GC (Gas Chromatography) detection method for degrading organic acid impurities in compound keto acid tablets

Abstract

The GC detection method for degrading the organic acid impurities in the compound keto acid tablets comprises the following steps: (1) dissolving reference substances of 2-methyl propionic acid, 2-methyl butyric acid and 3-methyl butyric acid in a solvent to obtain a reference substance solution of impurities of 2-methyl propionic acid, 2-methyl butyric acid and 3-methyl butyric acid, taking the solvent as a blank solvent, and injecting the blank solvent into a gas chromatograph; and (2) dissolving a sample in a trifluoroacetic acid solution, carrying out ultrasonic treatment, adding isopropyl ether, extracting, combining organic phases to obtain a test solution, injecting the test solution into a gas chromatograph, recording a chromatogram, and calculating the degraded organic acid impurities by using an external standard method. The invention relates to the technical field of determination of degraded organic acid impurities, some specific degraded organic acid impurities can be accurately detected and cannot be interfered by other components in a compound keto acid tablet, the separation degree of the impurities meets the requirements of the applicability of a chromatographic system, the sample determination result is accurate and reliable, and the sensitivity is high.

Description

Technical Field

[0001] The invention relates to the technical field of determination of degraded organic acid impurities, and in particular to a GC detection method for degraded organic acid impurities in a compound ketoacid tablet. Background Art

[0002] Under high humidity, high temperature and oxidative conditions, the active ingredient calcium ketovaline in compound ketoacid tablets will degrade to produce the organic acid impurity 2-methylpropionic acid, the active ingredient calcium racemic ketoisoleucine will degrade to produce the organic acid impurity 2-methylbutyric acid, and the active ingredient calcium ketoleucine will degrade to produce the organic acid impurity 3-methylbutyric acid. These known impurities lack ultraviolet absorption groups in their molecular structures and have very weak ultraviolet absorption. When detected using the common HPLC-UV method, the sensitivity is low and detection is very difficult.

[0003] In order to more objectively reflect the degradation of organic acid impurities in compound ketoacid tablets, the present invention attempts to establish a new GC method. 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 GC detection method for degraded organic acid impurities in compound keto acid tablets, establishes a more comprehensive, objective and efficient detection method for controlling degraded organic acid impurities in compound keto acid tablets, uses GC method to detect several specific degraded organic acid impurities, so that each impurity can be detected with high sensitivity, and is not affected by other components in the compound keto acid tablets. The separation degree between each impurity is good (greater than 1.5), which meets the requirements of the applicability of the chromatography system.

[0005] The technical solution adopted by the present invention is:

[0006] A GC detection method for degraded organic acid impurities in compound ketoacid tablets comprises the following steps:

[0007] Step (1): dissolving 2-methylpropionic acid, 2-methylbutyric acid and 3-methylbutyric acid reference substances in a solvent to obtain a degradation organic acid impurity reference substance solution, taking the solvent as a blank solvent, injecting the blank solvent into a gas chromatograph until the baseline is stable, and then injecting the degradation organic acid impurity reference substance solution into the gas chromatograph for detection;

[0008] Step (2): dissolving the sample in a trifluoroacetic acid solution, ultrasonically treating, adding isopropyl ether, extracting, combining the organic phases to obtain a test solution, injecting the test solution into a gas chromatograph, eluting by temperature ramping of the chromatographic column, recording the chromatogram, and calculating each known degradation organic acid impurity by an external standard method;

[0009] Wherein, the carrier gas is nitrogen or helium; and the capillary column uses 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid.

[0010] Preferably, the chromatographic column is programmed to heat as follows: the initial column temperature is 40°C, maintained for 3 minutes, heated to 85°C at a rate of 2.5°C per minute, maintained for 2 minutes, and then heated to 250°C at a rate of 50°C per minute, maintained for 15 minutes.

[0011] Preferably, the carrier gas flow rate is 0.5-5.0 ml / min, the injection volume is 0.1-1.0 μl, the inner diameter of the capillary column is 0.2 mm-0.5 mm, the column length is 20 m-60 m, the fixed liquid film thickness is 1 μm-5 μm, and detection is performed using an FID detector.

