A method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets

Abstract

The present application provides a method for detecting hydroxymethionine sulfoxide in compound α-keto acid tablets, and belongs to the technical field of impurity detection in compound α-keto acid tablets. The detection method of the present application selects a solvent with a specific pH value to dissolve the test solution and the system suitability solution, and further limits the mass volume concentration of the 5 keto (or hydroxy) amino acid calcium raw materials in the system suitability solution to 75%~120% of the mass volume concentration in the test solution, so that the two pairs of enantiomers of hydroxymethionine sulfoxide can be separated, and the peak of hydroxymethionine sulfoxide in the system suitability solution and the test solution can be kept consistent, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and calculating according to the self-control method through the chromatogram of the test solution and the chromatogram of the control solution, the detection method of the present application can effectively and accurately quantify the hydroxymethionine sulfoxide impurities in the compound α-keto acid tablets.

Description

Technical Field

[0001] The present application relates to the technical field of impurity testing in compound α-keto acid tablets, and in particular to a method for detecting hydroxymethionine sulfoxide in compound α-keto acid tablets. Background Art

[0002] Compound α-keto acid tablets are a drug with significant efficacy in the treatment of chronic kidney disease. They are used to prevent and treat damage caused by protein metabolism disorders due to chronic renal insufficiency. The drug is designed with essential amino acid active ingredients combined with α-keto or α-hydroxy acids of essential amino acids with carbon chain structures. The five essential amino acids are: L-acetate lysine, L-threonine, L-tryptophan, L-histidine and L-tyrosine; α-keto or α-hydroxy acids are 4 keto amino acid calciums and 1 hydroxy amino acid calcium, namely: racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, ketovaline calcium, racemic hydroxymethionine calcium.

[0003] In the prior art, it is known that hydroxymethionine sulfoxide impurities exist in compound α-ketoacid tablets, but when the hydroxymethionine sulfoxide impurities in the compound α-ketoacid tablets are detected by the existing liquid chromatography method, the peak position of the hydroxymethionine sulfoxide impurity cannot be accurately determined, and the peak cannot be accurately detected. Summary of the invention

[0004] The present application provides a method for detecting hydroxymethionine sulfoxide in compound α-keto acid tablets, which can accurately detect the hydroxymethionine sulfoxide impurity peak, thereby effectively and accurately quantifying the hydroxymethionine sulfoxide impurity in the compound α-keto acid tablets.

[0005] The embodiment of the present application is implemented as follows:

[0006] In a first aspect, the present application provides an example of a method for detecting hydroxymethionine sulfoxide in a compound α-keto acid tablet, which comprises: first preparing a system suitability solution, and injecting the system suitability solution into a liquid chromatograph for testing, wherein the separation degree between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram is ≥1.5, and then preparing a test solution and a control solution respectively, and injecting the test solution and the control solution into a liquid chromatograph for testing respectively, comparing the chromatogram of the system suitability solution with the chromatogram of the test solution to determine the peak position of hydroxymethionine sulfoxide, and determining the peak position of hydroxymethionine sulfoxide according to the chromatogram of the test solution and the chromatogram of the control solution. The chromatogram is calculated according to the self-control method; wherein, the test solution includes compound α-ketoacid tablets and a solvent, the control solution is prepared by diluting the test solution with a solvent, the system suitability solution includes hydroxymethionine sulfoxide, keto valine calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium and a solvent, and the mass volume concentration of keto valine calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium and ketophenylalanine calcium in the system suitability solution is 75%~120% of its mass volume concentration in the test solution; the pH value of the solvent is 2.4~2.6.

[0007] In the above technical scheme, the detection method of the present application can not only separate the two pairs of enantiomers of hydroxymethionine sulfoxide from the peaks by selecting a solvent with a pH value of 2.4 to 2.6 to dissolve the test solution and the system suitability solution, but also can make the separation degree between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram of the system suitability solution meet the requirements, and by further limiting the mass volume concentration of keto valine calcium, racemic hydroxymethionine calcium, racemic keto isoleucine calcium, keto leucine calcium and keto phenylalanine calcium in the system suitability solution to 75% to 120% of their mass volume concentration in the test solution, the peak of hydroxymethionine sulfoxide in the system suitability solution and the test solution can be kept consistent, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and calculating according to the self-control method through the chromatogram of the test solution and the chromatogram of the control solution, the detection method of the present application can effectively and accurately quantify the hydroxymethionine sulfoxide impurity in the compound α-keto acid tablets.

[0008] In some possible embodiments, the concentration of racemic hydroxymethionine calcium in the test solution is 0.75 mg / mL to 1.2 mg / mL.

[0009] In some possible embodiments, the concentrations of calcium ketovaline, calcium racemic hydroxymethionine, calcium racemic ketoisoleucine, calcium ketoleucine, and calcium ketophenylalanine in the system suitability solution are 1.1 mg / mL to 1.8 mg / mL, 0.75 mg / mL to 1.2 mg / mL, 0.83 mg / mL to 1.3 mg / mL, 1.3 mg / mL to 2.0 mg / mL, and 0.9 mg / mL to 1.4 mg / mL, respectively.

[0010] In the above technical scheme, on the basis of satisfying the concentration of racemic hydroxymethionine calcium in the test solution of 0.75 mg / mL to 1.2 mg / mL, by making the concentrations of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, and ketophenylalanine calcium in the system suitability solution within the above range, it is beneficial to keep the elution conditions of hydroxymethionine sulfoxide in the system suitability solution consistent with that in the test solution, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and making the separation degree between hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram of the system suitability solution meet the requirements.

