine calcium impurity reference solution (0.5 μg / ml): Accurately measure 1 ml of racemic ketoisoleucine calcium reference stock solution into a 100 ml volumetric flask, add acetonitrile:0.01 mol / L hydrochloric acid solution in a ratio of 3:7 to dilute to the mark; Accurately measure 5.0 ml into a 50 ml volumetric flask, add acetonitrile:0.01 mol / L hydrochloric acid solution in a ratio of 3:7 to dilute to the mark, and shake well.

[0016] Preparation of the test solution:

[0017] Take 20 pieces of sample and grind them into fine powder. Take 0.5g, accurately determine, put into a 100 volumetric flask, add acetonitrile: 0.01mol / L hydrochloric acid solution = 3:7 appropriate amount, dilute to the scale after ultrasonication for 10min, shake well, filter with 0.45μm filter, and use as the test solution.

[0018] The calculation formula for calculating the content of a single impurity in compound α-keto acid tablets is as follows

[0019]

[0020] in:

[0021] Cs: Concentration of a single impurity in the test solution (μg / ml)

[0022] C: Concentration of racemic ketoisoleucine calcium impurity reference solution (μg / ml)

[0023] As: Peak area of ​​a single impurity measured in the test solution

[0024] A: Average peak area of ​​racemic ketoisoleucine calcium impurity reference solution

[0025] The total amount of impurities can be obtained by adding up all the individual impurities measured.

[0026] The beneficial effects achieved by the present invention using the above structure are as follows: the present invention discloses a detection method for determining the total impurities of racemic ketoisoleucine calcium in compound α-keto acid tablets, adopts high performance liquid chromatography, sets certain chromatographic conditions, accurately takes a reference solution and a compound α-keto acid tablet sample solution with determination, respectively injects them into HPLC, records the chromatogram, calculates the peak area of ​​a single impurity by an external standard method, and calculates the total amount of impurities by summing them up, and for some specific racemic ketoisoleucine calcium degradation impurities, the method can also accurately detect them without being interfered by other components in the compound α-keto acid tablets, and can meet the requirements that the separation degree between various impurities meets the applicability of the chromatographic system; the detection method of the present invention has simple and convenient sample processing, accurate and reliable sample measurement results, high sensitivity, conventional reagents, and simple preparation of diluents and mobile phases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 A chromatogram of the main active ingredients such as calcium ketovaline in compound α-keto acid tablets according to a detection method for determining the total impurities of calcium racemic ketoisoleucine in compound α-keto acid tablets provided in the present application;

[0028] Figure 2 A chromatogram of the main active ingredients such as racemic isoleucine calcium in compound α-keto acid tablets according to a detection method for determining the total impurities of racemic ketoisoleucine calcium in compound α-keto acid tablets provided in the present application;

[0029] Figure 3 Example 4 of a detection method for determining the total impurities of racemic ketoisoleucine calcium in compound α-ketoacid tablets provided by the present application is a chromatogram of the impurities of racemic ketoisoleucine calcium in compound α-ketoacid tablets;

[0030] Figure 4 This is an overlapping diagram of the main active ingredients in the compound α-ketoacid tablets and the related impurities of racemic ketoisoleucine calcium, which is a detection method for determining the total impurities of racemic ketoisoleucine calcium in the compound α-ketoacid tablets provided in the present application.

[0031] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0033] Example 1

[0034] A method for detecting racemic ketoisoleucine calcium impurities in compound α-ketoacid tablets comprises the following steps:

[0035] (1) dissolving the racemic ketoisoleucine calcium reference substance in a solvent to obtain a racemic ketoisoleucine calcium impurity reference substance solution, using a solvent (acetonitrile: 0.01 mol / L hydrochloric acid solution = 3:7) as a blank solution, injecting 20 μl of the blank solvent into a liquid chromatograph until the baseline is stable, and then injecting the racemic ketoisoleucine calcium impurity reference substance solution 1 and the racemic ketoisoleucine calcium impurity reference substance solution 2 into the liquid chromatograph for detection, the racemic ketoisoleucine calcium impurity reference substance solution 1 into 5 needles, and the racemic ketoisoleucine calcium impurity reference substance solution 2 into 2 needles, wherein the racemic ketoisoleucine calcium impurity reference substance solution 1 and the reference substance working solution 2 are both reference substance working solutions of the same concentration, and the injection repeatability of the liquid chromatograph machine is tested by the reference substance working solution 1, and the RSD of the main peak area in the 5-needle reference solution shall not exceed 2.0%;

[0036] (2) Dissolve the sample in a solvent to obtain a test solution, inject 20 μl of the test solution into a liquid chromatograph, record the chromatogram, ignore the peak in the blank solvent, calculate the single impurity of racemic ketoisoleucine calcium using the main component external standard method, and calculate the total amount of impurities.

