A method for determining alanyl-glutamine related substances

Abstract

The present invention belongs to the field of analytical chemistry, and specifically relates to a method for determining related substances in alanyl-glutamine or its preparation, wherein the method adopts HPLC for detection; wherein the chromatographic column uses octadecyl amido bonded silica gel as a filler, and uses sodium octane sulfonate solution-acetonitrile (95:5) as a mobile phase. The method of the present invention can effectively separate 9 related substances of alanyl-glutamine, has good specificity, high sensitivity, and can realize accurate quantitative determination of alanyl-glutamine or its preparation.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for determining related substances in alanyl-glutamine and its preparations by using high performance liquid chromatography. Background Art

[0002] Alanyl-glutamine, the structural formula is as follows:

[0003]

[0004] It is a commonly used amino acid drug in clinical practice, which has the functions of supplementing essential amino acids, maintaining acid-base balance in the body, and enhancing the body's immunity. Many domestic companies have been approved to use this raw material in marketed preparations, such as: Evonik Meishi Simplified Co., Ltd., Shandong Chenlong Pharmaceutical Co., Ltd., Wuhan Da'an Pharmaceutical Co., Ltd., etc.

[0005] Impurity research is an important way to evaluate drug quality. Alanyl-glutamine may introduce residual raw materials and other related substances during the synthesis process, and may also produce degradation products during storage. It is necessary to quantitatively control its related substances to meet quality requirements and ensure drug safety.

[0006] The 2015 edition of the Chinese Pharmacopoeia and the 43rd edition of the USP both contain the quality standards for alanyl-glutamine. The HPLC method is used to determine its related substances. A chromatographic column (i.e., amino column) is used as the filler and potassium dihydrogen phosphate buffer-acetonitrile (35:65) is used as the mobile phase. The number of impurities controlled is relatively small.

[0007] Research has found that when amino columns are used, the durability of the system is very poor. A slight change in the mobile phase ratio will cause a significant change in the retention time of the chromatographic peaks of the relevant substances. In addition, amino columns require a long time to balance, and the mobile phase must have a high concentration of organic solvents (usually >50%), which shortens the service life of the column.

[0008] Therefore, it is very necessary to develop a new method for determining the related substances of alanyl-glutamine and its preparations to improve the problems existing in the prior art. Summary of the invention

[0009] In view of the shortcomings of the prior art in detecting the presence of impurities in alanyl-glutamine and related substances in its preparations, such as poor separation effect, too long retention time of individual impurities, drift of retention time, and poor durability of chromatographic columns, the present application provides a new HPLC determination method, which can accurately and effectively achieve the determination of alanyl-glutamine related substances by adjusting the chromatographic column and the mobile phase. In addition, the chromatographic conditions are simple, the durability is good, and the impurities and degradation products of alanyl-glutamine can be better controlled, which is of great significance in terms of its quality control.

[0010] According to the pharmacopoeia standards for alanylglutamine API and its preparations and combined with its synthesis process, the following impurities may be present in alanylglutamine or its preparations, and their impurity numbers, names and structural formulas are shown in Table 1. Among them, impurity 1, impurity 3, impurity 4, impurity 6, impurity 7, impurity 10 and SM1 are all included in the pharmacopoeia standards.

[0011] Table 1 Alanyl-glutamine related substances

[0012]

[0013] The technical problem to be solved by the present application is to provide a method for determining related substances in alanyl-glutamine and its preparations, and to effectively separate and quantitatively determine the impurities in the above table.

[0014] The determination method provided in the present application is an HPLC method, and its chromatographic conditions include: the chromatographic column uses octadecyl amido bonded silica gel as a filler, and the mobile phase is a mixed solution of sodium octane sulfonate solution and acetonitrile in a volume ratio of 95:5.

[0015] In some embodiments, in the determination method provided by the present application, the chromatographic column is an ACE Excel chromatographic column.

[0016] In some preferred embodiments, the chromatographic column is an ACE Excel 5 C18-Amide column; more preferably, the chromatographic column is an ACE Excel 5 C18-Amide 250 mm×4.6 mm.

[0017] In the determination method provided in the present application, a chromatographic column with similar performance to the ACE Excel 5 C18-Amide column can also be selected.

[0018] In some embodiments, in the determination method provided by the present application, the sodium octane sulfonate solution is an aqueous phosphoric acid solution of sodium octane sulfonate, and the concentration of sodium octane sulfonate is 0.0034 mol / L to 0.0040 mol / L.

