Detection method of acarbose drug related substances

Abstract

The invention discloses a method for detecting related substances of acarbose drugs. The method for detecting the related substances of the acarbose medicine comprises the following steps: carrying out isocratic elution by adopting a high performance liquid chromatography, and detecting an acarbose product to be detected, and the chromatographic conditions are as follows: a chromatographic column is an amino chromatographic column; a mobile phase is a mixed solution of acetonitrile and a phosphate buffer solution; the pH (Potential of Hydrogen) of the mobile phase is 6.0 to 7.2; the acarbose to-be-detected product contains acarbose and / or acarbose related substances. According to the detection method, impurities E, F and G can be better detected, and the separation degree is good.

Description

Technical Field

[0001] The present invention relates to a method for detecting acarbose-related substances. Background Art

[0002] The molecular formula of acarbose is C 25 H 43 NO 18 , molecular weight is 645.63, and its structural formula is as follows:

[0003]

[0004] Acarbose is an α-glucosidase inhibitor and a complex oligosaccharide with a structure similar to oligosaccharides. This non-oligosaccharide "pseudo-oligosaccharide" can compete with oligosaccharides at the brush border of cells in the upper small intestine and reversibly bind to α-glucosidase, inhibiting the activity of various α-glucosidases such as maltase, isomaltase, glucoamylase and sucrase, slowing down the decomposition of starch into oligosaccharides such as maltose (disaccharide), maltotriose and dextrin (oligosaccharide) and then into glucose, and slowing down the decomposition of sucrose into glucose and fructose, thus slowing down the absorption of intestinal glucose, thereby alleviating postprandial hyperglycemia and achieving the effect of lowering blood sugar. Long-term use can reduce fasting blood sugar and glycosylated hemoglobin concentrations.

[0005] As a hypoglycemic drug, the quality control of acarbose is particularly important in the production of raw materials. The detection of related substances is a key indicator of drug quality control, such as starting materials, intermediates, isomers, by-products and other substances introduced in the production of raw materials.

[0006] According to literature reports and national drug standards, the analytical methods for acarbose and acarbose tablets include the United States Pharmacopoeia method USP43, the European Pharmacopoeia method EP10.0, and the HPLC method included in the Chinese Pharmacopoeia 2020 edition. These three pharmacopoeia methods for HPLC analysis are very similar, as shown in the following table:

[0007]

[0008]

[0009]

[0010] The current acarbose detection method cannot effectively detect impurities E, F, and G. In addition, using this method, the main peak tailing is relatively serious, which will affect the detection of impurities. In view of this, a new method needs to be developed to detect acarbose and related substances in acarbose tablets. Summary of the invention

[0011] The purpose of the present invention is to solve the problem that the existing detection method for acarbose-related substances cannot detect impurities E, F, and G, and to provide a detection method for acarbose-related substances. The detection method of the present invention can better detect impurities E, F, and G with good separation.

[0012] The present invention mainly solves the above technical problems through the following technical solutions.

[0013] The present invention provides a method for detecting acarbose-related substances, which comprises the following steps: using high performance liquid chromatography to perform isocratic elution to detect acarbose to be tested, and the chromatographic conditions include:

[0014] The chromatographic column is an amino chromatographic column;

[0015] The mobile phase was a mixed solution of acetonitrile-phosphate buffer;

[0016] The pH of the mobile phase is 6.0-7.2;

[0017] The acarbose test product contains acarbose and / or acarbose-related substances.

[0018] In the present invention, the pH of the mobile phase is 7.2.

[0019] In the present invention, the filler of the amino chromatographic column may be aminopropylsilane bonded silica gel.

[0020] In the present invention, the specifications of the chromatographic column are selected according to the conventional techniques in the art. The length of the amino chromatographic column can be 150 mm-250 mm, for example, 250 mm; the inner diameter of the amino chromatographic column can be 3.0-4.6 mm, for example, 3.0 mm, 3.9 mm, 4.0 mm or 4.6 mm; the particle size of the filler in the amino chromatographic column can be 3.5 μm-5 μm, for example, 5 μm.

[0021] In the present invention, the amino chromatographic column may be Welch Ultimate XB-NH2 4.6×250 mm, 5 μm.

