Method for detecting gluconic acid related substances in compound calcium gluconate oral solution

Abstract

The invention relates to a method for detecting gluconic acid related substances in a compound calcium gluconate oral solution, which belongs to the field of detection, and adopts correction factor determination to realize accurate quantification of D-glucose-gamma-lactone and D-glucose-delta-lactone. Through the determination of the correction factor, a reference substance solution is not needed in the subsequent detection process, so that the problem that the detection accuracy is still difficult to meet when the reference substance solution is prepared at present is solved, and the detection efficiency of a large number of samples in actual production is remarkably improved. The method can be used as a method for detecting D-glucose-gamma-lactone and D-glucose-delta-lactone in gluconic acid-containing samples such as a compound calcium gluconate oral solution, has the characteristics of high specificity, high sensitivity, high accuracy, good durability and the like, has the advantages of universality, practicability and the like, and is simpler, more convenient and more efficient to operate.

Description

Technical Field

[0001] The invention belongs to the field of detection, and in particular relates to a method for detecting gluconic acid-related substances in a compound calcium gluconate oral solution. Background Art

[0002] Compound calcium gluconate oral solution is used for the prevention and auxiliary treatment of calcium deficiency, such as osteoporosis, rickets, etc. It can also be used for calcium supplementation for children, lactating women, menopausal women and the elderly. Calcium gluconate is the raw material drug of compound calcium gluconate oral solution, which is involved in bone formation and reconstruction of bone tissue after fracture, as well as muscle contraction, nerve transmission, coagulation mechanism and reduces capillary permeability. Calcium lactate is the raw material drug of compound calcium gluconate oral solution, which has the effects of promoting the calcification of bones and teeth, maintaining the normal excitability of nerves and muscles and reducing capillary permeability.

[0003] Compound calcium gluconate oral solution is a compound preparation whose main ingredients are calcium gluconate and calcium lactate. During the production process, calcium gluconate will produce two substances, D-gluconate-γ-lactone and D-gluconate-δ-lactone, which are related substances of gluconic acid.

[0004] When D-glucose-γ-lactone and D-glucose-δ-lactone in compound calcium gluconate oral solution are detected by liquid chromatography, if the external standard method is used, reference solutions of D-glucose-γ-lactone and D-glucose-δ-lactone need to be prepared. Although D-glucose-γ-lactone and D-glucose-δ-lactone are soluble in water, they are unstable in aqueous solution. D-glucose-γ-lactone, D-glucose-δ-lactone and gluconic acid are mutually converted, resulting in that even if the reference solution is prepared on the spot, it is still difficult to meet the accuracy of the detection. For the detection of a large number of samples in actual production, the preparation of the reference solution on the spot significantly reduces the detection efficiency of the samples.

[0005] Compound calcium gluconate oral solution is a chemical compound preparation with national drug quality standards. The current quality standards include tests for properties, identification, inspection, and content determination, but no determination of gluconic acid-related substances. Other standards only have methods for controlling D-glucose-γ-lactone and D-glucose-δ-lactone separately, but no method for simultaneously determining D-glucose-γ-lactone and D-glucose-δ-lactone, which does not fully control the overall quality of the product. Summary of the invention

[0006] The present invention provides a method for detecting gluconic acid related substances in a compound calcium gluconate oral solution. The present invention adopts a correction factor determination to achieve accurate quantification of D-glucose-γ-lactone and D-glucose-δ-lactone. Through the determination of the correction factor, there is no need to use a reference solution in the subsequent detection process, which solves the problem of using a reference solution prepared on the spot and the problem of instability of the reference solution prepared on the spot, which affects the accuracy of detecting gluconic acid related substances in the compound calcium gluconate oral solution. The present invention improves the separation degree of D-glucose-γ-lactone and D-glucose-δ-lactone by adjusting different pH values ​​of a dipotassium hydrogen phosphate solution in a mobile phase.

[0007] The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution of the present invention is carried out according to the following steps:

[0008] Step 1, test solution: accurately measure the compound calcium gluconate oral solution, place it in a volumetric bottle, dilute it to the scale with a solvent, and shake it well; the solvent is 0.01-0.08 mol / L potassium hydrogen phosphate solution;

[0009] Step 2, control solution: accurately measure the test solution, place it in a volumetric bottle, dilute it to the scale with a solvent, and shake it well; accurately measure the above solution, place it in a volumetric bottle, dilute it to the scale with a solvent, and shake it well; the solvent is 0.01-0.08 mol / L potassium dihydrogen phosphate solution;

[0010] Step 3, chromatographic conditions: amide bonded silica gel is used as filler; 0.01-0.08 mol / L potassium dihydrogen phosphate solution-acetonitrile is used as mobile phase A, and 0.01-0.08 mol / L potassium dihydrogen phosphate solution-acetonitrile is used as mobile phase B; detection wavelength is 190-220 nm; injection volume is 10 μL; flow rate is 0.2-0.8 mL per minute; column temperature is 25-35°C; gradient elution mode is:

[0011]

[0012] Among them, the pH value of dipotassium hydrogen phosphate solution is 4.5 to 5.5;

[0013] Step 4. Calculate the content of gluconic acid related substances: Calculate the content of gluconic acid related substances in the compound calcium gluconate oral solution by the peak area using the principal component self-reference method with the addition of a correction factor.