[0019] In some embodiments, in the determination method provided by the present application, the concentration of the sodium octane sulfonate solution is 0.0037 mol / L.

[0020] In the determination method provided in the present application, the sodium octane sulfonate solution can be prepared by the following method: take 0.795g to 0.935g of sodium octane sulfonate, add 1000ml of water to dissolve it, add 0.5ml of phosphoric acid, mix well, and obtain the solution.

[0021] In some embodiments, in the determination method provided in the present application, the chromatographic conditions also include: a detection wavelength of 210 nm, a flow rate of 0.8 to 1.1 ml per minute, and a column temperature of 23 to 30°C.

[0022] In some preferred embodiments, the flow rate is 1.0 ml per minute.

[0023] In some preferred embodiments, the column temperature is 27°C.

[0024] In the determination method provided in the present application, the chromatographic running time is 45 minutes.

[0025] In some embodiments, the determination method provided in the present application includes the following steps: detection by HPLC; the chromatographic conditions include: the chromatographic column is filled with octadecyl amido bonded silica gel, the mobile phase is a mixed solution of sodium octane sulfonate solution and acetonitrile with a volume ratio of 95:5, the detection wavelength is 210nm, the flow rate is 0.8-1.1ml per minute, and the column temperature is 23-30°C.

[0026] In some preferred embodiments, the determination method provided by the present application, the chromatographic conditions include: the chromatographic column is an ACEExcel 5 C18-Amide column, the mobile phase is a mixed solution of 0.0034 mol / L to 0.0040 mol / L sodium octane sulfonate solution and acetonitrile with a volume ratio of 95:5, the detection wavelength is 210 nm, the flow rate is 1.0 ml per minute, and the column temperature is 27°C.

[0027] In the determination method provided in the present application, the preparation of alanyl-glutamine is selected from alanyl-glutamine injection and alanyl-glutamine for injection.

[0028] The present application provides a method for determining related substances in alanyl-glutamine and its preparations by high performance liquid chromatography, which has simple chromatographic conditions, convenient operation, high sensitivity, good durability, can effectively separate various impurities, and quickly and accurately achieves the determination of alanyl-glutamine related substances, ensuring its quality control.

[0029] The present application uses octadecyl amido bonded silica gel as a chromatographic column filler, which has a strong retention capacity for all relevant substances, can tolerate 100% aqueous phase, has good durability, a long average service life of the chromatographic column, and a wider range of applications;

[0030] The present application adopts a mixed solution of sodium octane sulfonate solution and acetonitrile as the mobile phase, which can completely separate the relevant substances, and the system operation time is only 45 minutes, which is greatly shortened compared with the existing technology, which is conducive to improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1The assay chromatogram of the initial use of the amino column in Comparative Example 1;

[0032] Figure 2 Chromatogram of the amino column in Comparative Example 1 after using it for a period of time;

[0033] Figure 3 The chromatogram was detected using an Amide column;

[0034] Figure 4 The chromatogram was detected using a HILIC column;

[0035] Figure 5 Chromatogram of the system suitability test conducted using the chromatographic conditions of this application;

[0036] Figure 6 Sodium dodecyl sulfate solution-acetonitrile (95:5) was used as the mobile phase buffer salt to detect the chromatogram. DETAILED DESCRIPTION

[0037] The present invention is further illustrated below through the description of specific implementation modes, but this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the invention, but as long as they do not deviate from the basic idea of ​​the invention, they are all within the scope of the present invention.

[0038] Experimental Example 1 Screening of Ion Pair Reagents in Mobile Phase

[0039] 1) Solution preparation:

[0040] Diluent: water-acetonitrile (95:5) (v / v), used for preparation of test solution;

[0041] Sodium octane sulfonate solution: Take 0.865g of sodium octane sulfonate, add 1000ml of water to dissolve, add 0.5ml of phosphoric acid, mix well, and obtain a sodium octane sulfonate solution with a concentration of 0.0037mol / L;

[0042] Sodium dodecyl sulfate solution: Take 0.865 g of sodium dodecyl sulfate, add 1000 ml of water to dissolve, add 0.5 ml of phosphoric acid, mix well, and obtain a sodium dodecyl sulfate solution with a concentration of 0.0032 mol / L;

[0043] Mixed impurity stock solution: Accurately weigh about 10 mg each of impurity 1, impurity 3, impurity 4, impurity 6, impurity 8, impurity 10, SM1, and intermediate 1 reference substances, place them in 10 ml volumetric flasks, dissolve and dilute to the scale with diluent, and shake well; accurately weigh about 20 mg each of impurity 7 and impurity 9 reference substances, place them in 5 ml volumetric flasks, dissolve and dilute to the scale with diluent, and shake well to prepare mixed impurity stock solutions.

