Method for detecting glucuronic acid in calcium gluconate bulk drug

Abstract

The invention discloses a method for detecting glucuronic acid in a calcium gluconate bulk drug. The method has high system applicability, has incomparable advantages in specificity, quantitation limit, detection limit, linear range, repeatability and durability, and has high precision.

Description

Technical Field

[0001] This invention relates to the field of chemical drug analysis and detection technology, specifically to a method for detecting glucuronic acid in calcium gluconate raw material. Background Technology

[0002] Salbutamol (calcium gluconate) is a calcium supplement. It can maintain normal neuromuscular excitability, improve cell membrane permeability, promote bone and teeth calcification, and has anti-allergic effects. This product is listed in the Chinese Pharmacopoeia, the European Pharmacopoeia, and the United States Pharmacopeia. From the perspective of raw materials and processes, the production of calcium gluconate may generate the byproduct glucuronic acid. The structural formulas of calcium gluconate and D-glucuronic acid are as follows:

[0003]

[0004] To ensure the purity of calcium gluconate raw materials, developing a method for detecting residual glucuronic acid in calcium gluconate is of significant practical importance. Currently, the detection of glucuronic acid in calcium gluconate typically employs liquid chromatography (LC) with a UV detector. However, during reproducibility analysis, it was found that the peaks of the main components of calcium gluconate interfere with the glucuronic acid peaks, and the glucuronic acid response is weak. This invention employs an IC method to determine the glucuronic acid content in calcium gluconate. The method exhibits good peak shape, strong specificity, is unaffected by other impurities, is reliable, and can calculate the specific impurity content. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a method for detecting glucuronic acid in calcium gluconate raw materials, which fully meets the standards in terms of specificity, limit of quantitation, limit of detection, linear range, repeatability, and accuracy, and has high precision.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a method for detecting glucuronic acid in calcium gluconate raw material, specifically comprising the following steps:

[0007] 1) Preparation of the test solution of the active pharmaceutical ingredient

[0008] Take calcium gluconate raw material, add solvent to dissolve it, and prepare test solution;

[0009] 2) Preparation of reference solution

[0010] Dissolve D-glucuronic acid reference standard in a solvent to prepare a reference standard solution.

[0011] 3) Accurately measure the test solution and the reference solution separately, inject them into the ion chromatograph, record the chromatograms, and calculate the content of glucuronic acid in the test solution by peak area using the external standard method.

[0012] Specifically, the solvent is purified water.

[0013] Specifically, the preparation method of the test solution is as follows: take about 100 mg of calcium gluconate raw material, accurately weigh it, put it in a 10 ml volumetric flask, add an appropriate amount of water, warm it slightly to dissolve it, dilute it with water to the mark, and shake well.

[0014] Specifically, the preparation method of the reference solution is as follows: take an appropriate amount of D-glucuronic acid reference standard, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing about 50 μg per 1 ml, as the reference standard stock solution; accurately transfer 1 mL of the reference standard stock solution into a 10 ml volumetric flask, dilute it with water to the mark, and prepare a solution containing about 5 μg of glucuronic acid per 1 ml.

[0015] Specifically, the calculation formula in step 3) is:

[0016]

[0017] in:

[0018] Where: f—response factor; m S —Weigh the amount of glucuronic acid in the reference solution; c S —The content of glucuronic acid; V S —Total dilution volume of glucuronic acid in the reference solution; A S —The peak area f of glucuronic acid in the chromatogram of the reference solution 平均 —The average value of the response factor; A i —Peak area of ​​glucuronic acid in the test sample; m i —Sample weight of the test sample; V i —Dilution volume of the test sample.

[0019] Specifically, the detection conditions for the ion chromatography are as follows:

[0020] Column: Dionex IonPac TM AS11-HC RFIC TM (4*250mm) Analytical;

[0021] Rinse solution: 9 mmol / L sodium hydroxide solution;

[0022] Elution method: isocratic elution;

[0023] Flow rate: 1.0 ml / min;

[0024] Detector: Conductivity detector;

[0025] Detection method: Suppressed conductivity detection;

[0026] Suppressor: Thermo Scientific Dionex AERS500 4mm 085029;

[0027] Injection volume: 20 μl.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) This invention proposes using ultrapure water as a solvent, which minimizes the influence of the solvent itself on the solubility of calcium gluconate. Simultaneously, micro-temperature treatment achieves a solubility of calcium gluconate of 10 mg / mL. Furthermore, the use of water in the ion chromatography detection system meets the stringent requirements for solvent purity, ensuring the accuracy and stability of the detection results. With a small injection volume, the detection limit is achieved at 60 ng / ml, the sensitivity is 6 ppm, and the signal-to-noise ratio is greater than 3. This simultaneously satisfies both sensitivity and lower limit requirements.

