Method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and preparation thereof by high performance liquid chromatography

Abstract

The invention discloses a method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and a preparation thereof by high performance liquid chromatography. An adopted chromatographic column is an octadecyl silane bonded silica gel chromatographic column or an octane bonded silica gel chromatographic column; carrying out isocratic elution by adopting a mobile phase A and a mobile phase B as a mixed mobile phase; the mobile phase A is a monopotassium phosphate solution; the mobile phase B is acetonitrile or methanol. According to the method, the content of the specific impurity 5-hydroxymethyl-2-furancarboxylic acid in the calcium gluconate and the preparation thereof can be measured, so that the product quality of the calcium gluconate and the preparation thereof can be better monitored to make up the blank in the field, and the method is good in specificity, good in linearity, relatively high in sensitivity and good in repeatability, and is suitable for industrial production. The precision is good, and the accuracy is high.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and in particular relates to a method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations by high performance liquid chromatography. Background Art

[0002] Calcium gluconate and its preparations are calcium ion supplements, which can promote the calcification of bones and teeth. They are mainly used clinically for: (1) treatment of calcium deficiency, acute hypocalcemia, alkalosis and tetany caused by hypoparathyroidism; (2) allergic diseases; (3) rescue of magnesium poisoning; (4) rescue of fluoride poisoning; (5) application during cardiac resuscitation.

[0003] At present, the preparation methods of calcium gluconate mainly adopt glucose oxidation method and glucono-δ-lactone hydrolysis method, among which 5-hydroxymethyl-2-furancarboxylic acid is a degradation product in the preparation process of calcium gluconate.

[0004] However, neither the "Pharmacopoeia of the People's Republic of China (2020 Edition)" nor the common foreign pharmacopoeias have added an inspection item for the content of impurity 5-hydroxymethyl-2-furancarboxylic acid under the items of calcium gluconate and its preparations. Studying the content of 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations is crucial to improving the product quality of calcium gluconate and its preparations. Summary of the invention

[0005] The object of the present invention is to solve the above problems and provide a method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations by high performance liquid chromatography to fill the gap in this field.

[0006] The technical scheme for achieving the purpose of the present invention is: a method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations by high performance liquid chromatography, wherein the chromatographic column adopted is an octadecylsilane bonded silica gel chromatographic column or an octane bonded silica gel chromatographic column; a mobile phase A and a mobile phase B are used as a mixed mobile phase for isocratic elution; the mobile phase A is a potassium dihydrogen phosphate solution; and the mobile phase B is acetonitrile or methanol.

[0007] The concentration of the potassium dihydrogen phosphate solution is 0.01-0.10 mol / L, preferably 0.03 mol / L.

[0008] The pH value of the potassium dihydrogen phosphate solution is 2.5-3.5, preferably 2.8.

[0009] The isocratic elution conditions are as follows: mobile phase A: mobile phase B = 78:22-88:12, preferably mobile phase A: mobile phase B = 80:20.

[0010] Other chromatographic conditions of the HPLC are as follows: The column temperature is 20-40°C, preferably 35°C.

[0011] The ultraviolet detection wavelength is 200-300 nm, preferably 260 nm.

[0012] The flow rate of the mobile phase is 0.5 to 1.5 mL / min, preferably 1.0 mL / min.

[0013] The present invention has the positive effects that the method of the present invention can determine the content of the specific impurity 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations, so as to better monitor the product quality of calcium gluconate and its preparations to fill the gap in the field, and the method of the present invention has good specificity, good linearity, high sensitivity, good repeatability, good precision and high accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the HPLC chromatogram of the blank solution in Example 1.

[0015] Figure 2 It is the HPLC chromatogram of the blank auxiliary material solution in Example 1.

[0016] Figure 3 It is the HPLC chromatogram of the reference substance solution in Example 1.

[0017] Figure 4 It is the HPLC chromatogram of the test solution in Example 1.

[0018] Figure 5 This is the standard curve of 5-hydroxymethyl-2-furancarboxylic acid in Experimental Example 1.

[0019] Figure 6 This is the HPLC chromatogram of the quantitative limit solution in Experimental Example 2.

[0020] Figure 7 This is the HPLC chromatogram of the detection limit solution in Experimental Example 2. DETAILED DESCRIPTION

[0021] (Example 1) 1. Instruments and chromatographic conditions.

[0022] Chromatographic column: Octadecylsilane bonded silica gel chromatographic column (250 mm × 4.6 mm × 3 μm).

[0023] Column temperature: 35°C.

[0024] UV detection wavelength: 260nm.

[0025] Mobile phase flow rate: 1.0 mL / min.

[0026] Mobile phase A was 0.03 mol / L potassium dihydrogen phosphate solution, and the pH value was adjusted to 2.8 with phosphoric acid.

[0027] Mobile phase B was methanol.