[0012] Preferably, the concentration of the degraded organic acid impurity reference solution is 1.0-20.0 μg / ml.

[0013] Among them, the preparation method of the degradation organic acid impurity reference solution is as follows:

[0014] Step (1): Preparation of reference substance stock solution (500 μg / ml):

[0015] Take about 50 mg of each of 2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid reference substances, accurately weigh them, place them in the same 100 ml volumetric flask, add isopropyl ether to dissolve and dilute to the scale, and shake well;

[0016] Step (2): Preparation of reference solution (5 μg / ml):

[0017] Accurately measure 1 ml of the reference stock solution, place it in a 100 ml volumetric flask, dilute to the mark with isopropyl ether, and shake well;

[0018] The preparation method of the test solution is as follows:

[0019] Take about 250 mg of fine powder of this product, weigh accurately, add 5 ml of trifluoroacetic acid solution accurately, shake well, ultrasonicate for 5 minutes, add 10 ml of isopropyl ether accurately, ultrasonicate for 5 minutes, extract twice in the same way, combine the organic phases, add an appropriate amount of anhydrous sodium sulfate to dry, filter, and take the filtrate as the test solution;

[0020] The calculation formula for calculating the content of known impurities in compound ketoacid tablets is as follows:

[0021]

[0022] in:

[0023] Cs: concentration of known impurities in the test solution (μg / ml);

[0024] C: Concentration of known impurity reference solution (μg / ml);

[0025] As: peak area of ​​known impurities measured in the test solution;

[0026] A: Average peak area of ​​known impurity reference solution.

[0027] The beneficial effects achieved by the present invention using the above structure are as follows: This scheme provides a GC detection method for degraded organic acid impurities in compound keto acid tablets, which adopts gas chromatography, sets certain chromatographic conditions, accurately takes a reference solution and a sample solution of the compound keto acid tablet to be measured and injects them into the GC, records the chromatogram, and calculates the content of known impurities according to the external standard method. For specific degradation impurities, this method can accurately detect them without being interfered by other components in the compound keto acid tablets. The separation degree between each impurity meets the requirements of the applicability of the chromatographic system. It is a detection method with simple and convenient sample processing, accurate and reliable sample test results, high sensitivity, and conventional reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0029] Figure 1 is the GC chromatogram of blank solvent;

[0030] Figure 2 GC chromatograms showing the detection limits of 2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid;

[0031] Figure 3 GC chromatograms of 2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid reference substance solutions;

[0032] Figure 4 This is the GC chromatogram of the test solution of compound ketoacid tablets. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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.

[0034] Example 1

[0035] A GC detection method for degraded organic acid impurities in compound ketoacid tablets comprises the following steps:

[0036] Step (1): dissolving 2-methylpropionic acid, 2-methylbutyric acid and 3-methylbutyric acid reference substances in isopropyl ether to obtain a degraded organic acid impurity reference substance solution, using isopropyl ether as a blank solvent, injecting 0.5 μl of the blank solvent into a gas chromatograph, and then injecting the degraded organic acid impurity reference substance solution 1 and the reference substance solution 2 into the gas chromatograph for detection, injecting 5 needles into the degraded organic acid impurity reference substance solution 1, and injecting 2 needles into the reference substance solution 2, wherein the reference substance solution 1 and the reference substance solution 2 are both reference substance solutions of the same concentration, and testing the injection repeatability of the gas chromatograph machine by using the reference substance solution 1, and the RSD of the main peak area of ​​5 needles in the reference substance solution 1 shall not exceed 5.0%;

[0037] Step (2): dissolving the sample in trifluoroacetic acid solution, ultrasonically treating, adding isopropyl ether, extracting, combining the organic phases to obtain a test solution, injecting 0.5 μl of the test solution into a gas chromatograph, recording the chromatogram, ignoring the peak in the blank solvent, and calculating using the external standard method;

[0038] The formula for calculating the content of known impurities is as follows:

[0039]

[0040] in:

[0041] Cs: concentration of known impurities in the test solution (μg / ml);

[0042] C: Concentration of known impurity reference solution (μg / ml);

[0043] As: Peak area of ​​known impurities measured in the test solution; A: Average peak area of ​​known impurity reference solution.