[0037] The calculation formula for calculating the content of a single impurity in an amino acid solution is as follows

[0038]

[0039] in:

[0040] Cs: Concentration of a single impurity in the test solution (μg / ml)

[0041] C: Concentration of racemic ketoisoleucine calcium impurity reference solution (μg / ml)

[0042] As: Peak area of ​​a single impurity measured in the test solution

[0043] A: Average peak area of ​​racemic ketoisoleucine calcium impurity reference solution

[0044] The total amount of impurities can be obtained by adding up all the individual impurities measured.

[0045] Example 2

[0046] This embodiment provides a method for detecting racemic ketoisoleucine calcium impurities in compound α-ketoacid tablets, comprising the following steps:

[0047] (1) Dissolve the racemic ketoisoleucine calcium reference substance in a solvent to obtain a racemic ketoisoleucine calcium impurity reference substance solution, take the solvent as a blank solution, inject 20 μl of the blank solvent into a liquid chromatograph until the baseline is stable, then inject the racemic ketoisoleucine calcium impurity reference substance solution 1 and the racemic ketoisoleucine calcium impurity reference substance solution 2 into the liquid chromatograph for detection, inject 5 needles into the racemic ketoisoleucine calcium impurity reference substance solution 1, inject 2 needles into the racemic ketoisoleucine calcium impurity reference substance solution 2, and the RSD of the main peak area in the 5-needle reference solution shall not exceed 2.0%;

[0048] (2) Dissolve the sample in a solvent to obtain a test solution, inject 20 μl of the test solution into a liquid chromatograph, record the chromatogram, ignore the peak in the blank solvent, calculate the single impurity of racemic ketoisoleucine calcium using the main component external standard method, and calculate the total amount of impurities.

[0049] The mobile phase flow rate is 1.0 ml / min, the injection volume is 20 μl, the column temperature is 30°C, and the chromatographic column is C18-amide, 5 μm 150×4.6 mm. The mobile phase A and the mobile phase B are used for gradient elution, and the ultraviolet detector is used for detection, and the detection wavelength is 205 nm; the mobile phase A is potassium hexafluorophosphate-phosphoric acid aqueous solution (0.2 mol / L to 0.5 mol / L potassium hexafluorophosphate, 0.1% to 0.15% phosphoric acid), and the mobile phase B is acetonitrile, the potassium hexafluorophosphate and phosphoric acid are analytical grade, and the acetonitrile is chromatographic grade.

[0050] The gradient elution program of the mobile phase is as follows:

[0051] Time / min Mobile phase A / % Mobile phase B / % 0 95 5 5 95 5 15 70 30 30 70 30 31 95 5 38 95 5

[0052] The reference solution and test solution in the method are prepared as follows:

[0053] Preparation of racemic ketoisoleucine calcium impurity reference solution:

[0054] (1) Preparation of racemic ketoisoleucine calcium reference stock solution (0.5 mg / ml): weigh about 50 mg of racemic ketoisoleucine calcium reference into a 100 ml volumetric flask, add acetonitrile: 0.01 mol / L hydrochloric acid solution in a ratio of 3:7, sonicate for 10 min, dilute to scale, and shake well;

[0055] (2) Preparation of racemic ketoisoleucine calcium impurity reference solution (0.5 μg / ml): Accurately measure 1 ml of racemic ketoisoleucine calcium reference stock solution into a 100 ml volumetric flask, add acetonitrile:0.01 mol / L hydrochloric acid solution in a ratio of 3:7 to dilute to the mark; Accurately measure 5.0 ml into a 50 ml volumetric flask, add acetonitrile:0.01 mol / L hydrochloric acid solution in a ratio of 3:7 to dilute to the mark, and shake well.

