A high performance liquid chromatography method for determining related substances in alanyl glutamine bulk drug

Abstract

The application provides a high performance liquid chromatography method for determining related substances in alanyl glutamine bulk drug, adopts a chromatographic column with octadecylsilane bonded silica gel as a filler, can effectively separate alanyl glutamine related substances, including degradation impurities and process impurities, and the method has high precision, long service life of the column, and can better control the product quality of the alanyl glutamine bulk drug.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection technology and relates to a high-performance liquid chromatography method for determining related substances in alanylglutamine raw material. Background Technology

[0002] L-Alanyl-L-glutamine, also known as alanyl-glutamine dipeptide, has the molecular formula C8H10. 15 N3O4 is a white or off-white, loose, lumpy substance. Alanylglutamine is a component of parenteral nutrition and is suitable for patients who need glutamine supplementation, such as those in a catabolic or hypermetabolic state.

[0003] The quality standard for alanylglutamine raw material is included in the 2020 edition (Part II) of the Chinese Pharmacopoeia. Its related substance detection method uses a chromatographic column packed with amino-bonded silica gel. The current challenges of the Chinese Pharmacopoeia method are: short column lifespan, poor method reproducibility, and poor or even impossible separation of impurities. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a high-performance liquid chromatography (HPLC) method for determining related substances in alanylglutamine raw material. The method utilizes an octadecylsilane-bonded silica column as the packing material, which has a longer service life, better reproducibility, better separation effect for impurities, and higher detection sensitivity.

[0005] To achieve the purpose of the invention, the present invention adopts the following technical solution:

[0006] A high-performance liquid chromatography method for related substances in alanylglutamine active pharmaceutical ingredient, comprising the following steps:

[0007] (1) Preparation of solution:

[0008] a. Diluent: Purified water;

[0009] b. Test solution: Take an appropriate amount of alanylglutamine raw material, dilute with solvent to prepare the test solution; c. Reference solution: Take an appropriate amount of alanylglutamine raw material, dilute with solvent to prepare the reference solution; d. System suitability solution: Take an appropriate amount of alanylglutamine impurity (i.e., alanylglutamine), dilute with solvent to prepare the system suitability solution.

[0010] (2) Detection:

[0011] Inject the test solution, reference solution, and system suitability solution into the liquid chromatograph, record the chromatograms, and calculate the results using the principal component external standard method with correction factors.

[0012] The chromatographic conditions are as follows:

[0013] Column: Octadecylsilane-bonded silica gel as the packing material;

[0014] Detection wavelength: 200nm±2nm;

[0015] Flow rate: 0.9–1.1 ml / min;

[0016] Column temperature: 32~34℃;

[0017] Injector temperature: 2~8℃

[0018] Injection volume: 10–50 μL;

[0019] Mobile phase: phosphate buffer solution - acetonitrile

[0020] Elution method: isocratic elution.

[0021] Further, the method for preparing the mobile phase is as follows: take 1.36g of potassium dihydrogen phosphate, add 1000ml of water to dissolve it, adjust the pH value to 2.1±0.05 with phosphoric acid solution, add 11.5ml±0.5ml of acetonitrile, add 0.108g of sodium octane sulfonate, dissolve and mix well.

[0022] Further, in step (1), the concentration of the test solution is 1-3 mg / ml; the concentration of the reference solution is 0.8-4 μg / ml; the concentration of the main component of the system suitability solution is 1-4 mg / ml, and the concentration of impurities is 0.8-4 μg / ml.

[0023] Preferably, the concentration of the main component in the system suitability solution is 2 mg / ml.

[0024] Preferably, in step (2), the flow rate is 1.0 ml / min, the column temperature is 33°C, the injector temperature is 5°C, and the injection volume is 20 μL.