[0030] (2) The method of the present invention has high selectivity for glucuronic acid in calcium gluconate, which can ensure good separation between calcium gluconate and other components, avoid mutual interference, and improve the accuracy of analysis. The peak shape is good, the specificity is strong, the separation degree from the main component meets the requirements, the method is reliable, and the specific content value of glucuronic acid can be calculated.

[0031] (3) The present invention exhibits unparalleled advantages in specificity, limit of quantitation, limit of detection, linear range, accuracy and precision.

[0032] (3) The present invention uses the IC method to determine glucuronic acid in calcium gluconate, and provides other methods for detecting glucuronic acid residues in calcium gluconate, which can complement and improve other methods. Attached Figure Description

[0033] Figure 1 This refers to the examination of the blank solvent ion chromatogram in the method of this invention;

[0034] Figure 2 This is the ion chromatogram of the reference solution in the method of this invention;

[0035] Figure 3 This refers to the ion chromatogram of the test sample solution examined in the method of this invention;

[0036] Figure 4 This is the ion chromatogram of the spiked test sample solution used in the method of this invention to check the standard limit;

[0037] Figure 5 This is the ion chromatogram of the fructose solution examined in the method of this invention;

[0038] Figure 6This refers to the ion chromatogram of the arabinose solution examined in the method of this invention;

[0039] Figure 7 This is the ion chromatogram of the D-anhydrous glucose solution examined in the method of this invention;

[0040] Figure 8 This is the ion chromatogram of the 5-hydroxymethylfurfural solution examined in the method of this invention;

[0041] Figure 9 This is a standard curve of glucuronic acid in the method of this invention. Detailed Implementation

[0042] To enhance understanding of the present invention, the present invention will be further described in detail below with reference to embodiments and accompanying drawings. These embodiments are only used to explain the present invention and do not constitute a limitation on the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] The detection method described in this invention is mainly for the detection of glucuronic acid, and the external standard method is used for calculation.

[0045] The information on the instruments and reagents used in the implementation of the detection method of the present invention is shown in the table below:

[0046]

[0047] Example 1

[0048] This embodiment provides a method for detecting glucuronic acid in calcium gluconate raw material.

[0049] I. Chromatographic conditions

[0050] Instrument: Ion chromatograph;

[0051] Column: Dionex IonPac TM AS11-HC RFIC TM (4*250mm) Analytical;

[0052] Detector: Conductivity detector

[0053] Detection method: Suppressed conductivity detection

[0054] Rinse solution: 9 mmol / L sodium hydroxide aqueous solution;

[0055] Elution method: isocratic elution;

[0056] Flow rate: 1.0 ml / min;

[0057] Suppressor: Thermo Scientific Dionex AERS500 4mm 085029;

[0058] Injection volume: 20 μl.

[0059] II. Detection Methods

[0060] (1) Blank solvent: purified water

[0061] (2) Preparation of test solution

[0062] Weigh approximately 100 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of water, warm slightly to dissolve, dilute with water to the mark, and shake well.

[0063] (3) Preparation of reference solution

[0064] Take an appropriate amount of glucuronic acid reference standard and add water to prepare a solution containing approximately 5 μg of glucuronic acid per 1 ml.

[0065] (4) Accurately measure the test solution and the reference solution respectively, inject them into the liquid chromatograph, record the chromatogram, and calculate the content of glucuronic acid in the test solution by peak area using the external standard method.

[0066] III. Formula for Calculating Glucuronic Acid Content

[0067] 1.

[0068] Where: f—response factor;

[0069] ms — The amount of glucuronic acid in the reference solution;

[0070] The content of cs-glucuronic acid;

[0071] Vs—Total dilution volume of glucuronic acid in the reference solution;

[0072] As—the peak area of ​​glucuronic acid in the chromatogram of the reference solution.

[0073] 2.

[0074] Where: f_average — the average value of the response factors;

[0075] Ai—Peak area of ​​glucuronic acid in the test sample;

[0076] mi—sample weight;

[0077] Vi—Dilution volume of the test sample.

[0078] Example 2: Specificity Test

[0079] (1) Blank solution: purified water.

[0080] (2) Reference solution: Take an appropriate amount of reference standard, dissolve it in water and dilute it quantitatively to prepare a solution containing about 50 μg per ml. Accurately transfer 1 mL into a 10 ml volumetric flask, dilute with water to the mark, and prepare a solution containing about 5 μg of glucuronic acid per ml.