[0011] In some possible embodiments, the system suitability solution further comprises tyrosine and tryptophan, and the concentrations of tyrosine and tryptophan in the system suitability solution are 0.38 mg / mL to 0.6 mg / mL and 0.3 mg / mL to 0.48 mg / mL, respectively.

[0012] In some possible implementation schemes, the control solution is a 500-1000-fold dilution of the test solution.

[0013] In some possible embodiments, the solvent is prepared by the following method:

[0014] Prepare a 0.05 mol / L sodium dihydrogen phosphate solution, and adjust its pH value to 2.4-2.6 with phosphoric acid.

[0015] In some possible implementation schemes, the detection temperature is 20°C to 30°C.

[0016] In the above technical solution, by making the detection temperature within the above range, it is beneficial to keep the peak conditions of hydroxymethionine sulfoxide in the system suitability solution and the test solution consistent, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide.

[0017] In some possible embodiments, the chromatographic column of the liquid chromatograph is an organic acid column, and the organic acid column includes Prevail organic acid 4.6mm×250mm, 5µm.

[0018] In some possible embodiments, a mobile phase is used for gradient elution in a liquid chromatography test, the mobile phase includes a mobile phase A and a mobile phase B, the mobile phase A is a mixture of a sodium dihydrogen phosphate solution with a pH value of 2.4 to 2.6 and acetonitrile, the mobile phase B includes acetonitrile, the flow rate of the mobile phase is 0.9 mL / min to 1.1 mL / min, the injection volume is 15 μL to 25 μL, the column temperature is 23° C. to 27° C., the detection wavelength is 205 nm, and the gradient elution method is as follows:

[0019] At 0 min, the volume of mobile phase A accounts for 100% of the volume of the mobile phase;

[0020] At 10 min, the volume of mobile phase A accounts for 100% of the volume of mobile phase;

[0021] At 10 min to 27 min, the volume of mobile phase A accounts for 100% to 88% of the volume of the mobile phase, and the volume of mobile phase B accounts for 0% to 12% of the volume of the mobile phase;

[0022] At 27-35 min, the volume of mobile phase A accounts for 88%-100% of the volume of the mobile phase, and the volume of mobile phase B accounts for 12%-0% of the volume of the mobile phase;

[0023] At 35min~40min, the volume of mobile phase A accounts for 100% of the volume of mobile phase.

[0024] In some possible implementation schemes, the content of hydroxymethionine sulfoxide in the test solution is calculated as follows:

[0025] C = E*F*G / H;

[0026] Wherein E is the peak area of ​​hydroxymethionine sulfoxide in the test solution, F is the correction factor, the correction factor is the slope ratio of racemic hydroxymethionine calcium to hydroxymethionine sulfoxide, G is the reciprocal of the dilution multiple of the control solution, and H is the peak area of ​​racemic hydroxymethionine calcium in the control solution.

[0027] In the above technical scheme, other ingredients in the compound α-ketoacid tablets, such as calcium ketovaline, racemic calcium hydroxymethionine, racemic calcium ketoisoleucine, calcium ketoleucine and calcium ketophenylalanine, will affect the peak shape of the hydroxymethionine sulfoxide peak, that is, it is impossible to quantify it according to the external standard method. The present application is calculated according to the chromatogram of the test solution and the chromatogram of the control solution according to the self-control method.

[0028] This application has at least the following beneficial effects:

[0029] In order to solve the defects and drawbacks of the existing analytical detection methods of hydroxymethionine sulfoxide impurities in compound α-keto acid tablets and related preparations, and to achieve the purpose of accurate and effective quantitative detection of hydroxymethionine sulfoxide in compound α-keto acid tablets, the present application provides a detection method for hydroxymethionine sulfoxide in compound α-keto acid tablets, which can separate the two pairs of enantiomers of hydroxymethionine sulfoxide through liquid chromatography, and can also make the separation between hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram meet the requirements, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and calculating according to the self-control method through the chromatogram of the test solution and the chromatogram of the control solution. The detection method of the present application can effectively and accurately quantify the hydroxymethionine sulfoxide impurities in the compound α-keto acid tablets, provide good quality assurance for the compound α-keto acid tablets, and allow the general public to use the medicine with confidence. Moreover, the detection method of the present application can effectively determine the hydroxymethionine sulfoxide impurity in the compound α-keto acid tablets on the liquid phase instrument commonly used in current pharmaceutical production factories, avoiding the use of more expensive ultra-high pressure liquid chromatography that is not commonly used in factories, reducing the detection cost, and making the detection more routine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0031] Figure 1 is a chromatogram of the system suitability solution of Example 1 of the present application;

[0032] Figure 2 The chromatogram of the test solution of Example 1 of the present application;

[0033] Figure 3 is a chromatogram of the control solution of Example 1 of the present application;

[0034] Figure 4 is a chromatogram of the system suitability solution of Example 2 of the present application;

[0035] Figure 5 is a chromatogram of the system suitability solution of Example 3 of the present application;

[0036] Figure 6 is a chromatogram of the system suitability solution of Example 4 of the present application;

[0037] Figure 7 The chromatogram of the hydroxymethionine sulfoxide reference solution of Comparative Example 1 of the present application;

[0038] Figure 8 The chromatogram of the test solution of Comparative Example 1 of the present application;

[0039] Fig. 9 The chromatogram of the hydroxymethionine sulfoxide reference substance solution of Comparative Example 2 of the present application;