[0044] System suitability solution: Take about 80 mg of alanyl-glutamine, weigh it accurately, and place it in a 20 ml volumetric flask. Accurately measure 0.25 ml of each of the stock solutions of impurity 1, impurity 3, impurity 4, impurity 6, impurity 7, impurity 8, impurity 9, impurity 10, SM1, and intermediate 1, place them in the same 20 ml volumetric flask, dissolve and dilute to the scale with diluent, shake well, and use as the system suitability solution.

[0045] 2) Chromatographic conditions:

[0046] Mobile phase 1: sodium octane sulfonate solution-acetonitrile = 95:5 (v / v);

[0047] Mobile phase 2: sodium dodecyl sulfate solution-acetonitrile = 95:5 (v / v);

[0048] Chromatographic column: octadecyl amido bonded silica gel as filler (ACE Excel 5 C18-Amide 250mm×4.6mm);

[0049] Flow rate: 1.0 mL / min, detection wavelength: 210 nm, column temperature: 27 °C;

[0050] Accurately measure 20 μl of the above system suitability solution, inject it into the liquid chromatograph, and perform the determination under two chromatographic conditions, mobile phase 1 and mobile phase 2, respectively, to examine the effect of the ion pair reagent on the separation of each impurity.

[0051] 3) Test results

[0052] Table 2 Investigation of mobile phase buffer salt types

[0053]

[0054] It can be seen from the above experiments that the use of sodium octane sulfonate as an ion pair reagent to prepare a mobile phase can meet the separation requirements of various impurities under the above chromatographic conditions.

[0055] Experimental Example 2 Screening of buffer salt concentration in mobile phase

[0056] The chromatographic conditions and system suitability solution of the above-mentioned Experimental Example 1 were used, wherein sodium octane sulfonate was used as an ion pair reagent to prepare buffer salts of different concentrations as mobile phases, and the effects of mobile phases of various concentrations on the separation of impurities were investigated. The experimental scheme and results are shown in the following table.

[0057] Table 3 Investigation of mobile phase buffer salt concentration

[0058]

[0059] Under the above chromatographic conditions, the separation between the main peak and its adjacent impurities, between impurity 7 and impurity 9, and between impurity 8 and impurity 6 need to be paid special attention to, and the remaining impurities can be effectively separated. Therefore, the separation between the above components is statistically analyzed. From the test results in the above table, it can be seen that when the concentration of sodium octane sulfonate is in the range of 0.0034mol / l to 0.0040mol / l, the minimum separation between the main peak and its adjacent impurities reaches 3.6, and the minimum separation between impurity 8 and impurity 6 is not less than 1.5, and the separation meets the requirements.

[0060] It can be seen that the use of sodium octane sulfonate as an ion pair reagent to prepare the mobile phase significantly improves the column efficiency, the separation degree and sensitivity between various impurities, and achieves complete separation of various components.

[0061] Test Example 3 System Applicability Test Investigation

[0062] The chromatographic conditions and system suitability solution of the above-mentioned Experimental Example 1 were used, wherein 0.0037 mol / L sodium octane sulfonate solution-acetonitrile = 95:5 (v / v) was used as the mobile phase of this experiment.

[0063] Accurately measure 20 μl of the system suitability solution of Test Example 1, inject it into the liquid chromatograph, and record the chromatogram. The test data and results are shown in the table below and the spectrum is shown in Figure 5 .

[0064] Table 4 System suitability test results

[0065] Impurity No. Retention time (min) Separation Impurity 1 3.616 / Impurity 4 4.032 2.9 Impurity 3 4.585 3.6 Intermediate 1 7.297 12.9 SM1 8.227 3.7 Impurity 7 11.052 9.8 Impurity 9 13.155 4.5 Main Peak 16.236 3.5 Impurity 10 19.610 3.7 Impurity 8 23.433 6.0 Impurity 6 24.746 1.8

[0066] It can be seen from the above table and the chromatogram that under the chromatographic conditions of this experiment, the peak shapes of alanyl-glutamine and its 10 impurities were determined, and the separation between the main component and the impurities and between the impurities was greater than 1.5, which met the standard requirements of a separation between the main peak and the impurities greater than 1.5 and a separation between the impurities greater than 1.0, and the separation effect was good.