[0081] (3) Test solution: Weigh about 100 mg of calcium gluconate accurately, place it in a 10 ml volumetric flask, add an appropriate amount of water, warm slightly to dissolve, dilute to the mark with water, and shake well.

[0082] (4) Standard limit spiking solution: Take an appropriate amount of glucuronic acid reference standard, dissolve it in water and dilute quantitatively to prepare a solution containing approximately 50 μg per ml, as the reference standard stock solution. Accurately weigh approximately 100 mg of calcium gluconate, place it in a 10 ml volumetric flask, add an appropriate amount of water, warm slightly to dissolve, add 1 mL of the reference standard stock solution, and dilute with water to the mark to prepare a standard limit spiking solution containing approximately 10 mg of calcium gluconate and approximately 5 μg of glucuronic acid per ml.

[0083] (5) Fructose solution: Weigh an appropriate amount of fructose accurately, add water to dissolve and dilute to prepare a solution containing about 50 μg of fructose per 1 mL.

[0084] (6) Arabic sugar solution: Weigh an appropriate amount of arabinose accurately, add water to dissolve and dilute to prepare a solution containing about 50 μg of arabinose per 1 mL.

[0085] (7) D-anhydrous glucose solution: Weigh an appropriate amount of D-anhydrous glucose, accurately weigh it, add water to dissolve and dilute it to prepare a solution containing about 50 μg of D-anhydrous glucose per 1 mL.

[0086] (8) 5-Hydroxymethylfurfural solution: Weigh an appropriate amount of 5-hydroxymethylfurfural, accurately weigh it, add water to dissolve and dilute it to prepare a solution containing about 50 μg of 5-hydroxymethylfurfural per 1 mL.

[0087] Inject blank solution, system suitability solution, reference solution, test solution, and each blank excipient solution according to the chromatographic conditions of Example 1, and record the chromatograms (see attached instructions). Figures 1-8 .

[0088] Specificity tests showed that blank solvent, main component of test sample, adjacent peaks, and blank excipients did not interfere with the detection of glucuronic acid.

[0089] Example 3: Limit of Quantitation and Limit of Detection Test

[0090] The limits of detection (LOD) and quantitation (LOQ) were determined using the signal-to-noise ratio method. The reference solution was serially diluted, and the measured signal was compared with the baseline noise to calculate the lowest concentration that could be reliably detected. The results are shown in Table 1.

[0091] Table 1: Results of Limit of Quantitation and Limit of Detection

[0092]

[0093] Note: Sensitivity = Limit of Quantitation or Limit of Detection of Glucuronite Concentration / Sample Concentration (10 mg / ml)

[0094] The experimental results show that the quality control limit of glucuronic acid in the sample is 0.05%, and the sensitivity of the quantitation limit and detection limit is less than 1 / 25 of the quality control limit of glucuronic acid, proving that the sensitivity of the present invention is good.

[0095] Example 4: Linearity and Range Detection

[0096] Take an appropriate amount of glucuronic acid reference standard, dissolve it in water, and quantitatively dilute it to prepare a solution containing approximately 50 μg per ml, as the reference standard stock solution. Take the reference standard stock solution and quantitatively dilute it to prepare solutions with concentrations of 0.002%, 0.010%, 0.025%, 0.050%, 0.075%, and 0.100% relative to the test sample concentration, as solutions for each linear gradient concentration. The linear relationship is plotted as a function of the measured peak area and the analyte concentration. Linear regression is performed using the least squares method, requiring the linear regression coefficient r to be no less than 0.990. The results are shown in Table 2. Figure 9 .

[0097] Table 2: Linearity Measurement Results

[0098]

[0099] Note: Intercept ratio = Linear equation intercept / 100% concentration peak area

[0100] As shown in the table above, the detection method of the invention has a linear correlation coefficient r of 0.9991 for glucuronic acid in the range of 0.002% to 0.100% relative to the concentration of the test sample, which proves that it has a good linear relationship.

[0101] Example 5: Precision detection of reference solution injection

[0102] The reference solution from Example 1 was measured six times consecutively, and the relative standard deviation of the peak area was examined. The results are shown in Table 3.

[0103] Table 3: Results of Precision Test for Injection of Reference Solution

[0104]

[0105] As shown in the table above, the detection method described in this invention has good precision in determining the injection precision of the reference solution, with a peak area RSD of less than 2%.