[0014] Furthermore, the volume ratio of the dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase A is 30:70.

[0015] Furthermore, the volume ratio of the dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase B is 55:45.

[0016] Furthermore, the solvent is a 0.02 mol / L dipotassium hydrogen phosphate solution.

[0017] Furthermore, the pH value of the dipotassium hydrogen phosphate solution is 5.0.

[0018] Furthermore, the detection wavelength is 190 nm.

[0019] Further, the flow rate was 0.5 mL per minute.

[0020] Furthermore, the correction factor is the correction factor of D-glucose-γ-lactone and D-glucose-δ-lactone; wherein the correction factor of D-glucose-γ-lactone is 1.17, and the correction factor of D-glucose-δ-lactone is 1.06.

[0021] The present invention has the following beneficial effects:

[0022] The invention provides a method for detecting gluconic acid-related substances in a compound calcium gluconate oral solution. The method controls the preparation and injection time of D-glucose-γ-lactone and D-glucose-δ-lactone reference substance solutions within 10 minutes, accurately determines the correction factors of D-glucose-γ-lactone and D-glucose-δ-lactone, adopts the correction factor determination, realizes the accurate quantification of D-glucose-γ-lactone and D-glucose-δ-lactone, reduces the dosage of the reference substance and increases the stability of the detection.

[0023] By determining the correction factor, there is no need to use reference solution in the subsequent detection process, which solves the problem that the reference solution used for fresh preparation is still difficult to meet the detection accuracy, and significantly improves the detection efficiency of a large number of samples in actual production.

[0024] The mobile phases used in the existing methods for detecting D-glucose-γ-lactone and D-glucose-δ-lactone are all ion pair reagents, and there is no detection method using dipotassium hydrogen phosphate and acetonitrile aqueous solution as the mobile phase. The present invention uses dipotassium hydrogen phosphate and acetonitrile aqueous solution as the mobile phase, and by adjusting the pH value of the dipotassium hydrogen phosphate solution, the separation degree is significantly higher than that of other mobile phase detection systems.

[0025] The present invention can be used as a method for detecting D-glucose-γ-lactone and D-glucose-δ-lactone in samples containing gluconic acid such as compound calcium gluconate oral solution. The method has the characteristics of strong specificity, high sensitivity, high accuracy, good durability, etc., and has the advantages of universality and practicality, and is simpler and more efficient in operation.

[0026] The present invention adds the content determination of D-glucose-γ-lactone and D-glucose-δ-lactone on the basis of the existing quality standards, improves the quality control standard, can more comprehensively and effectively control the quality of the product, can be used as an effective technical means for quality control and process stability inspection, and is of great significance to improving product quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The chromatogram of the D-glucose-γ-lactone reference substance with retention time 8.004 minutes and 0 minutes;

[0028] Figure 2 The chromatogram of the D-glucose-γ-lactone reference substance with a retention time of 8.007 minutes and a 5-minute period;

[0029] Figure 3 The chromatogram of the D-glucose-γ-lactone reference substance with a retention time of 8.006 minutes and a 10-minute period;

[0030] Figure 4 The chromatogram of the D-glucose-γ-lactone reference substance with a retention time of 8.009 minutes and a 20-minute period;

[0031] Figure 5 The chromatogram of the D-glucose-γ-lactone reference substance with a retention time of 8.009 minutes and a 30-minute period;

[0032] Figure 6 The chromatogram of the D-glucose-δ-lactone reference substance with retention time 10.151 minutes and 0 minutes;

[0033] Figure 7 The chromatogram of the D-glucose-δ-lactone reference substance with a retention time of 10.147 minutes and a 5-minute period;

[0034] Figure 8 The chromatogram of the D-glucose-δ-lactone reference substance with a retention time of 10.147 minutes and a 10-minute period;

[0035] Fig. 9 The chromatogram of the D-glucose-δ-lactone reference substance with a retention time of 10.147 minutes and a 20-minute period;

[0036] Fig.10 This is the chromatogram of the D-glucose-δ-lactone reference substance with a retention time of 10.150 minutes and a 30-minute period.