[0040] Fig.10 The chromatogram of the test solution of Comparative Example 2 of the present application;

[0041] Fig.11 This is a chromatogram of the system suitability solution of Comparative Example 3 of the present application;

[0042] Fig.12 This is a chromatogram of the system suitability solution of Comparative Example 4 of the present application;

[0043] Fig.13 This is a chromatogram of the system suitability solution of Comparative Example 5 of the present application. DETAILED DESCRIPTION

[0044] The embodiments of the present application will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. In the examples, if specific conditions are not specified, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0045] The structural formula of hydroxymethionine sulfoxide is as follows:

[0046] ;

[0047] Hydroxymethionine sulfoxide is a chiral compound with two chiral centers in the molecule. There are four isomers, which are two pairs of enantiomers. The two pairs of enantiomers are diastereoisomers of each other. Therefore, the performance in the non-chiral chromatographic column is more complicated. There should be two peaks corresponding to one pair of enantiomers.

[0048] When liquid chromatography is used to detect hydroxymethionine sulfoxide impurities in compound α-ketoacid tablets, it is easily affected by various factors, resulting in the inability to accurately determine the peak position of the hydroxymethionine sulfoxide impurity and the inability to accurately identify the peak.

[0049] Based on this, the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0050] S1. Prepare solvent

[0051] A 0.05 mol / L sodium dihydrogen phosphate solution was adjusted to a pH value of 2.4-2.6 using phosphoric acid to prepare a solvent.

[0052] As an example, the pH of the solvent may be 2.40, 2.41, 2.42, 2.43, 2.44, 2.45, 2.46, 2.47, 2.48, 2.49, 2.50, 2.51, 2.52, 2.53, 2.54, 2.55, 2.56, 2.57, 2.58, 2.59, or 2.60.

[0053] Optionally, the pH value of the solvent is 2.45-2.55.

[0054] Optionally, the pH value of the solvent is 2.5.

[0055] 0.05 mol / L sodium dihydrogen phosphate solution can be prepared by the following method:

[0056] Sodium dihydrogen phosphate was dissolved in deionized water, and the mass volume ratio of sodium dihydrogen phosphate to deionized water was 0.0078 g / mL.

[0057] S2. Prepare the test solution, control solution, sensitivity solution and system suitability solution respectively.

[0058] The compound α-keto acid tablet powder is weighed and dissolved in the prepared solvent and quantitatively diluted to prepare a test solution, wherein the concentration of racemic hydroxymethionine calcium in the test solution is 0.75 mg / mL to 1.2 mg / mL.

[0059] As an example, the concentration of racemic calcium hydroxymethionine in the test solution may be 1 mg / mL.

[0060] It should be noted that the test solution has poor stability and needs to be freshly prepared before use.

[0061] The prepared test solution was measured and diluted with the prepared solvent to prepare a control solution, and the control solution was diluted 500~1000 times of the test solution.

[0062] As an example, the control solution can be a 500-fold dilution of the test solution.

[0063] The prepared control solution is measured and diluted with the prepared solvent to obtain a sensitivity solution, and the sensitivity solution is diluted 3 to 5 times of the control solution.

[0064] As an example, the sensitivity solution is a 4-fold dilution of the control solution.

[0065] Weigh hydroxymethionine sulfoxide, keto-valine calcium, racemic hydroxymethionine calcium, racemic keto-isoleucine calcium, keto-leucine calcium, keto-phenylalanine calcium, tyrosine and tryptophan respectively and dissolve them in the prepared solvent so that the concentration of hydroxymethionine sulfoxide is the maximum allowable value and the mass volume concentration of keto-valine calcium, racemic hydroxymethionine calcium, racemic keto-isoleucine calcium, keto-leucine calcium, keto-phenylalanine calcium, tyrosine and tryptophan in the system suitability solution is 75% to 120% of their mass volume concentration in the test solution.

[0066] As an example, the mass volume concentration of calcium ketovaline, racemic calcium hydroxymethionine, racemic calcium ketoisoleucine, calcium ketoleucine, calcium ketophenylalanine, tyrosine and tryptophan in the system suitability solution can be 100% of their mass volume concentration in the test solution.

[0067] Optionally, the concentrations of calcium ketovaline, calcium racemic hydroxymethionine, calcium racemic ketoisoleucine, calcium ketoleucine, calcium ketophenylalanine, tyrosine and tryptophan in the system suitability solution are 1.1 mg / mL to 1.8 mg / mL, 0.75 mg / mL to 1.2 mg / mL, 0.83 mg / mL to 1.3 mg / mL, 1.3 mg / mL to 2.0 mg / mL, 0.9 mg / mL to 1.4 mg / mL, 0.38 mg / mL to 0.6 mg / mL and 0.3 mg / mL to 0.48 mg / mL, respectively.

[0068] As an example, the concentration of calcium ketovaline in the system suitability solution may be 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, or 1.8 mg / mL; the concentration of calcium racemic hydroxymethionine in the system suitability solution may be 0.75 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, or 1.2 mg / mL; the concentration of calcium racemic ketoisoleucine in the system suitability solution may be 0.83 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, or 1.3 mg / mL; the concentration of calcium ketoleucine in the system suitability solution may be The concentration of calcium ketophenylalanine in the system suitability solution may be 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2.0 mg / mL; the concentration of calcium ketophenylalanine in the system suitability solution may be 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL or 1.4 mg / mL; the concentration of tyrosine in the system suitability solution may be 0.38 mg / mL, 0.4 mg / mL, 0.5 mg / mL or 0.6 mg / mL; the concentration of tryptophan in the system suitability solution may be 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL or 0.48 mg / mL.