[0028] The isocratic elution conditions were as follows: mobile phase A: mobile phase B = 80:20.

[0029] Diluent: Water.

[0030] Injection volume: 20 μL.

[0031] 2. The experimental methods are as follows: 1. Solution preparation.

[0032] 1.1. Blank solution (diluent): water.

[0033] 1.2. Blank auxiliary material solution: aqueous solution of calcium glucarate in hydrochloric acid.

[0034] 1.3. Reference solution (10 μg / mL): Accurately weigh 1 mg of 5-hydroxymethyl-2-furancarboxylic acid reference substance and place it in a 100 mL volumetric flask. First add a small amount of diluent to dissolve it, then dilute it to the scale with diluent, shake well, and use it as the reference solution.

[0035] 1.4. Test solution (100 mg / mL): calcium gluconate injection.

[0036] 2. Test method.

[0037] Accurately measure 20 μL of the blank solution, blank excipient solution, reference solution, and test solution, respectively, and inject them into the chromatograph. Perform HPLC analysis according to the above chromatographic conditions and record the chromatograms. The results are shown in Table 1. Figure 1 to Figure 4 .

[0038] Depend on Figure 1 to Figure 4 It can be seen that the blank solution and the blank auxiliary material solution of the present invention do not interfere with the determination of 5-hydroxymethyl-2-furancarboxylic acid, indicating that the method of the present invention has good specificity.

[0039] (Test Example 1) This test example is a linearity and range test.

[0040] Linear stock solution (0.5 mg / mL): Accurately weigh 50 mg of 5-hydroxymethyl-2-furancarboxylic acid and place it in a 100 mL volumetric flask. First add a small amount of diluent to dissolve it, then dilute it to the scale with the diluent, shake well, and use it as the linear stock solution.

[0041] 10% linear solution: Take 1.0mL of linear stock solution, place it in a 250mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0042] 20% linear solution: Take 2.0mL of linear stock solution, place it in a 250mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0043] 50% linear solution: Take 5.0mL of linear stock solution, place it in a 250mL volumetric flask, add diluent to the scale, shake well, and the solution is ready.

[0044] 100% linear solution (20 μg / mL): Take 2.0 mL of the linear stock solution, place it in a 50 mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0045] 150% linear solution: Take 3.0mL of linear stock solution, place it in a 50mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0046] 200% linear solution: Take 4.0mL of linear stock solution, place it in a 50mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0047] 0.5% linear solution (quantitation limit solution): Take 1.0 mL of 100% linear solution, place it in a 200 mL volumetric flask, add diluent to the scale, shake well, and obtain.

[0048] Accurately measure 20 μL of each of the above linear solutions and inject them into the chromatograph respectively. Perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1 respectively, record the chromatograms, calculate the peak areas, and the results are shown in Table 1.

[0049] The standard curve was drawn with the concentration of the quantitative limit solution and each linear solution (μg / mL) as the horizontal axis and the peak area of ​​5-hydroxymethyl-2-furancarboxylic acid as the vertical axis (see Figure 5 ) and proposed a linear regression equation, and the linear regression equation was: y=1.6224x+0.0124, R²=0.9999, r=1.0000.

[0050] Table 1 5-Hydroxymethyl-2-furancarboxylic acid concentration (μg / mL) 5-Hydroxymethyl-2-furancarboxylic acid peak area 0.5% linear solution 0.0991 0.0155 10% linear solution 1.9812 3.2104 20% linear solution 3.9624 6.4379 50% linear solution 9.906 16.3057 100% linear solution 19.812 32.3463 150% linear solution 29.7121 47.8765 200% linear solution 39.6240 64.4073

[0051] The results showed that 5-hydroxymethyl-2-furancarboxylic acid had good linearity in the concentration range of 0.0991 to 39.6240 μg / ml.

[0052] (Test Example 2) This test example is a detection limit and quantification limit test.

[0053] The 0.5% linear solution in Test Example 1 was used as the quantification limit solution, and the quantification limit solution was diluted 2 times as the detection limit solution.

[0054] Accurately measure 20 μL of the quantitative limit solution and the detection limit solution, respectively, and inject them into the chromatograph, and perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, and record the chromatograms. The results are shown in Figure 6 and Figure 7 And Table 2.

[0055] Table 2

[0056] Depend on Figure 6 and Figure 7 As can be seen from Table 2, the quantitative limit is 0.0001%, the quantitative limit signal-to-noise ratio is greater than 10, the detection limit is 0.00005%, and the detection limit signal-to-noise ratio is greater than 3, indicating that the method of the present invention has high sensitivity.

[0057] (Test Example 3) This test example is a repeatability test of the test solution.

[0058] Accurately measure 20 μL of each of 6 test sample solutions and inject them into the chromatograph respectively. Perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, calculate the peak area and content of 5-hydroxymethyl-2-furancarboxylic acid, and calculate the relative standard deviation. The results are shown in Table 3.