[0022] In the present invention, in the mobile phase, the phosphate buffer is a mixed solution of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate. Preferably, the concentration of potassium dihydrogen phosphate is 0.4 mg / mL-0.8 mg / mL, such as 0.6 mg / mL. Preferably, the concentration of anhydrous disodium hydrogen phosphate is 0.2 mg / mL-0.3 mg / mL, such as 0.279 mg / mL.

[0023] In the present invention, the volume ratio of acetonitrile to phosphate buffer is (70-75):(30-25), preferably 70:30, 71:29, 73:27 or 75:25.

[0024] In the present invention, the column temperature of the chromatographic column can be 25°C-45°C according to the conventional art, preferably 30°C, 35°C or 40°C.

[0025] In the present invention, the flow rate of the mobile phase can be 1 ml / min-2 ml / min, preferably 2 ml / min, according to the conventional practice in the art.

[0026] In the present invention, in the high performance liquid chromatography method, the injection volume is conventional in the art, and can be 10-20 μL, preferably 10 μL.

[0027] In the present invention, in the high performance liquid chromatography, the detection wavelength is conventional in the art, generally 200nm-260nm, preferably 210nm.

[0028] In the present invention, the inner diameter of the amino chromatographic column can be 4.6 mm, the filler particle size of the amino chromatographic column can be 5 μm; the length of the amino chromatographic column can be 150 mm-250 mm; the pH of the mobile phase is 7.2; the mobile phase is a mixed solution of acetonitrile-phosphate buffer; the phosphate buffer is a mixed solution of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate;

[0029] Preferably, the concentration of potassium dihydrogen phosphate is 0.4 mg / mL-0.8 mg / mL, such as 0.6 mg / mL; the concentration of anhydrous disodium hydrogen phosphate is 0.2 mg / mL-0.3 mg / mL, such as 0.279 mg / mL.

[0030] In the present invention, the acarbose test product can be various acarbose products, such as acarbose bulk drug or acarbose preparation.

[0031] In the present invention, the related substances are related substances of acarbose in the art, such as one or more of the following structures:

[0032]

[0033] In the present invention, the acarbose sample to be tested may also contain inorganic impurities.

[0034] In the present invention, the acarbose sample to be tested may further contain residual solvent.

[0035] In the present invention, the acarbose sample to be tested may further contain pharmaceutical excipients.

[0036] In the present invention, the acarbose sample to be tested may be pretreated before testing, and the pretreatment step comprises the following steps: dissolving the acarbose sample to be tested with a diluent and placing it.

[0037] The diluent may be any diluent solvent that can dissolve the acarbose test product and has no effect on subsequent detection, such as water.

[0038] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0039] The reagents and raw materials used in the present invention are commercially available.

[0040] The positive improvement effect of the present invention is that the detection method of the present invention can simultaneously detect impurities A, B, C, D, E, F and G in acarbose-related substances with good separation degree. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Example 1

[0042] Figure 2 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Example 2

[0043] Figure 3 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Comparative Example 1

[0044] Figure 4 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Comparative Example 2

[0045] Figure 5 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Comparative Example 3

[0046] Figure 6 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Comparative Example 4

[0047] Figure 7 This is the liquid chromatogram of the acarbose peak identification solution obtained by analysis in Comparative Example 5 DETAILED DESCRIPTION

[0048] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0049] The names and structural formulas of the related substances of acarbose in the present invention are shown in the following table

[0050]

[0051] Example 1

[0052] Chromatographic conditions

[0053] Chromatographic column: Welch Ultimate XB-NH2 4.6×250mm, 5μm

[0054] Mobile phase: acetonitrile-buffer (71:29), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolve in water and dilute to 1000 ml; adjust pH to 6.0 with 2 mol / L sodium hydroxide solution

[0055] Column temperature: 35°C

[0056] Flow rate: 2.0ml / min

[0057] Injection volume: 10 μl

[0058] Detection wavelength: 210nm

[0059] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance (EP standard substance), add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0060] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 1 And as shown in Table 1.