[0069] On the basis of satisfying that the concentration of racemic hydroxymethionine calcium in the test solution is 0.75 mg / mL~1.2 mg / mL, by making the concentrations of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan in the system suitability solution within the above range, it is beneficial to maintain the consistency of the elution conditions of hydroxymethionine sulfoxide in the system suitability solution and the test solution, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and ensuring that the separation degree between hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram of the system suitability solution meets the requirements.

[0070] S3. Inject the prepared test solution, control solution, sensitivity solution and system suitability solution into the liquid chromatograph for testing.

[0071] Inject the prepared system suitability solution into the liquid chromatograph for testing and record the chromatogram until the separation degree between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram is ≥1.5.

[0072] The prepared sensitivity solution was injected into a liquid chromatograph for testing, and the chromatogram was recorded. The signal-to-noise ratio of the peak height of racemic hydroxymethionine calcium in the chromatogram was ≥10.

[0073] Inject the prepared test solution into the liquid chromatograph for testing, record the chromatogram, and compare the chromatogram of the system suitability solution with the chromatogram of the test solution to determine the peak position of hydroxymethionine sulfoxide.

[0074] Inject the prepared control solution into the liquid chromatograph for testing and record the chromatogram.

[0075] The conditions for liquid chromatography are as follows:

[0076] The test temperature is 20℃~30℃.

[0077] As an example, the test temperature may be 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C.

[0078] By keeping the detection temperature within the above range, it is helpful to keep the elution conditions of hydroxymethionine sulfoxide in the system suitability solution and the test solution consistent, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide.

[0079] Chromatographic conditions: The chromatographic column was an organic acid column, a mixture of sodium dihydrogen phosphate solution with a pH value of 2.4 to 2.6 and acetonitrile was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the steps in Table 1. The flow rate of the mobile phase was 0.9 mL / min to 1.1 mL / min, the injection volume was 15 μL to 25 μL, the column temperature was 23°C to 27°C, and the detection wavelength was 205 nm.

[0080] Optionally, the chromatographic column is a Prevail organic acid 4.6mm×250mm, 5µm or a chromatographic column of equivalent performance.

[0081] Optionally, the volume ratio of sodium dihydrogen phosphate solution to acetonitrile in mobile phase A is 948:52-955:45, and the sodium dihydrogen phosphate solution is prepared by the following method:

[0082] A sodium dihydrogen phosphate solution with a concentration of 0.045 mol / L to 0.055 mol / L is prepared, and its pH value is adjusted to 2.4 to 2.6 with phosphoric acid.

[0083] As an example, the flow rate of the mobile phase can be 0.9mL / min, 0.92mL / min, 0.95mL / min, 0.98mL / min, 1mL / min, 1.02mL / min, 1.05mL / min, 1.08mL / min or 1.1mL / min; the injection volume can be 15μL, 18μL, 20μL, 22μL or 25μL; the column temperature can be 23°C, 24°C, 25°C, 26°C or 27°C.

[0084] Table 1 Gradient elution method

[0085]

[0086] S4. Calculation

[0087] Determine the detection limit and the quantification limit, determine the linear relationship, and calculate the correction factor, which is the slope ratio of racemic hydroxymethionine calcium and hydroxymethionine sulfoxide, determine the precision and accuracy, and determine the range of the hydroxymethionine sulfoxide content in combination with the precision, accuracy and linear relationship, and finally calculate the content of hydroxymethionine sulfoxide in the test solution. The calculation formula is as follows;

[0088] C = E*F*G / H;

[0089] Wherein E is the peak area of ​​hydroxymethionine sulfoxide in the test solution, F is the correction factor, the correction factor is the slope ratio of racemic hydroxymethionine calcium to hydroxymethionine sulfoxide, G is the reciprocal of the dilution multiple of the control solution, and H is the peak area of ​​racemic hydroxymethionine calcium in the control solution.

[0090] Since the peak shape of the reference solution of hydroxymethionine sulfoxide is different from that of the compound α-ketoacid tablets, the concentrations of other ingredients in the compound α-ketoacid tablets, such as calcium ketovaline, racemic calcium hydroxymethionine, racemic calcium ketoisoleucine, calcium ketoleucine, calcium ketophenylalanine, tyrosine and tryptophan, will affect the peak shape of the hydroxymethionine sulfoxide peak, and therefore it is impossible to quantify according to the external standard method. Therefore, the present application is calculated according to the chromatogram of the test solution and the chromatogram of the reference solution according to the self-control method.

[0091] The detection method of the present application selects a solvent with a pH value of 2.4 to 2.6 to dissolve the test solution and the system suitability solution, which can not only separate the two pairs of enantiomers of hydroxymethionine sulfoxide, but also make the separation degree between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram of the system suitability solution meet the requirements, and by further limiting the mass volume concentration of keto valine calcium, racemic hydroxymethionine calcium, racemic keto isoleucine calcium, keto leucine calcium, keto phenylalanine calcium, tyrosine and tryptophan in the system suitability solution to 75% to 120% of their mass volume concentration in the test solution, the peak of hydroxymethionine sulfoxide in the system suitability solution and the test solution can be kept consistent, thereby determining the two peak positions of the two pairs of enantiomers of hydroxymethionine sulfoxide, and calculating according to the self-control method through the chromatogram of the test solution and the chromatogram of the control solution, the detection method of the present application can effectively and accurately quantify the hydroxymethionine sulfoxide impurity in the compound α-keto acid tablets.