[0025] Furthermore, the chromatographic column specifications for the detection method in step (2) are Inertsil ODS-3V, 5μm, 4.6×250mm;

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] The method for detecting related substances in alanylglutamine raw material provided by this invention can effectively separate alanylglutamine related substances, including degradation impurities and process impurities. Moreover, this method has high precision, long column life, and can better control the product quality of alanylglutamine raw material. Attached Figure Description

[0028] Figure 1 This is a liquid chromatogram of the method described in the Chinese Pharmacopoeia in Example 1;

[0029] Figure 2 The liquid chromatogram of the method of this application in Example 1;

[0030] Figure 3 The liquid chromatogram of the system suitability solution in Example 2;

[0031] Figure 4 This is the liquid chromatogram of the test solution in Example 2. Detailed Implementation

[0032] To make the objectives, technical solutions, and technical effects of the embodiments of the present invention clearer, the technical solutions and crystal forms in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention. In conjunction with the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] The various instruments and materials involved in this invention are described below:

[0034] 1. Materials

[0035] Alanylglutamine reference standard (National Institutes for Food and Drug Control, batch number 140702-202005); Alanylglutamine raw material (Jingjing Pharmaceutical Co., Ltd., batch number C582204006); Cyclo-(L-alanyl-L-glutamine) (National Institutes for Food and Drug Control, batch number 140702-202005); Cyclo-(L-alanyl-L-glutamic acid) (National Institutes for Food and Drug Control, batch number 140761-201903); L-pyroglutamyl-L-alanine (National Institutes for Food and Drug Control, batch number 140766-201803); L-pyroglutamic acid (National Institutes for Food and Drug Control, batch number 140762- 202103); D-alanyl-L-glutamine (China National Institutes for Food and Drug Control, batch number 140763-201602); L-alanyl-L-glutamic acid (China National Institutes for Food and Drug Control, batch number 140765-201702); L-alanyl-L-alanyl-L-glutamine (Shanghai Kewei Chemical Technology Co., Ltd., batch number 30175-A6); cyclic imine (QCC, batch number 13-JAN-22-33); alanyl-glutamine-alanyl-glutamine (QCC, batch number 13-JAN-22-27); alanyl-(γ-glutamine)-alanyl-glutamine (QCC, batch number 24-NOV-20-28).

[0036] 2. Main Instruments

[0037] Agilent 1260 high performance liquid chromatograph; DAD detector.

[0038] 3. The chemical structural formula of the known impurity

[0039]

[0040]

[0041] Example 1: Comparison of Detection Results of Related Substances of Alanylglutamine

[0042] Table 1 Chinese Pharmacopoeia - System Suitability Solutions

[0043]

[0044]

[0045] Table 2 Application Methods - System Suitability Solutions

[0046]

[0047] Experimental results show that in the system suitability solution chromatogram using the Chinese Pharmacopoeia method, impurities 7 and 8 were not separated, and the resolutions of the main peak and the preceding and following peaks were 1.4 and 0.6, respectively, which do not meet the requirement of a resolution of not less than 1.5. In the system suitability solution chromatogram using the proposed method, all peaks were completely separated, with a resolution of not less than 2.0, indicating excellent separation.

[0048] Example 2: Detection of related substances in alanylglutamine raw material

[0049] (1) High performance liquid chromatography conditions:

[0050] Column: Inertsil ODS-3V, 5μm, 4.6×250mm;

[0051] Detection wavelength: 200nm;

[0052] Flow rate: 1.0 ml / min;

[0053] Column temperature: 33℃;

[0054] Injector temperature: 5℃

[0055] Injection volume: 20 μL;

[0056] Mobile phase: Phosphate buffer solution (dissolve 1.36 g of potassium dihydrogen phosphate in 1000 ml of water, adjust the pH to 2.1 ± 0.05 with phosphoric acid solution, add 11.5 ml ± 0.5 ml of acetonitrile, add 0.108 g of sodium octane sulfonate, dissolve and mix well);

[0057] Elution method: isocratic elution.

[0058] (2) Sample and solution preparation:

[0059] Diluent: Purified water;

[0060] Blank solution: diluent;

[0061] Test solution: Accurately weigh alanylglutamine raw material and dilute with solvent to a solution of 2 mg / ml; System suitability solution: Accurately weigh alanylglutamine reference standard, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, and impurity 10, and dilute with solvent to a solution containing 2 mg / ml alanylglutamine, 4 μg / ml impurity 1 impurity 2 impurity 0.8 μg / ml impurity 3 impurity 2 μg / ml impurity 4 impurity 1 μg / ml impurity 5 impurity 1 μg / ml impurity 6 impurity 4 μg / ml impurity 5 impurity 1 μg / ml impurity 6 impurity 4 μg / ml impurity 7 impurity 2 μg / ml impurity 8 impurity 2 μg / ml impurity 9 impurity 2 μg / ml impurity 10 impurity 2 μg / ml impurity;

[0062] (3) Determination method

[0063] Take blank solution, test solution, and system suitability solution respectively, inject them under the above chromatographic conditions, record the chromatograms, and calculate the principal component external standard method with correction factor.