[0037] Fig.11 The chromatogram of compound calcium gluconate oral solution is shown in Figure 1, with a retention time of 7.990 minutes for D-glucose-γ-lactone and 10.083 minutes for D-glucose-δ-lactone. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0039] The exemplary embodiments of the present invention and the description thereof are used to explain the present invention but are not intended to limit the present invention.

[0040] Example 1

[0041] In this example, D-glucose-γ-lactone and D-glucose-δ-lactone reference substances were accurately weighed, dissolved in solvents and diluted into solutions of a certain concentration, and 10 μL of the reference substance solutions were accurately measured at 0, 5, 10, 20, and 30 minutes, respectively, injected into a liquid chromatograph, and the chromatograms were recorded to examine the peak areas of the D-glucose-γ-lactone and D-glucose-δ-lactone reference substance solutions. The results are shown in the attached Figures 1 to 10 and Table 1.

[0042] Table 1 Results of stability tests on D-glucose-γ-lactone and D-glucose-δ-lactone

[0043]

[0044] By the attached Figures 1 to 10 From the measurement results in Table 1, it can be seen that the peak areas of D-glucose-γ-lactone and D-glucose-δ-lactone are basically unchanged from 0 to 10 minutes, and the peak areas are significantly reduced from 10 to 30 minutes. D-glucose-γ-lactone, D-glucose-δ-lactone and gluconic acid are converted into each other, indicating that the D-glucose-γ-lactone and D-glucose-δ-lactone reference solution is stable within 10 minutes.

[0045] In this example, the correction factors of D-glucose-γ-lactone and D-glucose-δ-lactone were determined according to the above-determined conclusion that the D-glucose-γ-lactone and D-glucose-δ-lactone reference substance solutions were stable within 10 minutes.

[0046] Accurately weigh appropriate amounts of sodium gluconate reference substance, D-glucose-γ-lactone reference substance and D-glucose-δ-lactone reference substance, and prepare solutions of different concentrations respectively. The preparation and injection time of D-glucose-γ-lactone and D-glucose-δ-lactone reference substance solutions were controlled within 10 minutes, and the samples were injected and the chromatograms were recorded. The regression curves of gluconic acid, D-glucose-γ-lactone and D-glucose-δ-lactone concentrations versus their peak areas were drawn respectively. The correction factors of D-glucose-γ-lactone and D-glucose-δ-lactone were calculated by the ratio of the slope of the regression line of gluconic acid to the slope of the regression line of D-glucose-γ-lactone and D-glucose-δ-lactone, respectively. The correction factors of D-glucose-γ-lactone and D-glucose-δ-lactone were accurately determined. The correction factor of D-glucose-γ-lactone was 1.17, and the correction factor of D-glucose-δ-lactone was 1.06. The correction factor is used to achieve accurate quantification of D-glucose-γ-lactone and D-glucose-δ-lactone, reduce the amount of reference substances and increase the stability of detection.

[0047] Example 2

[0048] This embodiment uses dipotassium hydrogen phosphate and acetonitrile aqueous solution as mobile phases A and B, and examines the separation degree of D-glucose-γ-lactone and D-glucose-δ-lactone by adjusting the pH value of the dipotassium hydrogen phosphate solution in the mobile phases A and B. The results are shown in Table 2. As can be seen from Table 2, under the condition of a pH value of 5.0, the separation degree of D-glucose-γ-lactone and D-glucose-δ-lactone can be completely separated. The separation degree of D-glucose-γ-lactone and D-glucose-δ-lactone in the method of this embodiment is significantly higher than that of other mobile phase detection systems.

[0049] Table 2 Separation degree of D-glucose-γ-lactone and D-glucose-δ-lactone at different pH values

[0050] pH value of potassium hydrogen phosphate solution Separation 4.0 Unseparated 5.0 Separation 5.5 Not completely separated

[0051] Example 3

[0052] The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution of the present embodiment is as follows:

[0053] D-glucose-γ-lactone and D-glucose-δ-lactone were determined according to high performance liquid chromatography (General Rules 0512 of Part IV of the Chinese Pharmacopoeia 2020 Edition).

[0054] Test solution: Accurately measure 5 mL of the product, place it in a 20 mL volumetric flask, dilute to the scale with solvent, and shake well.

[0055] Control solution: Accurately measure 1 mL of the test solution, place it in a 20 mL volumetric flask, dilute it to the mark with solvent, and shake well; Accurately measure 1 mL of the above solution, place it in a 10 mL volumetric flask, dilute it to the mark with solvent, and shake well.