[0067] Test Example 4 System Durability Investigation (Column Temperature)

[0068] The chromatographic conditions and system suitability solution of the above-mentioned Test Example 1 were adopted, wherein 0.0037 mol / L sodium octane sulfonate solution-acetonitrile = 95:5 (v / v) was adopted as the mobile phase of this test, and the separation of each impurity was investigated under different chromatographic column temperature conditions (23°C, 25°C, 27°C, 30°C and 35°C).

[0069] Accurately measure 20 μl of the system suitability solution of Test Example 1, inject it into the liquid chromatograph, and record the chromatogram. The test data and results are shown in the table below.

[0070] Table 5 Column temperature investigation

[0071] Column temperature Minimum separation between main peak and impurities Minimum separation (separation of impurity 6 and impurity 8) T＝23℃ 3.6 1.7 T＝25℃ 3.6 1.5 T＝27℃ 3.6 1.3 T＝30℃ 3.5 1.1 T＝35℃ 3.6 0.3

[0072] From the above test data, it can be seen that when the column temperature is in the range of 23℃~30℃, the minimum separation between the main peak and its adjacent impurities is greater than 1.5, and the separation between the more difficult impurities 6 and 8 is greater than 1.0, which can achieve effective separation and the separation meets the requirements. At the same time, it shows that under this chromatographic condition, the column temperature is in the range of 23℃~30℃, and the durability is good.

[0073] Test Example 5 System Durability Investigation (Flow Rate)

[0074] The chromatographic conditions and test solution of the above-mentioned Test Example 1 were adopted, wherein 0.0037 mol / L sodium octane sulfonate solution-acetonitrile = 95:5 (v / v) was used as the mobile phase of this test, and the separation of each impurity under different flow rate conditions (0.8 ml / min, 0.9 ml / min and 1.0 ml / min) was investigated.

[0075] Accurately measure 20 μl of the system suitability solution of Test Example 1, inject it into the liquid chromatograph, and record the chromatogram. The test data and results are shown in the table below.

[0076] Table 6 Flow rate investigation

[0077] Flow rate Minimum separation between main peak and impurities Minimum separation between impurities (separation between impurity 6 and impurity 8) 0.8ml / min 3.7 1.5 0.9ml / min 3.7 1.4 1.0ml / min 3.6 1.3 1.1ml / min 3.6 1.4

[0078] From the test data in the table above, it can be seen that when other conditions remain unchanged, the flow rate is in the range of 0.8ml / min to 1.1ml / min, the minimum separation between the main peak and the impurity is greater than 1.5, and the separation between the more difficult impurities 6 and 8 is greater than 1.0, and the separation meets the requirements. Therefore, the flow rate of this determination method is selected to be 0.8ml / min to 1.1ml / min, and the flow rate durability is good. Test Example 6 Alanyl Glutamine API Detection

[0079] The chromatographic conditions of the above-mentioned Test Example 1 were adopted, wherein 0.0037 mol / L sodium octane sulfonate solution-acetonitrile = 95:5 (v / v) was used as the mobile phase of this test to detect the related substances in the alanyl-glutamine bulk drug. The test data are shown in the table below.

[0080] Alanyl-glutamine API Source: Homemade

[0081] Preparation of test solution: Take about 100 mg of alanyl-glutamine raw material, place it in a 25 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as the test solution.

[0082] Determination method: Accurately measure 20 μl of the test solution, inject it into the liquid chromatograph, record the chromatogram, and calculate the impurity content by the area normalization method.

[0083] Table 7 Measurement results

[0084] Impurity No. Retention time (min) Peak area% Separation Impurity 8 20.024 0.03 4.7 Impurity 6 20.671 0.04 1.3

[0085] The test results showed that impurities 8 and 6 were detected in the alanylglutamine API, and no other known impurities were detected.

[0086] The method for related substances in alanylglutamine in the 2015 edition of the Chinese Pharmacopoeia was adopted, with a chromatographic column (i.e., amino column) filled with amino-bonded silica gel, potassium dihydrogen phosphate buffer-acetonitrile (35:65) as the mobile phase, a detection wavelength of 215nm, a flow rate of 0.7ml per minute, and a column temperature of 30°C. Accurately measure 20μl of the system suitability solution of the above-mentioned test example 1, inject it into the liquid chromatograph, measure it under the chromatographic conditions, and record the chromatogram, see Figure 1 .