[0092] The following is a further detailed description of the method for detecting hydroxymethionine sulfoxide in a compound α-keto acid tablet of the present application in conjunction with the examples.

[0093] Example 1

[0094] The present application embodiment provides a method for detecting hydroxymethionine sulfoxide in a compound α-keto acid tablet, which is determined according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition Part IV General Rules 0512), comprising the following steps:

[0095] S1. Prepare solvent

[0096] 7.8 g of sodium dihydrogen phosphate was taken and dissolved in 1000 mL of deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. The pH value of the sodium dihydrogen phosphate solution was adjusted to 2.5 with phosphoric acid to prepare a solvent.

[0097] S2. Prepare the test solution, control solution, sensitivity solution and system suitability solution respectively.

[0098] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared solvent and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0099] Take an appropriate amount of the test solution and quantitatively dilute it with the prepared solvent to make a solution containing about 2 µg of racemic hydroxymethionine calcium per 1 ml to prepare a control solution.

[0100] Take an appropriate amount of the control solution and quantitatively dilute it with the prepared solvent to make a solution containing about 0.5 µg of racemic hydroxymethionine calcium per 1 ml to prepare a sensitivity solution.

[0101] Weigh 20 mg of hydroxymethionine sulfoxide reference substance and place it in a 20 ml volumetric flask, add the prepared solvent to dissolve and dilute to the scale, shake well to obtain the hydroxymethionine sulfoxide stock solution; weigh appropriate amounts of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan reference substances, and transfer appropriate amounts of hydroxymethionine sulfoxide stock solution, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare system suitability solutions containing 5 µg of hydroxymethionine sulfoxide, 1.5 mg of ketovaline calcium, 1 mg of racemic hydroxymethionine calcium, 1.1 mg of racemic ketoisoleucine calcium, 1.7 mg of ketoleucine calcium, 1.2 mg of ketophenylalanine calcium, 0.4 mg of tyrosine and 0.5 mg of tryptophan per 1 ml.

[0102] S3. Inject the prepared test solution, control solution, sensitivity solution and system suitability solution into the liquid chromatograph for testing.

[0103] Inject the prepared system suitability solution into the liquid chromatograph for testing and record the chromatogram until the separation degree between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram is ≥1.5.

[0104] The prepared sensitivity solution was injected into a liquid chromatograph for testing, and the chromatogram was recorded. The signal-to-noise ratio of the peak height of racemic hydroxymethionine calcium in the chromatogram was ≥10.

[0105] Inject the prepared test solution into the liquid chromatograph for testing, record the chromatogram, and compare the chromatogram of the system suitability solution and the chromatogram of the test solution to determine the peak position of hydroxymethionine sulfoxide. The peak elution order of the system suitability solution is hydroxymethionine sulfoxide (double peak), tyrosine, ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, tryptophan, and ketophenylalanine calcium.

[0106] Inject the prepared control solution into the liquid chromatograph for testing and record the chromatogram.

[0107] The conditions for liquid chromatography are as follows:

[0108] The test temperature is 25℃.

[0109] Chromatographic conditions: The chromatographic column was Prevail organic acid 4.6mm×250mm, 5µm, a mixture of acetonitrile and 0.05mol / L sodium dihydrogen phosphate solution was used as mobile phase A, acetonitrile was used as mobile phase B, and gradient elution was performed according to the steps in Table 1. The flow rate of the mobile phase was 1mL / min, the injection volume was 20μL, the column temperature was 25℃, and the detection wavelength was 205nm.

[0110] Among them, mobile phase A was prepared by the following method: 7.8 g of sodium dihydrogen phosphate was taken, and 1000 mL of deionized water was added to dissolve it to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L, and the pH value of the sodium dihydrogen phosphate solution was adjusted to 2.5 with phosphoric acid, and then the sodium dihydrogen phosphate solution with adjusted pH value was mixed with acetonitrile at a volume ratio of 950:50.

[0111] S4. Calculation

[0112] The content of hydroxymethionine sulfoxide in the test solution was calculated using the following formula:

[0113] C = E*F*G / H;

[0114] Wherein E is the peak area of ​​hydroxymethionine sulfoxide in the test solution, F is the correction factor, the correction factor is the slope ratio of racemic hydroxymethionine calcium to hydroxymethionine sulfoxide, G is the reciprocal of the dilution multiple of the control solution, and H is the peak area of ​​racemic hydroxymethionine calcium in the control solution.

[0115] The chromatograms of the system suitability solution, the test solution, and the reference solution are shown in Figure 1. Figures 1 to 3 shown.

[0116] Example 2

[0117] The embodiment of the present application provides a method for detecting hydroxymethionine sulfoxide in a compound α-ketoacid tablet. Example 2, based on Example 1, only changes the concentrations of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan in the system suitability solution, so that each 1 ml of the system suitability solution contains 5 μg of hydroxymethionine sulfoxide, 1.1 mg of ketovaline calcium, 0.75 mg of racemic hydroxymethionine calcium, 0.83 mg of racemic ketoisoleucine calcium, 1.3 mg of ketoleucine calcium, 0.9 mg of ketophenylalanine calcium, 0.38 mg of tyrosine and 0.3 mg of tryptophan, respectively, and the rest remain unchanged.

[0118] The chromatogram of the system suitability solution is as follows: Figure 4 shown.