[0061] Table 1

[0062]

[0063] Example 2

[0064] Chromatographic conditions

[0065] Chromatographic column: Welch Ultimate XB-NH2 4.6×250mm, 5μm

[0066] Mobile phase: acetonitrile-buffer (71:29), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolve in water and dilute to 1000 ml; adjust the pH to 7.2 with 2 mol / L sodium hydroxide solution.

[0067] Column temperature: 35°C

[0068] Flow rate: 2.0ml / min

[0069] Injection volume: 10 μl

[0070] Detection wavelength: 210nm

[0071] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance (EP standard substance), add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0072] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 2 And as shown in Table 2.

[0073] Table 2

[0074]

[0075]

[0076] After the pH of the buffer in the mobile phase was adjusted to 7.2, impurity A became a single peak, and the separation degree between impurity A and acarbose was 1.7; the theoretical plate number of acarbose was 3984, which was greater than 2000 and qualified; the separation degrees of the remaining impurities and the main peak were all greater than 2.0, which were qualified. This method is suitable for the detection of related substances.

[0077] Comparative Example 1 Pharmacopoeia Method Reproduction-1

[0078] Chromatographic conditions:

[0079] Chromatographic column: Welch Ultimate XB-NH2 4.6×250mm, 5μm

[0080] Mobile phase: acetonitrile-buffer (75:25), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml, pH 6.8.

[0081] Column temperature: 35°C

[0082] Flow rate: 2ml / min

[0083] Injection volume: 10 μl

[0084] Detection wavelength: 210nm

[0085] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance, add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0086] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 3 And as shown in Table 3.

[0087] Table 3

[0088]

[0089] Comparative Example 2 Pharmacopoeia Method Reproduction-2

[0090] Chromatographic conditions:

[0091] Chromatographic column: Agilent Zorbox NH2 4.6×250mm, 5μm

[0092] Mobile phase: acetonitrile-buffer (75:25), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml, pH 6.8.

[0093] Column temperature: 35°C

[0094] Flow rate: 2ml / min

[0095] Injection volume: 10 μl

[0096] Detection wavelength: 210nm

[0097] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance, add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0098] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 4 And as shown in Table 4.

[0099] Table 4

[0100]

[0101] Comparative Example 3 Pharmacopoeia Method Reproduction-3

[0102] Chromatographic conditions:

[0103] Chromatographic column: Thermo Fisher Hypersil APS-2 4.0mm×250mm, 5μm

[0104] Mobile phase: acetonitrile-buffer (75:25), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml, pH 6.8.

[0105] Column temperature: 35°C

[0106] Flow rate: 2ml / min

[0107] Injection volume: 10 μl

[0108] Detection wavelength: 210nm

[0109] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance, add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0110] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 5 And as shown in Table 5.

[0111] Table 5

[0112]

[0113] Comparative Example 4 Pharmacopoeia Method Reproduction-4

[0114] Chromatographic conditions:

[0115] Chromatographic column: Waters Spherisorb Amino 4.6mm×150mm, 5μm

[0116] Mobile phase: acetonitrile-buffer (75:25), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml, pH 6.8.

[0117] Column temperature: 35°C

[0118] Flow rate: 1.0ml / min

[0119] Injection volume: 10 μl

[0120] Detection wavelength: 210nm

[0121] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance, add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0122] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 6 and as shown in Table 6.

[0123] Table 6

[0124]

[0125] Comparative Example 5 Pharmacopoeia Method Reproduction-5

[0126] Chromatographic conditions:

[0127] Chromatographic column: Fei Nuo Mei Luna NH2 4.6mm×250mm, 5μm

[0128] Mobile phase: acetonitrile-buffer (75:25), buffer: 0.6 g potassium dihydrogen phosphate and 0.279 g anhydrous disodium hydrogen phosphate, dissolved in water and diluted to 1000 ml, pH 6.8.

[0129] Column temperature: 35°C

[0130] Flow rate: 2.0ml / min

[0131] Injection volume: 10 μl

[0132] Detection wavelength: 210nm

[0133] Preparation of test solution: Take 20 mg of acarbose peak identification reference substance, add 1 ml of purified water to dissolve, and the acarbose peak identification solution is obtained.