[0064] Experimental results: The liquid chromatogram of the system suitability solution is shown in the figure. Figure 1 The liquid chromatogram of the test solution is shown in [reference needed]. Figure 2 .

[0065] Table 3 Related Substance Validation - System Suitability - System Suitability Solution - 2

[0066]

[0067] Experimental results show that in the system suitability solution chromatogram, the resolution between each peak is greater than 1.5, and the theoretical plate number of each impurity is greater than 5000. No impurities were detected in the chromatogram of the alanylglutamine test solution.

[0068] Example 3: Methodological Validation

[0069] 1. Exclusivity

[0070] The instruments and chromatographic conditions are the same as in Example 1.

[0071] Examine the separation between peaks

[0072] Take appropriate amounts of alanylglutamine reference standard, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, and impurity 10, dissolve and dilute them with purified water to prepare a mixed solution, take 20 μl, and inject it into a high-performance liquid chromatograph. The results show that the resolution between each peak meets the requirements.

[0073] 2. Limit of detection and limit of quantitation

[0074] Accurately weigh appropriate amounts of alanylglutamine reference standard, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, and impurity 10. Dissolve in deionized water and dilute stepwise. Inject into a high-performance liquid chromatograph (HPLC). The limit of quantitation (LOQ) is set at a signal-to-noise ratio (SNR) of approximately 10, and the limit of detection (LOD) is set at a SNR of approximately 3. The LOQ results are as follows: the concentrations of all known impurities and alanylglutamine are between 0.0399 μg / ml and 0.197 μg / ml, relative to the concentration of the test solution, between 0.002% and 0.01%. The SNR of all known impurity peaks and the alanylglutamine peak are between 17 and 752. The detection limits were as follows: the detection limits for all known impurities and alanylglutamine were 0.0120 ug / ml to 0.0591 ug / ml, relative to the concentration of the test sample, which were 0.0006% to 0.003%; the signal-to-noise ratios of the known impurity peaks and alanylglutamine peaks were 6 to 144.

[0075] 3. Linear relationship

[0076] Accurately weigh appropriate amounts of alanylglutamine reference standard, and reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Dissolve and dilute with purified water to LOQ, 50%, 100%, 150%, and 200% limit concentrations, respectively. Inject 20 μl of each solution into a high-performance liquid chromatograph (HPLC) and measure the peak area. Using peak area as the total efficiency index (Y) and injection concentration as the abscissa (X), the linear regression equation was obtained. The specific results are shown in Table 2.

[0077] Table 4 Related Material Validation - Linearity Results

[0078]

[0079]

[0080]

[0081]

[0082] The results showed that the peak area and concentration of each impurity exhibited a good linear relationship within the range of the limit of quantitation to twice the limit, with correlation coefficients (r) of 1.000 for all impurities. The response factor (A / C) RSD ranged from 0.74% to 8.18%. The ratio of the Y-axis intercept to the 100% response value ranged from 0.12% to 1.96%.

[0083] 4. Stability

[0084] Reference solution: Accurately weigh an appropriate amount of alanylglutamine reference standard and dilute it to prepare a solution containing 2 μg of alanylglutamine in 1 ml.

[0085] Test solution: Accurately weigh an appropriate amount of alanylglutamine raw material and dilute it to a solution containing 2 mg of alanylglutamine in 1 ml.