[0044] Example 2

[0045] This embodiment provides a GC detection method for degraded organic acid impurities in compound ketoacid tablets, comprising the following steps:

[0046] Step (1): dissolving 2-methylpropionic acid, 2-methylbutyric acid and 3-methylbutyric acid reference substances in a solvent to obtain a degradation organic acid impurity reference substance solution, using isopropyl ether as a blank solvent, injecting 0.5 μl of the blank solvent into a gas chromatograph, and then injecting degradation organic acid impurity reference substance solution 1 and reference substance solution 2 into the gas chromatograph for detection, injecting 5 needles into degradation organic acid impurity reference substance solution 1, injecting 2 needles into reference substance solution 2, and the RSD of the main peak area of ​​5 needles in reference substance solution 1 shall not exceed 5.0%;

[0047] Step (2): dissolving the sample in trifluoroacetic acid solution, ultrasonically treating, adding isopropyl ether, extracting, combining the organic phases to obtain a test solution, injecting 0.5 μl of the test solution into a gas chromatograph, recording the chromatogram, ignoring the peak in the blank solvent, and calculating using the external standard method;

[0048] The capillary column was a stationary phase of 6% cyanopropylphenyl-94% dimethylpolysiloxane, with a programmed temperature rise of 40°C for the initial column temperature, maintained for 3 minutes, then raised to 85°C at a rate of 2.5°C per minute, maintained for 2 minutes, and then raised to 250°C at a rate of 50°C per minute, maintained for 15 minutes, with a carrier gas flow rate of 3.0 ml / min, injection volume of 0.5 μl, an inner diameter of the capillary column of 0.53 mm, a column length of 30 m, a stationary phase film thickness of 3.0 μm, and a FID detector for detection;

[0049] The reference solution and test solution in the method are prepared as follows:

[0050] Preparation of reference solution for degradation of organic acid impurities:

[0051] Step (1): Preparation of reference substance stock solution (500 μg / ml): Take approximately 50 mg of each of 2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid reference substances, accurately weigh, place in the same 100 ml volumetric flask, add isopropyl ether to dissolve and dilute to the mark, and shake well;

[0052] Step (2): Preparation of reference substance solution (5 μg / ml): Accurately measure 1 ml of reference substance stock solution, place in a 100 ml volumetric flask, dilute to scale with isopropyl ether, and shake well;

[0053] Preparation of test solution:

[0054] Take about 250 mg of fine powder of this product, weigh accurately, add 5 ml of trifluoroacetic acid solution accurately, shake well, ultrasonicate for 5 minutes, add 10 ml of isopropyl ether accurately, ultrasonicate for 5 minutes, extract twice in the same way, combine the organic phases, add an appropriate amount of anhydrous sodium sulfate to dry, filter, and take the filtrate as the test solution;

[0055] This method has high sensitivity and the limit of quantification (S / N ≥ 10) can reach 0.5 μg / ml. The typical chromatogram of the test solution of this method is as follows: Figure 1 to Figure 4 .

[0056] Example 3

[0057] The three known methods for detecting degraded organic acid impurities are the same as those in Example 2;

[0058] Three known degradation organic acid impurities are shown in the table below:

[0059] Table 1 Three known degradation organic acid impurities

[0060]

[0061] Several degraded organic acid impurities can be successfully detected and separated by the detection method of the present invention.

[0062] Embodiment 4:

[0063] This example determines the linear ranges of the three components "2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid". The results are shown in Tables 2 to 4.

[0064] Table 2 2-Methylpropionic acid linear test data

[0065]

[0066] Table 3 2-Methylbutyric acid linear test data

[0067]

[0068] Table 4 3-Methylbutyric acid linear test data

[0069]

[0070] Embodiment 5:

[0071] This example verifies the quantitative limit of the method. Take the quantitative limit solution under the linear item, accurately measure 0.5 μl and inject it into the gas chromatograph, inject 6 times continuously, record the chromatogram, calculate its signal-to-noise ratio, and the signal-to-noise ratio S / N≥10 is the quantitative limit concentration, and calculate the relative standard deviation of each peak area.