[0056] Chromatographic conditions: amide-bonded silica gel was used as filler; 0.02 mol / L dipotassium hydrogen phosphate solution (adjusted to pH 5.0 with 3 mol / L phosphoric acid)-acetonitrile (30:70) was used as mobile phase A, and 0.02 mol / L dipotassium hydrogen phosphate solution (adjusted to pH 5.0 with 3 mol / L phosphoric acid)-acetonitrile (55:45) was used as mobile phase B; the detection wavelength was 190 nm; the injection volume was 10 μL; the flow rate was 0.5 mL per minute; the column temperature was 30°C; the solvent was 0.02 mol / L dipotassium hydrogen phosphate solution (adjusted to pH 5.0 with 3 mol / L phosphoric acid); gradient elution was performed according to the table below.

[0057]

[0058]

[0059] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. According to the table below, use the main component self-reference method with correction factors to calculate the content of D-glucose-γ-lactone and D-glucose-δ-lactone by peak area.

[0060] Glucose related substances Relative (gluconic acid) retention time Correction Factor D-glucose-γ-lactone 0.87 1.17 D-Gluco-δ-lactone 1.10 1.06

[0061] (1) D-glucose-γ-lactone:

[0062] Chemical structure:

[0063]

[0064] Molecular formula: C6H 10 O6

[0065] Molecular weight: 178.14

[0066] (2) D-glucose-δ-lactone:

[0067] Chemical structure:

[0068]

[0069] Molecular formula: C6H 10 O6

[0070] Molecular weight: 178.14.

[0071] The retention time of D-glucose-γ-lactone measured in this example is 7.990 minutes, and the retention time of D-glucose-δ-lactone is 10.083 minutes. The chromatogram of compound calcium gluconate oral solution is as follows: Fig.11 As shown, it can be seen that in the detection process of this embodiment, no reference solution is needed. The problem that the reference solution is still difficult to meet the accuracy of the detection when it is prepared on the spot is solved by the determination of the correction factor, and the content of D-glucose-γ-lactone and D-glucose-δ-lactone is accurately measured. The method of this embodiment can be used as a detection method for D-glucose-γ-lactone and D-glucose-δ-lactone in samples containing gluconic acid such as compound calcium gluconate oral solution. The method has the characteristics of strong specificity, high sensitivity, high accuracy, good durability, etc., and has more advantages such as universality and practicality, and is simpler and more efficient in operation.

Claims

1. A method for detecting gluconic acid-related substances in compound calcium gluconate oral solution, wherein the gluconic acid-related substances are D-glucose-γ-lactone and D-glucose-δ-lactone, characterized in that It is carried out according to the following steps: Step 1. Test solution: Accurately measure compound calcium gluconate oral solution, place in a volumetric bottle, dilute to the mark with solvent, and shake well; Step 2, control solution: accurately measure the test solution, place it in a volumetric bottle, dilute it to the mark with solvent, and shake well; accurately measure the above solution, place it in a volumetric bottle, dilute it to the mark with solvent, and shake well; The solvents described in steps 1 and 2 are both 0.01-0.08 mol / L potassium hydrogen phosphate solution; Step 3, chromatographic conditions: amide bonded silica gel is used as filler; 0.01-0.08 mol / L potassium dihydrogen phosphate solution-acetonitrile is used as mobile phase A, and 0.01-0.08 mol / L potassium dihydrogen phosphate solution-acetonitrile is used as mobile phase B; detection wavelength is 190-220 nm; injection volume is 10 μL; flow rate is 0.2-0.8 mL per minute; column temperature is 25-35°C; gradient elution mode is: Among them, the pH value of dipotassium hydrogen phosphate solution is 4.5 to 5.5; Step 4. Calculate the content of gluconic acid related substances: Calculate the content of gluconic acid related substances in the compound calcium gluconate oral solution by the peak area using the principal component self-reference method with the addition of a correction factor.

2. The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution according to claim 1, characterized in that The volume ratio of dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase A is 30:

70.

3. The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution according to claim 1, characterized in that The volume ratio of dipotassium hydrogen phosphate solution to acetonitrile in the mobile phase B is 55:

45.

4. The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution according to claim 1, characterized in that The solvent is 0.02 mol / L dipotassium hydrogen phosphate solution.

5. The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution according to claim 1 or 4, characterized in that The pH value of the dipotassium hydrogen phosphate solution is 5.

0.

6. The method for detecting gluconic acid-related substances in the compound calcium gluconate oral solution according to claim 1, characterized in that The detection wavelength is 190nm.

7. The method for detecting gluconic acid-related substances in compound calcium gluconate oral solution according to claim 1, characterized in that The flow rate was 0.5 mL per minute.

8. The method for detecting gluconic acid-related substances in compound calcium gluconate oral solution according to claim 1, characterized in that The correction factors are the correction factors of D-glucose-γ-lactone and D-glucose-δ-lactone; wherein the correction factor of D-glucose-γ-lactone is 1.17, and the correction factor of D-glucose-δ-lactone is 1.06.

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