[0056] Preparation of test solution:

[0057] Take 20 pieces of sample and grind them into fine powder. Take 0.5g, accurately determine, put into a 100 volumetric flask, add acetonitrile: 0.01mol / L hydrochloric acid solution = 3:7 appropriate amount, dilute to the scale after ultrasonication for 10min, shake well, filter with 0.45μm filter, and use as the test solution.

[0058] This method has high sensitivity and the limit of quantification (S / N ≥ 10) can reach 0.01 μg / ml. The typical chromatogram of the test solution of this method is Figure 1 .

[0059] Example 3 Method Mass Conservation Experiment

[0060] This embodiment provides a method for detecting racemic ketoisoleucine calcium impurities in compound α-ketoacid tablets, comprising the following steps:

[0061] (1) dissolving the racemic ketoisoleucine calcium reference substance in a solvent to obtain a racemic ketoisoleucine calcium impurity reference substance solution, using a solvent (acetonitrile: 0.01 mol / L hydrochloric acid solution = 3:7) as a blank solution, injecting 20 μl of the blank solvent into a liquid chromatograph until the baseline is stable, and then injecting the racemic ketoisoleucine calcium impurity reference substance solution 1 and the racemic ketoisoleucine calcium impurity reference substance solution 2 into the liquid chromatograph for detection, the racemic ketoisoleucine calcium impurity reference substance solution 1 into 5 needles, and the racemic ketoisoleucine calcium impurity reference substance solution 2 into 2 needles, wherein the racemic ketoisoleucine calcium impurity reference substance solution 1 and the reference substance working solution 2 are both reference substance working solutions of the same concentration, and the injection repeatability of the liquid chromatograph machine is tested by the reference substance working solution 1, and the RSD of the main peak area in the 5-needle reference solution shall not exceed 2.0%;

[0062] (2) Dissolve the sample in a solvent to obtain a test solution, inject 20 μl of the test solution into a liquid chromatograph, record the chromatogram, ignore the peak in the blank solvent, calculate the single impurity of racemic ketoisoleucine calcium using the main component external standard method, and calculate the total amount of impurities.

[0063] The mobile phase flow rate is 1.0 ml / min, the injection volume is 20 μl, the column temperature is 30°C, and the chromatographic column is C18-amide, 5 μm 150×4.6 mm. The mobile phase A and the mobile phase B are used for gradient elution, and the ultraviolet detector is used for detection, and the detection wavelength is 205 nm; the mobile phase A is potassium hexafluorophosphate-phosphoric acid aqueous solution (0.2 mol / L to 0.5 mol / L potassium hexafluorophosphate, 0.1% to 0.15% phosphoric acid), and the mobile phase B is acetonitrile, potassium hexafluorophosphate and phosphoric acid are analytical grade, and acetonitrile is chromatographic grade.

[0064] According to the prescription of compound α-ketoacid tablets, tablets containing no amino acid components except racemic ketoisoleucine calcium were prepared, as shown in Table 1. Considering the actual production conditions, the tablets were placed under different conditions for forced degradation. The degradation conditions and the corresponding racemic ketoisoleucine calcium content are shown in Table 2. The reference working solution was the same as the racemic ketoisoleucine calcium impurity reference solution of Example 2, and the racemic ketoisoleucine calcium forced degradation solution was used as the test solution.

[0065] Table 1 Prescription of racemic ketoisoleucine calcium tablets

[0066] Element Addition amount (g) Calcium racemic ketoisoleucinate 67 Corn starch 45 Crospovidone 10 Povidone K30 8 talcum powder 20 Colloidal Silica 3 Magnesium Stearate 16 talcum powder 12

[0067] Table 2 Forced degradation conditions of racemic ketoisoleucine calcium

[0068]

[0069]

[0070] The HPLC method was used to detect the experiments 1 to 5 and the mass conservation was calculated. The results are shown in Table 3:

[0071] Table 3 Mass conservation calculation table for different tests

[0072]

[0073] The sum of the content of racemic ketoisoleucine calcium and the impurities is between 99.0% and 101.0%, and the mass conservation is applicable to all tests. After testing, it is found that the mass conservation verification of the content of racemic ketoisoleucine calcium and the total impurity content is met.