[0119] Example 3

[0120] The present application embodiment provides a method for detecting hydroxymethionine sulfoxide in a compound α-ketoacid tablet. Example 3, based on Example 1, only changes the concentrations of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan in the system suitability solution, so that each 1 ml of the system suitability solution contains 5 μg of hydroxymethionine sulfoxide, 1.8 mg of ketovaline calcium, 1.2 mg of racemic hydroxymethionine calcium, 1.3 mg of racemic ketoisoleucine calcium, 2.0 mg of ketoleucine calcium, 1.4 mg of ketophenylalanine calcium, 0.6 mg of tyrosine and 0.48 mg of tryptophan, respectively, and the rest remain unchanged.

[0121] Chromatography of System Suitability Solutions Figure 5 shown.

[0122] Example 4

[0123] The present application provides a method for detecting hydroxymethionine sulfoxide in a compound α-keto acid tablet. In Example 3, based on Example 1, only the dilution multiple of the control solution is changed, an appropriate amount of the test solution is measured, and the prepared solvent is used to quantitatively dilute the solution to prepare a solution containing approximately 1 μg of racemic hydroxymethionine calcium per 1 ml to prepare a control solution.

[0124] The chromatogram of the system suitability solution is as follows: Figure 6 shown.

[0125] Comparative Example 1

[0126] The comparative example of the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0127] S1. Prepare mobile phase A

[0128] Take 7.8 g of sodium dihydrogen phosphate, add 1000 mL of deionized water to dissolve, and prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. Use phosphoric acid to adjust the pH value of the sodium dihydrogen phosphate solution to 2.5, and then mix the sodium dihydrogen phosphate solution with adjusted pH value with acetonitrile in a volume ratio of 950:50.

[0129] S2. Prepare the test solution and hydroxymethionine sulfoxide reference solution respectively.

[0130] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared mobile phase A and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0131] Weigh 20 mg of hydroxymethionine sulfoxide reference substance, dissolve and dilute it in mobile phase A to prepare a solution containing about 5 μg of hydroxymethionine sulfoxide per ml.

[0132] S3. Inject the prepared hydroxymethionine sulfoxide reference solution and the test solution into the liquid chromatograph for testing.

[0133] The test was carried out according to the method of Example 1. The chromatograms of the hydroxymethionine sulfoxide reference solution and the test solution were as follows: Figure 7-8 shown.

[0134] Depend on Figure 7 and Figure 8 By comparison, it can be seen that the chromatogram of the test solution does not have a peak shape corresponding to the chromatogram of the hydroxymethionine sulfoxide reference solution, and the peak suspected to be hydroxymethionine sulfoxide in the chromatogram of the hydroxymethionine sulfoxide reference solution is a single peak. Therefore, using mobile phase A as the solvent, the peak position of hydroxymethionine sulfoxide in the chromatogram of the test solution cannot be determined, and hydroxymethionine sulfoxide cannot be detected.

[0135] Comparative Example 2

[0136] The comparative example of the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0137] S1. Prepare solvent

[0138] 7.8 g of sodium dihydrogen phosphate was taken and dissolved in 1000 mL of deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. The pH value of the sodium dihydrogen phosphate solution was adjusted to 2.5 with phosphoric acid to prepare a solvent.

[0139] S2. Prepare the test solution and hydroxymethionine sulfoxide reference solution respectively.

[0140] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared mobile phase A and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0141] Weigh 20 mg of the hydroxymethionine sulfoxide reference substance, dissolve and dilute it with the above solvent to prepare a solution containing about 5 μg of hydroxymethionine sulfoxide per ml.

[0142] S3. Inject the prepared hydroxymethionine sulfoxide reference solution and the test solution into the liquid chromatograph for testing.

[0143] The test was carried out according to the method of Example 1. The chromatograms of the hydroxymethionine sulfoxide reference solution and the test solution were as follows: Figures 9-10 shown.

[0144] Depend on Fig. 9 and Fig.10By comparison, it can be seen that the chromatogram of the test solution does not have a peak shape corresponding to the chromatogram of the hydroxymethionine sulfoxide reference solution. Although the peak suspected of hydroxymethionine sulfoxide in the chromatogram of the hydroxymethionine sulfoxide reference solution is a double peak, it is small at first and then large, while the double peak suspected of hydroxymethionine sulfoxide in the chromatogram of the test solution is large at first and then small, and the peak shapes are inconsistent. Therefore, it is impossible to quantify hydroxymethionine sulfoxide according to the external standard method.

[0145] Comparative Example 3

[0146] The comparative example of the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0147] S1. Prepare solvent

[0148] 7.8 g of sodium dihydrogen phosphate was taken and dissolved in 1000 mL of deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. The pH value of the sodium dihydrogen phosphate solution was adjusted to 2.5 with phosphoric acid to prepare a solvent.

[0149] S2. Prepare the test solution and system suitability solution separately

[0150] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared solvent and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0151] Weigh 20 mg of hydroxymethionine sulfoxide reference substance and place it in a 20 ml volumetric flask, add the prepared solvent to dissolve and dilute to the scale, shake well to obtain the hydroxymethionine sulfoxide stock solution; weigh appropriate amounts of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan reference substances, and transfer appropriate amounts of hydroxymethionine sulfoxide stock solution, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare system suitability solutions containing 5 µg of hydroxymethionine sulfoxide, 0.75 mg of ketovaline calcium, 0.5 mg of racemic hydroxymethionine calcium, 0.55 mg of racemic ketoisoleucine calcium, 0.85 mg of ketoleucine calcium, 0.6 mg of ketophenylalanine calcium, 0.25 mg of tyrosine and 0.2 mg of tryptophan per 1 ml.