[0134] According to the above chromatographic conditions, accurately measure 10 μL of the test solution and inject it into the liquid chromatograph, isocratically elute, record the chromatogram, and the test results are as follows. Figure 7 And as shown in Table 7.

[0135] Table 7

[0136]

[0137]

Claims

1. A method for detecting acarbose-related substances, characterized in that: The method comprises the following steps: using high performance liquid chromatography to perform isocratic elution to detect acarbose to be tested, and the chromatographic conditions include: The chromatographic column is an amino chromatographic column; The mobile phase was a mixed solution of acetonitrile-phosphate buffer; The pH of the mobile phase is 6.0-7.2; The acarbose test product contains acarbose and / or acarbose-related substances.

2. The method for detecting acarbose-related substances according to claim 1, characterized in that: The detection method meets one or more of the following conditions: (1) The pH of the mobile phase is 7.2; (2) The filler of the amino chromatographic column is aminopropylsilane bonded silica gel; (3) The length of the amino chromatographic column is 150 mm to 250 mm; (4) The inner diameter of the amino chromatographic column is 3.0-4.6 mm; and (5) The filler particle size in the amino chromatographic column is 3.5 μm-5 μm.

3. The method for detecting acarbose-related substances according to claim 2, characterized in that: The detection method meets one or more of the following conditions: (1) The amino chromatographic column is 250 mm in length; (2) The inner diameter of the amino chromatographic column is 3.0 mm, 3.9 mm, 4.0 mm or 4.6 mm; and (3) The filler particle size in the amino chromatographic column is 5 μm.

4. The method for detecting acarbose-related substances according to claim 1, characterized in that: The detection method meets one or more of the following conditions: (1) The volume ratio of acetonitrile to phosphate buffer is (70-75):(30-25); (2) The phosphate buffer is a mixed solution of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate; (3) The column temperature of the amino chromatographic column is 25°C-45°C; (4) the flow rate of the mobile phase is 1 ml / min-2 ml / min; and (5) The amino chromatography column is Ultimate XB-NH2 4.6×250 mm, 5 μm.

5. The method for detecting acarbose-related substances according to claim 4, characterized in that: The detection method meets one or more of the following conditions: (1) The volume ratio of acetonitrile to phosphate buffer is 70:30, 71:29, 73:27 or 75:25; (2) The concentration of potassium dihydrogen phosphate is 0.4 mg / mL-0.8 mg / mL; (3) The concentration of the anhydrous disodium hydrogen phosphate is 0.2 mg / mL-0.3 mg / mL; (4) the column temperature of the chromatographic column is 30°C, 35°C or 40°C; and (5) The flow rate of the mobile phase is 2 ml / min.

6. The method for detecting acarbose-related substances according to claim 5, characterized in that: The detection method meets one or both of the following conditions: (1) the concentration of potassium dihydrogen phosphate is 0.6 mg / mL; and (2) The concentration of the anhydrous disodium hydrogen phosphate is 0.279 mg / mL.

7. The method for detecting acarbose-related substances according to any one of claims 1 to 6, characterized in that: The inner diameter of the amino chromatographic column is 4.6 mm; the filler particle size of the amino chromatographic column is 5 μm; the length of the amino chromatographic column is 150 mm-250 mm; the mobile phase is a mixed solution of acetonitrile-phosphate buffer; the phosphate buffer is a mixed solution of potassium dihydrogen phosphate and anhydrous disodium hydrogen phosphate; the pH of the mobile phase is 7.

2.

8. The method for detecting acarbose-related substances according to claim 7, characterized in that: The concentration of the potassium dihydrogen phosphate is 0.6 mg / mL; the concentration of the anhydrous disodium hydrogen phosphate is 0.279 mg / mL.

9. The method for detecting acarbose-related substances according to claim 1, characterized in that: The detection method meets one or both of the following conditions: (1) the acarbose test product is an acarbose bulk drug or an acarbose preparation; and (2) The acarbose sample to be tested contains inorganic impurities, residual solvents or pharmaceutical excipients.

10. The method for detecting acarbose-related substances according to claim 1, characterized in that: The related substances are one or more of the following structures:

Images