[0074] Example 4 Testing of three typical racemic ketoisoleucine calcium impurities

[0075] The detection methods of three typical racemic ketoisoleucine calcium impurities are the same as those in Example 2.

[0076] Three typical impurities of racemic ketoisoleucine calcium are shown in Table 4 below:

[0077] Table 4 3 Typical Impurities of Racemic Ketoisoleucine Calcium

[0078]

[0079]

[0080] The detection method of the present invention can successfully detect and separate several specific impurities of racemic ketoisoleucine calcium. The peaks of racemic ketoisoleucine calcium impurities and reference substances in the spectrum of compound α-ketoacid tablets are shown in Figure 2. Figure 2 .

[0081] Embodiment 5:

[0082] This example determines the linear ranges of the five components of "2-butylene glycol, 2-methylbutyric acid, glycol, valeric acid, and calcium ketophenylalanine", and the results are shown in Tables 5 to 9.

[0083] Table 5 Hein linear test data

[0084]

[0085] Table 6 2-Methylbutyric acid linear test data

[0086]

[0087] Table 7 Valeric acid linear test data

[0088]

[0089] Table 8 Ketophenylalanine calcium linear test data

[0090]

[0091] Table 9 2-butylene glycol linear test data

[0092]

[0093] Embodiment 6:

[0094] This example verifies the quantitative limit of the method. Take the quantitative limit solution under the linear item, accurately measure 10 μl and inject it into the liquid chromatograph, inject it 6 times continuously, and record the chromatogram. Calculate its signal-to-noise ratio. If the signal-to-noise ratio S / N ≥ 10, it is the quantitative limit concentration. And calculate the relative standard deviation of each peak area.

[0095] Table 10 Limit of quantitation

[0096]

[0097]

[0098] Embodiment 7:

[0099] This example verifies the detection limit of the method. Take the quantitative limit concentration solution, dilute it about 3 times and inject it, record the chromatogram, calculate the signal-to-noise ratio, and the sample concentration with a signal-to-noise ratio S / N ≥ 3 is the detection limit concentration.

[0100] Table 11 Detection Limits

[0101]

[0102]

[0103] Embodiment 8:

[0104] In this example, six samples were measured in parallel according to the description of the above experimental method to verify the precision.

[0105] Table 12 Precision

[0106]

[0107] Embodiment 9:

[0108] This example verifies the stability of the sample dissolution solution. Place the sample solution at room temperature without light protection, and accurately measure 10μl for sample injection and detection at 0, 4, 8, 12, and 24 hours, and record the chromatogram; take out each impurity stock solution and place it in a refrigerator (2-8°C), take it out at different times, and after it reaches room temperature, accurately measure 10μl for sample injection and detection, and record the chromatogram. Investigate the stability of the solution.

[0109] Table 13 Impurity reference solution room temperature stability results

[0110]

[0111]

[0112] Table 14 Stability results of impurity reference solution in refrigerator (2~8℃)

[0113]

[0114] Embodiment 10:

[0115] This example verifies the accuracy of the HPLC method of the present invention through a recovery experiment. Three solutions of each component at 80%, 100%, and 120% and a blank control are selected, and the recovery rate of each component is determined according to the description of the above experimental method.

[0116] Table 152 - Methylbutyric acid recovery test data

[0117]

[0118]

[0119] Table 162-Test data of recovery rate of butylene glycol

[0120]

[0121] Table 17 Hydantoin recovery test data

[0122]

[0123] Table 18 Valeric acid recovery test data

[0124]

[0125] The invention discloses a detection method for determining racemic ketoisoleucine calcium related substances in compound α-keto acid tablets. The method adopts high performance liquid chromatography, sets certain chromatographic conditions, accurately takes a reference solution and a compound α-keto acid tablet sample solution and injects them into HPLC respectively, records the chromatogram, calculates the peak area of ​​a single impurity by an external standard method, and calculates the total amount of impurities by summing up. Moreover, the method can accurately detect some specific racemic ketoisoleucine calcium degradation impurities without being interfered by other components in the compound amino acid, and can meet the requirement that the separation degree between various impurities meets the applicability of the chromatographic system. The detection method of the invention has simple and convenient sample processing, accurate and reliable sample measurement results, high sensitivity, conventional reagents, and simple preparation of diluents and mobile phases.