[0087] After the amino column was used for one and a half months under normal frequency conditions, the test was repeated again according to the test conditions. The spectrum is shown in Figure 2 .

[0088] Figure 1 It shows that when the method of related substances in the 2015 edition of the Chinese Pharmacopoeia is used, impurities 3 and 7 overlap, and baseline separation cannot be achieved, which interferes with impurity detection; the retention time of impurity 6 is close to 80 minutes, and the peak time is too late, which is not conducive to detection efficiency;

[0089] Depend on Figure 2 It can be seen that the durability of this method is poor, especially the durability of the chromatographic column is poor. After a period of use, the retention time of the chromatographic peaks of the relevant substances drifts, and the impurity 10 and the main peak are not separated by baseline, which interferes with the detection of impurities.

[0090] Comparative Example 2: Amide column determination

[0091] A triple bonded amide chromatography column (Amide) (250mm×4.6mm, 5μm) was used, with 50mmol / L potassium dihydrogen phosphate buffer (adjusted to pH=4.5): acetonitrile = (28:72) as the mobile phase, detection wavelength 210nm, column temperature 35℃, flow rate 0.5ml per minute. Accurately measure 20μl of the system suitability solution of the above test example 1, inject it into the liquid chromatograph, measure under the chromatographic conditions, and record the chromatogram, see Figure 3 .

[0092] Figure 3 It shows that, using the above chromatographic conditions, 9 known impurities were detected, and impurity 8 overlapped with the main peak and could not be separated, thus failing to meet the detection requirements.

[0093] Comparative Example 3: HILIC column determination

[0094] A Sepax hydrophilic silica gel chromatography column (HILIC) (250 mm × 4.6 mm, 5 μm) was used, with 30 mmol / L potassium dihydrogen phosphate buffer (adjusted to pH = 4.0): methanol: acetonitrile = (25:5:70) as the mobile phase, detection wavelength 215 nm, column temperature 25 ° C, flow rate 1.0 ml per minute. Accurately measure 20 μl of the above system suitability solution, inject it into the liquid chromatograph, measure under the chromatographic conditions, and record the chromatogram, see Figure 4 .

[0095] Figure 4 It shows that when the above chromatographic conditions are used, the retention time of impurities and main peak is too early, and baseline separation cannot be achieved between impurities, which interferes with impurity detection.

Claims

1. A method for determining related substances in alanyl-glutamine and its preparations, characterized in that: HPLC was used for detection. The chromatographic conditions included: the chromatographic column was filled with octadecyl amido bonded silica gel, the mobile phase was a mixed solution of sodium octane sulfonate solution and acetonitrile in a volume ratio of 95:5, the concentration of sodium octane sulfonate in the sodium octane sulfonate solution was 0.0034 mol / L to 0.0040 mol / L, the column temperature was 23 to 30°C, Among them, the related substances include L-alanyl-L-alanine, R-2-chloropropionic acid and D-2-chloropropionyl-L-glutamine.

2. The measuring method according to claim 1, characterized in that The related substances also include one or more selected from the following substances: cyclo-(L-alanyl-L-glutamine), L-pyroglutamyl-L-alanine, L-pyroglutamic acid, L-alanyl-L-glutamic acid, L-alanine, L-alanyl-L-alanyl-L-glutamine, L-glutamine.

3. The measuring method according to claim 1, characterized in that The chromatographic column is an ACE Excel chromatographic column.

4. The measuring method according to claim 3, characterized in that The chromatographic column is ACE Excel 5C18-Amide 250 mm×4.6 mm.

5. The measuring method according to claim 1, characterized in that The sodium octane sulfonate solution is a phosphoric acid aqueous solution of sodium octane sulfonate.

6. The measuring method according to claim 1, characterized in that The chromatographic conditions also include: a detection wavelength of 210 nm and a flow rate of 0.8 to 1.1 ml per minute.

7. The measuring method according to claim 6, characterized in that The flow rate was 1.0 ml per minute.

8. The measuring method according to claim 6, characterized in that The column temperature was 27°C.

9. The measuring method according to claim 1, characterized in that The chromatographic run time was 45 minutes.

10. The measuring method according to any one of claims 1 to 9, characterized in that The alanyl-glutamine preparation is selected from alanyl-glutamine injection and alanyl-glutamine for injection.

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