[0059] Table 3

[0060] It can be seen from Table 3 that the relative standard deviation RSD (n=6) is less than 2.0%, indicating that the detection method of the present invention has good repeatability.

[0061] (Test Example 4) This test example is a repeatability test of the sample solution with 100% limit addition.

[0062] 100% limit loading sample solution: Accurately weigh 50 mg of 5-hydroxymethyl-2-furancarboxylic acid and place it in a 100 mL volumetric flask. Add the test solution to the mark and shake well to use as the limit loading stock solution (0.5 mg / mL).

[0063] Take 2.0 mL of the limit loading stock solution, place it in a 50 mL volumetric flask, add the test solution to the scale, shake well, and the 100% limit loading sample solution (20 μg / mL) is obtained.

[0064] Accurately measure 6 portions of 100% limit sample solution, 20 μL each, inject into the chromatograph, perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, calculate the recovery rate and relative standard deviation, and the results are shown in Table 4.

[0065] Table 4

[0066] It can be seen from Table 4 that the recovery rate is between 97.40% and 99.75%, the average value is 98.54%, and the relative standard deviation RSD (n=6) is 0.9%, which is less than 2.0%, indicating that the detection method of the present invention has good repeatability.

[0067] (Test Example 5) This test example is an intermediate precision test.

[0068] Different people, different days, and different instruments repeated the determination of Test Example 4 once (6 parallel copies), and calculated the average recovery rate and relative standard deviation with the 6 test results of Test Example 4. The results are shown in Table 5.

[0069] Table 5

[0070] It can be seen from Table 5 that the recovery rate is still between 97.40% and 99.75%, with an average value of 98.79%, and the relative standard deviation RSD (n=12) is 0.7%, which is less than 2.0%, indicating that the detection method of the present invention has good precision.

[0071] (Test Example 6) This test example is an accuracy test.

[0072] 50% limit loading sample solution: Take 1.0 mL of the limit loading stock solution of Test Example 4, place it in a 50 mL volumetric flask, add the test solution to the scale, shake well, and the 50% limit loading sample solution (10 μg / mL) is obtained.

[0073] 150% limit loading sample solution: Take 3.0 mL of the limit loading stock solution of Test Example 4, put it in a 50 mL volumetric flask, add the test solution to the scale, shake well, and the 150% limit loading sample solution (30 μg / mL) is obtained.

[0074] Accurately measure 20 μL of each of three 50% limit sample solutions and three 150% limit sample solutions, inject them into the chromatograph, perform high performance liquid chromatography analysis according to the chromatographic conditions of Example 1, record the chromatogram, calculate the recovery rate, and calculate the average recovery rate and relative standard deviation together with the 6 test results of Test Example 4. The results are shown in Table 6.

[0075] Table 6

[0076] It can be seen from Table 6 that the recovery rate is still between 97.40% and 99.75%, with an average value of 98.65%, and the relative standard deviation RSD (n=12) is 0.8%, which is less than 2.0%, indicating that the detection method of the present invention has good accuracy.

Claims

1. A method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations by high performance liquid chromatography, characterized in that: The chromatographic column used is an octadecylsilane bonded silica gel chromatographic column or an octane bonded silica gel chromatographic column; mobile phase A and mobile phase B are used as a mixed mobile phase for isocratic elution; the mobile phase A is a potassium dihydrogen phosphate solution; and the mobile phase B is acetonitrile or methanol.

2. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparation by high performance liquid chromatography according to claim 1, characterized in that: The concentration of the potassium dihydrogen phosphate solution is 0.01-0.10 mol / L, and the pH value of the potassium dihydrogen phosphate solution is 2.5-3.

5.

3. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparation by high performance liquid chromatography according to claim 2, characterized in that: The concentration of the potassium dihydrogen phosphate solution is 0.03 mol / L, and the pH value of the potassium dihydrogen phosphate solution is 2.

8.

4. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparation by high performance liquid chromatography according to claim 1, characterized in that: The isocratic elution conditions are as follows: mobile phase A: mobile phase B = 78:22-88:

12.

5. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparation by high performance liquid chromatography according to claim 4, characterized in that: The isocratic elution conditions are as follows: mobile phase A: mobile phase B = 80:

20.

6. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparation by high performance liquid chromatography according to claim 1, characterized in that: The temperature of the chromatographic column is 20-40°C; the ultraviolet detection wavelength is 200-300nm; and the flow rate of the mobile phase is 0.5-1.5mL / min.

7. The method for determining 5-hydroxymethyl-2-furancarboxylic acid in calcium gluconate and its preparations by high performance liquid chromatography according to claim 6, characterized in that: The column temperature was 35°C; the UV detection wavelength was 260 nm; and the mobile phase flow rate was 1.0 mL / min.

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