[0152] S3. Inject the prepared test solution and system suitability solution into the liquid chromatograph for testing.

[0153] The test was performed according to the method of Example 1. The chromatogram of the system suitability solution was as follows: Fig.11 shown.

[0154] Depend on Fig.10 and Fig.11By comparison, the chromatogram of the test solution does not have a peak shape corresponding to the chromatogram of the system suitability solution, indicating that other components in the system suitability solution, such as calcium ketovaline, calcium racemic hydroxymethionine, calcium racemic ketoisoleucine, calcium ketoleucine, calcium ketophenylalanine, tyrosine and tryptophan, will affect the peak shape of the hydroxymethionine sulfoxide peak, so the quantification of hydroxymethionine sulfoxide cannot be performed.

[0155] Comparative Example 4

[0156] The comparative example of the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0157] S1. Prepare solvent

[0158] 7.8 g of sodium dihydrogen phosphate was taken and dissolved in 1000 mL of deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. The pH value of the sodium dihydrogen phosphate solution was adjusted to 2.3 with phosphoric acid to prepare a solvent.

[0159] S2. Prepare the test solution and system suitability solution separately

[0160] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared solvent and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0161] Weigh 20 mg of hydroxymethionine sulfoxide reference substance and place it in a 20 ml volumetric flask, add the prepared solvent to dissolve and dilute to the scale, shake well to obtain the hydroxymethionine sulfoxide stock solution; weigh appropriate amounts of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan reference substances, and transfer appropriate amounts of hydroxymethionine sulfoxide stock solution, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare system suitability solutions containing 5 µg of hydroxymethionine sulfoxide, 1.5 mg of ketovaline calcium, 1 mg of racemic hydroxymethionine calcium, 1.1 mg of racemic ketoisoleucine calcium, 1.7 mg of ketoleucine calcium, 1.2 mg of ketophenylalanine calcium, 0.4 mg of tyrosine and 0.5 mg of tryptophan per 1 ml.

[0162] S3. Inject the prepared test solution and system suitability solution into the liquid chromatograph for testing.

[0163] The test was performed according to the method of Example 1. The chromatogram of the system suitability solution was as follows: Fig.12 shown.

[0164] Depend on Fig.12It can be seen that the separation between the hydroxymethionine sulfoxide and each keto (or hydroxy) amino acid calcium peak in the chromatogram of the system suitability solution does not meet the requirements.

[0165] Comparative Example 5

[0166] The comparative example of the present application provides a method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, which is determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the 2020 Edition of the Chinese Pharmacopoeia), and comprises the following steps:

[0167] S1. Prepare solvent

[0168] 7.8 g of sodium dihydrogen phosphate was taken and dissolved in 1000 mL of deionized water to prepare a sodium dihydrogen phosphate solution with a concentration of 0.05 mol / L. The pH value of the sodium dihydrogen phosphate solution was adjusted to 2.7 with phosphoric acid to prepare a solvent.

[0169] S2. Prepare the test solution and system suitability solution separately

[0170] Weigh the powder of compound α-ketoacid tablets, dissolve it in the prepared solvent and quantitatively dilute it to make a solution containing about 1 mg of racemic hydroxymethionine calcium per 1 ml, shake well, filter, and take the filtrate to prepare the test solution.

[0171] Weigh 20 mg of hydroxymethionine sulfoxide reference substance and place it in a 20 ml volumetric flask, add the prepared solvent to dissolve and dilute to the scale, shake well to obtain the hydroxymethionine sulfoxide stock solution; weigh appropriate amounts of ketovaline calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium, tyrosine and tryptophan reference substances, and transfer appropriate amounts of hydroxymethionine sulfoxide stock solution, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare system suitability solutions containing 5 µg of hydroxymethionine sulfoxide, 1.5 mg of ketovaline calcium, 1 mg of racemic hydroxymethionine calcium, 1.1 mg of racemic ketoisoleucine calcium, 1.7 mg of ketoleucine calcium, 1.2 mg of ketophenylalanine calcium, 0.4 mg of tyrosine and 0.5 mg of tryptophan per 1 ml.

[0172] S3. Inject the prepared test solution and system suitability solution into the liquid chromatograph for testing.

[0173] The test was performed according to the method of Example 1. The chromatogram of the system suitability solution was as follows: Fig.13 shown.

[0174] Depend on Fig.13 It can be seen that the hydroxymethionine sulfoxide in the chromatogram of the system suitability solution is a single peak, and the separation between it and the peaks of each ketone or hydroxyamino acid calcium does not meet the requirements.

[0175] Test Example 1

[0176] According to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0512), the detection limit and quantification limit were determined, the linear relationship was determined, and the correction factor was calculated. The correction factor was the slope ratio of racemic hydroxymethionine calcium and hydroxymethionine sulfoxide. The precision and accuracy were determined, and the range of hydroxymethionine sulfoxide content was determined in combination with the precision, accuracy and linear relationship. The durability of the control solution and the test solution was determined. The results are shown in Tables 2 to 8.

[0177] Table 2 Detection limit and quantification limit

[0178]

[0179] Table 3 Linearity

[0180]

[0181] Correction factor for hydroxymethionine sulfoxide = slope of racemic hydroxymethionine calcium / slope of hydroxymethionine sulfoxide = 14.6105 / 6.8792 = 2.12.