[0072] Table 5 Limit of quantitation

[0073]

[0074] Embodiment 6:

[0075] This example verifies the detection limit of the method. A quantitative limit concentration solution is taken, diluted about 3 times and injected, a chromatogram is recorded, and the signal-to-noise ratio is calculated. The sample concentration with a signal-to-noise ratio S / N ≥ 3 is the detection limit concentration.

[0076] Table 6 Detection limits

[0077]

[0078] Embodiment 7:

[0079] In this example, six samples were measured in parallel according to the description of the above experimental method to verify the precision.

[0080] Table 7 Precision

[0081]

[0082] Embodiment 8:

[0083] This example verifies the stability of the reference solution. Place the reference solution at room temperature without light protection, and accurately measure 0.5 μl of each solution for injection and detection at 0, 4, 8, 12, and 24 hours, and record the chromatogram; take another reference solution and place it in a refrigerator (2-8°C), take it out at different times, and after it reaches room temperature, accurately measure 0.5 μl of each solution for injection and detection, and record the chromatogram. Investigate the stability of the solution.

[0084] Table 8 Room temperature stability results of impurity reference solution

[0085]

[0086] Table 9 Stability results of impurity reference solution in refrigerator (2~8℃)

[0087]

[0088] Embodiment 9:

[0089] In this example, the accuracy of the GC method of the present invention was verified by a recovery experiment. Three solutions of each component at levels of 80%, 100%, and 120% and a blank control were selected, and the recovery rate of each component was determined according to the description of the above experimental method.

[0090] Table 10 2-Methylpropionic acid recovery test data

[0091]

[0092] Table 11 2-Methylbutyric acid recovery test data

[0093]

[0094] Table 12 3-Methylbutyric acid recovery test data

[0095]

[0096] 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.

Claims

1. A GC detection method for degraded organic acid impurities in compound ketoacid tablets, wherein the degraded organic acid impurities are degraded impurities including 2-methylpropionic acid, 2-methylbutyric acid, and 3-methylbutyric acid, characterized in that: The steps include: Step (1): dissolving 2-methylpropionic acid, 2-methylbutyric acid and 3-methylbutyric acid reference substances in a solvent to obtain a degradation organic acid impurity reference substance solution, taking the solvent as a blank solvent, injecting the blank solvent into a gas chromatograph until the baseline is stable, and then injecting the degradation organic acid impurity reference substance solution into the gas chromatograph for detection; Step (2): dissolving the sample in a trifluoroacetic acid solution, ultrasonically treating, adding isopropyl ether, extracting, combining the organic phases to obtain a test solution, injecting the test solution into a gas chromatograph, eluting by temperature ramping of the chromatographic column, recording the chromatogram, and calculating each known degradation organic acid impurity by an external standard method; Wherein, the carrier gas is nitrogen or helium; the capillary column uses 6% cyanopropylphenyl-94% dimethylpolysiloxane as the stationary liquid; The chromatographic column temperature was programmed as follows: the initial column temperature was 40°C, maintained for 3 minutes, then increased to 85°C at a rate of 2.5°C per minute, maintained for 2 minutes, then increased to 250°C at a rate of 50°C per minute, maintained for 15 minutes.

2. The GC detection method for degrading organic acid impurities in the compound ketoacid tablets according to claim 1, characterized in that: The carrier gas flow rate is 0.5-5.0 ml / min, the injection volume is 0.1-1.0 μl, the inner diameter of the capillary column is 0.2 mm-0.5 mm, the column length is 20 m-60 m, the fixed liquid film thickness is 1 μm-5 μm, and the FID detector is used for detection.

3. The GC detection method for degrading organic acid impurities in the compound ketoacid tablets according to claim 1, characterized in that: The concentration of the degraded organic acid impurity reference solution is 1.0-20.0 μg / ml.

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