[0126] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

[0127] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0128] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

[0129] The present invention and its implementation methods are described above, and such description is not restrictive. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design structures and embodiments similar to the technical solution without creativity, which should all fall within the protection scope of the present invention.

Claims

1. A method for detecting racemic ketoisoleucine calcium impurities in compound α-ketoacid tablets, characterized in that: The steps include: Step 1: dissolving the racemic ketoisoleucine calcium reference substance in a solvent to obtain a racemic ketoisoleucine calcium impurity reference substance solution, taking the solvent as a blank solution, injecting the blank solvent into a liquid chromatograph until the baseline is stable, and then injecting the racemic ketoisoleucine calcium impurity reference substance solution into the liquid chromatograph for detection; Step 2: dissolving the sample in a solvent to obtain a test solution, injecting the test solution into a liquid chromatograph, using mobile phase A and mobile phase B for gradient elution, recording the chromatogram, calculating the single impurity of racemic ketoisoleucine calcium by the main component external standard method, and calculating the total amount of impurities; Wherein, the mobile phase A is potassium hexafluorophosphate-phosphoric acid aqueous solution, the mobile phase B is acetonitrile, and the chromatographic column is C18-ami de, 5μm 150×4.6mm; The gradient elution process is as follows:

2. The method for detecting the impurity of racemic ketoisoleucine calcium in the compound α-ketoacid tablets according to claim 1, characterized in that: The flow rate of the mobile phase is 1.0-1.5 ml / min, the injection volume is 10-20 μl, the column temperature is 25° C.-35° C., and the detection is performed using an ultraviolet detector with a detection wavelength of 205 nm.

3. The method for detecting the impurity of racemic ketoisoleucine calcium in the compound α-ketoacid tablets according to claim 2, characterized in that: The concentration of the racemic ketoisoleucine calcium impurity reference solution is 0.3-0.6 μg / ml.

4. The method for detecting the impurity of racemic ketoisoleucine calcium in the compound α-ketoacid tablets according to claim 3, characterized in that: The racemic ketoisoleucine calcium impurities include 2-butylene hydantoin, 2-methylbutyric acid, hydantoin, and valeric acid.

5. The method for detecting the impurity of racemic ketoisoleucine calcium in the compound α-ketoacid tablets according to claim 4, characterized in that: The potassium hexafluorophosphate-phosphoric acid aqueous solution contains 0.2 mol / L to 0.5 mol / L potassium hexafluorophosphate and 0.1% to 0.15% phosphoric acid.

6. The method for detecting the impurity of racemic ketoisoleucine calcium in the compound α-ketoacid tablets according to claim 5, characterized in that: The preparation steps of the racemic ketoisoleucine calcium impurity reference solution are: Step 1: Preparation of racemic ketoisoleucine calcium reference stock solution (0.5 mg / ml): weigh about 50 mg of racemic ketoisoleucine calcium reference into a 100 ml volumetric flask, add acetonitrile: 0.01 mol / L hydrochloric acid solution = 3:7, sonicate for 10 min, dilute to scale, and shake well; Step 2: Preparation of racemic ketoisoleucine calcium impurity reference solution (0.5 μg / ml): Accurately measure 1 ml of racemic ketoisoleucine calcium reference stock solution to a 100 ml volumetric flask, add acetonitrile: 0.01 mol / L hydrochloric acid solution = 3:7 to dilute to the mark; Accurately measure 5.0 ml to a 50 ml volumetric flask, add acetonitrile: 0.01 mol / L hydrochloric acid solution = 3:7 to dilute to the mark, and shake well; The preparation steps of the test solution are: Grind 20 pieces of sample into fine powder, take 0.5g, accurately quantify, put into a 100 volumetric flask, add acetonitrile: 0.01mol / L hydrochloric acid solution = 3:7 appropriate amount, ultrasonicate for 10min, dilute to scale, shake well, filter with 0.45μm filter, and use as the test solution; The calculation formula for calculating the content of a single impurity in compound α-keto acid tablets is as follows: in: Cs: concentration of a single impurity in the test solution; C: concentration of racemic ketoisoleucine calcium impurity reference solution; As: peak area of ​​a single impurity measured in the test solution; A: Average peak area of ​​racemic ketoisoleucine calcium impurity reference solution; The total amount of impurities can be obtained by adding up all the individual impurities measured.

Images