[0086] System suitability solution: Accurately weigh appropriate amounts of alanylglutamine reference standard, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, and impurity 10 reference standards, and prepare a solution containing 2 mg of alanylglutamine, 4 μg of impurity 1, 0.8 μg of impurity 2, 2 μg of impurity 3, 1 μg of impurity 4, 1 μg of impurity 5, 4 μg of impurity 6, 2 μg of impurity 7, 2 μg of impurity 8, 2 μg of impurity 9, and 2 μg of impurity 10 per ml. Spiked test solution: Accurately weigh appropriate amounts of alanylglutamine raw material and reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and prepare a solution containing 2 mg of alanylglutamine, 4 μg of impurity 1, 0.8 μg of impurity 2, 2 μg of impurity 3, 1 μg of impurity 4, 1 μg of impurity 5, 4 μg of impurity 6, 2 μg of impurity 7, 2 μg of impurity 8, 2 μg of impurity 9, and 2 μg of impurity 10 per ml. Inject 20 μl of each of the alanylglutamine reference solution, test solution, system suitability solution, and spiked test solution into a high-performance liquid chromatograph (HPLC) at 5°C for 0, 10, 24, 46, 68, and 84 hours, and determine the peak area. The results are shown in Tables 3, 4, 5, and 6.

[0087] Table 5 System Suitability and Solution Stability (5℃)

[0088]

[0089]

[0090] Table 6. Stability of reference solution (5℃)

[0091] time 0h 10h 24h 52h 74h 84h RSD (%) Peak area 58.595 58.106 57.793 58.069 59.051 58.122 0.78

[0092] Table 7. Stability of the test sample solution (5℃)

[0093]

[0094] Table 8. Stability of the spiked solution of the test sample (5℃)

[0095]

[0096]

[0097]

[0098] The results showed that the peak area RSD of the system suitability solution was no greater than 5% after refrigeration for 5 days, indicating that the system suitability solution had good stability within 5 days of being stored at 5℃.

[0099] The peak area RSD of the main peak measured at each time point after refrigeration of the reference solution for 84 hours was not greater than 5.0%, indicating that the reference solution had good stability after refrigeration for 84 hours.

[0100] The difference between the content of each impurity in the test solution after 84 hours of refrigeration and the content of each impurity after 0 hours is not greater than 0.001%, indicating that the test solution has good stability after 84 hours of refrigeration at 5°C.

[0101] The difference between the content of each impurity in the spiked solution of the test sample after 84 hours of refrigeration and the content of each impurity after 0 hours is less than 0.01%, indicating that the spiked solution of the test sample has good stability after 84 hours of refrigeration.

[0102] 5. Precision

[0103] 5.1 Repeatability

[0104] Take 1 ml of the spiked test solution containing 2 mg alanine glutamine, 4 μg impurity 1, 0.8 μg impurity 2, 2 μg impurity 3, 1 μg impurity 4, 1 μg impurity 5, 4 μg impurity 6, 2 μg impurity 7, 2 μg impurity 8, 2 μg impurity 9, and 2 μg impurity 10. Take 20 μl of the solution each time and inject it into the high performance liquid chromatograph. Repeat the injection 6 times and measure the peak area. The results are shown in Table 7.

[0105] Table 9 Results of Repeatability Tests

[0106]

[0107]

[0108] The results showed that the average contents of impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 in the six spiked solutions were 0.20%, 0.04%, 0.10%, 0.05%, 0.05%, 0.18%, 0.10%, 0.10%, 0.10%, and 0.10%, respectively, with RSDs of 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 2.29%, 5.35%, 0.00%, 0.00%, and 0.00%, respectively.

[0109] 5.2 Intermediate Precision

[0110] Different researchers prepared the following solutions at different times:

[0111] Reference solution: Accurately weigh an appropriate amount of alanylglutamine reference standard and dilute it to prepare a solution containing 2 μg of alanylglutamine in 1 ml.

[0112] Test solution: Accurately weigh an appropriate amount of alanylglutamine raw material and dilute it to a solution containing 2 mg of alanylglutamine in 1 ml.

[0113] System suitability solution: Accurately weigh appropriate amounts of alanylglutamine reference standard, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, impurity 8, impurity 9, and impurity 10 reference standards, and prepare a solution containing 2 mg of alanylglutamine, 4 μg of impurity 1, 0.8 μg of impurity 2, 2 μg of impurity 3, 1 μg of impurity 4, 1 μg of impurity 5, 4 μg of impurity 6, 2 μg of impurity 7, 2 μg of impurity 8, 2 μg of impurity 9, and 2 μg of impurity 10 per ml. Spiked test solution: Accurately weigh appropriate amounts of alanylglutamine raw material and reference standards for impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and prepare a solution containing 2 mg of alanylglutamine, 4 μg of impurity 1, 0.8 μg of impurity 2, 2 μg of impurity 3, 1 μg of impurity 4, 1 μg of impurity 5, 4 μg of impurity 6, 2 μg of impurity 7, 2 μg of impurity 8, 2 μg of impurity 9, and 2 μg of impurity 10 per ml. Inject 20 μl of each of the alanylglutamine reference solution, test solution, system suitability solution, and spiked test solution into a high-performance liquid chromatograph (HPLC) at 5°C for 0, 10, 24, 46, 68, and 84 hours, and determine the peak area. The results are shown in Table 8.