[0182] Table 4 Precision

[0183]

[0184] Table 5 Accuracy

[0185]

[0186] Table 6 Range

[0187]

[0188] Table 7 Stability of control solution

[0189]

[0190] Table 8 Stability of test solution

[0191]

[0192] Test Example 2

[0193] The impurity contents of hydroxymethionine sulfoxide in the compound α-keto acid tablets of Examples 1 to 4 are shown in Table 9.

[0194] Table 9 Hydroxymethionine sulfoxide impurity content in the compound α-ketoacid tablets of Examples 1 to 4

[0195]

[0196] The detection method of the present application can effectively and accurately calculate the hydroxymethionine sulfoxide impurity in the compound α-ketoacid tablets according to the self-control method.

[0197] The above description is only a specific embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for detecting hydroxymethionine sulfoxide in compound α-ketoacid tablets, characterized in that: It includes: First, a system suitability solution is prepared, and the system suitability solution is injected into a liquid chromatograph for testing, and the separation degree between the peaks of hydroxymethionine sulfoxide and each ketoamino acid calcium and each hydroxyamino acid calcium in the chromatogram is ≥1.5, and then a test solution and a control solution are respectively prepared, and the test solution and the control solution are respectively injected into a liquid chromatograph for testing, and the chromatogram of the system suitability solution and the chromatogram of the test solution are compared to determine the peak position of hydroxymethionine sulfoxide, and calculation is performed according to the chromatogram of the test solution and the chromatogram of the control solution according to the self-control method; The test solution comprises a compound α-ketoacid tablet and a solvent, the control solution is prepared by diluting the test solution with the solvent, the system suitability solution comprises hydroxymethionine sulfoxide, keto valine calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium, ketophenylalanine calcium and the solvent, and the mass volume concentration of keto valine calcium, racemic hydroxymethionine calcium, racemic ketoisoleucine calcium, ketoleucine calcium and ketophenylalanine calcium in the system suitability solution is 75% to 120% of its mass volume concentration in the test solution; The pH value of the solvent is 2.4-2.

6.

2. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 1, characterized in that: The concentration of racemic hydroxymethionine calcium in the test solution is 0.75 mg / mL to 1.2 mg / mL.

3. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 2, characterized in that: The concentrations of calcium ketovaline, calcium racemic hydroxymethionine, calcium racemic ketoisoleucine, calcium ketoleucine and calcium ketophenylalanine in the system suitability solution are 1.1 mg / mL to 1.8 mg / mL, 0.75 mg / mL to 1.2 mg / mL, 0.83 mg / mL to 1.3 mg / mL, 1.3 mg / mL to 2.0 mg / mL and 0.9 mg / mL to 1.4 mg / mL, respectively.

4. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 3, characterized in that: The system suitability solution also includes tyrosine and tryptophan, and the concentrations of tyrosine and tryptophan in the system suitability solution are 0.38 mg / mL to 0.6 mg / mL and 0.3 mg / mL to 0.48 mg / mL, respectively.

5. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 1, characterized in that: The control solution is the test solution diluted 500 to 1000 times.

6. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 1, characterized in that: The solvent is prepared by the following method: Prepare a 0.05 mol / L sodium dihydrogen phosphate solution, and adjust its pH value to 2.4-2.6 with phosphoric acid.

7. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to claim 1, characterized in that: The detection temperature is 20℃~30℃.

8. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to any one of claims 1 to 7, characterized in that: The chromatographic column of the liquid chromatograph is an organic acid column, and the organic acid column includes Prevail organic acid 4.6mm×250mm, 5µm.

9. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to any one of claims 1 to 7, characterized in that: In the liquid chromatography test, a mobile phase is used for gradient elution, wherein the mobile phase includes a mobile phase A and a mobile phase B, wherein the mobile phase A is a mixture of a sodium dihydrogen phosphate solution having a pH value of 2.4 to 2.6 and acetonitrile, and the mobile phase B includes acetonitrile, the flow rate of the mobile phase is 0.9 mL / min to 1.1 mL / min, the injection volume is 15 μL to 25 μL, the column temperature is 23° C. to 27° C., and the detection wavelength is 205 nm; The method of the gradient elution is as follows: At 0 min, the volume of the mobile phase A accounts for 100% of the volume of the mobile phase; At 10 min, the volume of the mobile phase A accounts for 100% of the volume of the mobile phase; At 10 min to 27 min, the volume of the mobile phase A accounts for 100% to 88% of the volume of the mobile phase, and the volume of the mobile phase B accounts for 0% to 12% of the volume of the mobile phase; At 27-35 min, the volume of the mobile phase A accounts for 88%-100% of the volume of the mobile phase, and the volume of the mobile phase B accounts for 12%-0% of the volume of the mobile phase; At 35 min to 40 min, the volume of the mobile phase A accounts for 100% of the volume of the mobile phase.

10. The method for detecting hydroxymethionine sulfoxide in the compound α-ketoacid tablets according to any one of claims 1 to 7, characterized in that: The calculation formula for the content of hydroxymethionine sulfoxide in the test solution is as follows: C = E*F*G / H; Wherein E is the peak area of ​​hydroxymethionine sulfoxide in the test solution, F is a correction factor, the correction factor is the slope ratio of racemic hydroxymethionine calcium to hydroxymethionine sulfoxide, G is the reciprocal of the dilution multiple of the control solution, and H is the peak area of ​​racemic hydroxymethionine calcium in the control solution.

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