[0114] Table 10 Results of intermediate precision test

[0115]

[0116]

[0117] The results showed that the relative standard deviations (RSDs) of the contents of the six test sample solutions of each impurity were 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, 0.00%, and 0.00%, respectively, all of which were 0.00%.

[0118] 6. Accuracy Test

[0119] Impurity Mixed Solution: Accurately weigh appropriate amounts of impurities 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, and dilute them with solvent to prepare an impurity mixed solution. Accurately weigh alanylglutamine raw material, then add it to the impurity mixed solution and dilute to prepare solutions containing 2 mg / ml alanylglutamine and 80%, 100%, and 120% limit concentrations of other impurities, respectively. Prepare three parallel aliquots for each solution. Inject 20 μl of each solution into a high-performance liquid chromatograph (HPLC), determine the peak area, and calculate the recovery rate. The results are shown in Table 9.

[0120] Table 11 Accuracy Test Results

[0121]

[0122]

[0123]

[0124]

[0125]

[0126] The results showed that the average recoveries at known impurity quantitation limits ranged from 86.11% to 110.26%, with RSDs of the three recoveries all less than 6.67%. The average recoveries at 50%, 100%, and 150% concentrations ranged from 97.33% to 103.98%, with RSDs of the nine recoveries all not exceeding 1.12%, indicating good accuracy of the method.

Claims

1. A high performance liquid chromatography method for determining related substances in alanyl glutamine drug substance, characterized in that, It comprises the following steps: Prepare the solution: a. Diluent: purified water; b. Test solution: take a certain amount of alanyl glutamine raw material, dilute with solvent, as the test solution; c. Control solution: take a certain amount of alanyl glutamine raw material, dilute with solvent, as the control solution; d. System suitability solution: take a certain amount of alanyl glutamine impurities and alanyl glutamine, dilute with solvent, as the system suitability solution; Detection: Take the test solution, control solution, and system suitability solution into the liquid chromatograph respectively, record the chromatogram, and calculate according to the external standard method of the main component with a correction factor; The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel as the filler; Detection wavelength: 200 nm ± 2 nm; Flow rate: 0.9-1.1 ml / min; Column temperature: 32-34℃; Inj ector temperature: 2-8℃; Injection volume: 10-50µL; Mobile phase: phosphate buffer solution-acetonitrile; Elution mode: isocratic elution; The configuration method of the mobile phase is as follows: take 1.36 g of potassium dihydrogen phosphate, dissolve in 1000 ml of water, adjust the pH value to 2.1 ± 0.05 with phosphoric acid solution, add 11.5 ml ± 0.5 ml of acetonitrile, and 0.108 g of sodium octane sulfonate, and mix well after dissolution; The related substances in the alanyl glutamine raw material are: The alanyl glutamine raw material is from Jingjing Pharmaceutical Co., Ltd., batch number C582204006.

2. The HPLC method for determining the related substances in alanyl glutamine drug substance according to claim 1, characterized in that, The concentration of the test solution is 1-3 mg / ml; the concentration of the control solution is 0.8-4µg / ml; the concentration of the main component of the system suitability solution is 1-4 mg / ml, and the concentration of the impurity is 0.8-4µg / ml.

3. The HPLC method for determining the related substances in alanyl glutamine drug substance according to claim 2, characterized in that, The concentration of the main component of the system suitability solution is 2 mg / ml.

4. The HPLC method for determining the related substances in alanyl glutamine drug substance according to claim 1, characterized in that, The flow rate is 1.0 ml / min, the column temperature is 33℃, the injector temperature is 5℃, and the injection volume is 20µL.

5. The HPLC method for determination of related substances in alanyl glutamine drug substance as claimed in claim 1, wherein, The chromatographic column specification is ODS-3V, 5µm, 4.6×250mm.

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