[0106] Example 6: Stability detection of reference and test solutions

[0107] Inject 20 μl of the reference solution and test solution from Example 1 at 0 h, 1 h, 2 h, 6 h, 8 h and 24 h respectively, record the chromatograms, and calculate the relative standard deviation of the glucuronic acid peak area. The test results are shown in Table 4.

[0108] Table 4: Results of Solution Stability Test

[0109] time Reference Test sample Standard limit spiked test sample 0h 0.1498 Not detected 0.1386 1h 0.1526 Not detected 0.1505 2h 0.1514 Not detected 0.1502 6h 0.1471 Not detected 0.1446 8h 0.1448 Not detected 0.1426 24h 0.1452 Not detected 0.1396 average 0.1485 / 0.1444 RSD 2.6％ / 3.6

[0110] As shown in the table above, after 24 hours at room temperature, the peak area RSD of the reference solution was <6.0%, and glucuronic acid was not detected in the test solution at any time point. The RSD of the spiked test solution at the standard limit was <6.0%, indicating good solution stability.

[0111] Example 7: Repeatability Detection

[0112] The standard limit spiked test solution was taken and the detection method of Example 1 of this invention was repeated 6 times to verify the good repeatability of the method. The results are shown in Table 5.

[0113] Table 5: Repeatability Test Results

[0114] serial number Recovery rate (%) of spiked test solution at standard limit A 1 94.11 A 2 94.18 A 3 94.11 A 4 94.25 A 5 94.76 A 6 94.54 average 94.32 RSD 0.3％

[0115] As shown in the table above, the RSD of glucuronic acid recovery rate was <6.0% in 6 tests, proving that the method has good repeatability.

[0116] Example 8: Intermediate Precision Detection

[0117] The standard limit spiked test solution was taken and measured by different personnel at different times and on different instruments using the same repeatability method as in Example 7. The differences between the two measurement results were compared, and the results are shown in Table 6.

[0118] Table 6: Intermediate Precision Test Results

[0119] serial number Recovery rate (%) of spiked test solution at standard limit A 1 94.11 A 2 94.18 A 3 94.11 A 4 94.25 A 5 94.76 A 6 94.54 B 1 94.98 B 2 95.81 B 3 97.20 B 4 95.88 B 5 96.09 B 6 95.64 average 95.13 RSD 1.1％

[0120] As shown in the table above, the RSD of the recovery rate of glucuronide in the twelve standard limit spiked test solutions measured by different personnel at different times and on different instruments was less than 10.0%, indicating that the intermediate precision of the detection method was good.

[0121] Example 9: Accuracy Testing

[0122] The recovery method was used to determine the ratio between the actual measured amount and the theoretical amount of glucuronic acid in the spiked sample (recovery rate), expressed as a percentage (%). The recovery rate was required to be between 85% and 110% to confirm that the method has good accuracy. The results are shown in Table 7.

[0123] Table 7: Accuracy Test Results

[0124]

[0125] As shown in the table above, the recovery rate of glucuronic acid ranged from 85.38% to 96.58%, which met the validation requirements (85% to 110%), confirming that the method has good accuracy; the RSD value of the recovery rate was less than 10%, indicating good accuracy.

[0126] In summary, in specific tests, blank solvent, main component of the test sample, and adjacent peaks do not interfere with the detection of glucuronic acid.

[0127] In the limit of quantitation test, the concentration of glucuronic acid at the limit of quantitation was less than 1 / 25 of the quality control limit (20 ppm).

[0128] In the linearity test, glucuronic acid showed good linearity in the range of 20 ppm (limit of quantitation) to 1000 ppm relative to the concentration of the test sample, with a correlation coefficient r of not less than 0.990 and the percentage of the peak area of ​​the y-axis intercept relative to the limit concentration reference standard was much less than 25%.

[0129] In the accuracy test, the recovery rate of glucuronic acid was between 85% and 110%, with RSD ≤ 10%.

[0130] In the repeatability test, the recovery rates of glucuronic acid in all six spiked samples met the validation requirements (85%–110%).

[0131] In the intermediate precision test, the recovery rates of glucuronic acid in six spiked samples measured by different personnel on different dates using different instruments were all within the validation requirements (85%–110%), with RSD ≤ 10%.

[0132] Therefore, this invention employs an isocratic elution system with 9 mmol / L sodium hydroxide solution, which effectively separates glucuronic acid from the main component of the test sample and other impurities, resulting in good peak symmetry, which is beneficial for the detection of glucuronic acid and demonstrates high system applicability. Furthermore, it exhibits unparalleled advantages in specificity, limit of quantitation, limit of detection, linear range, and repeatability, demonstrating high precision.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the invention.

Claims

1. A method for detecting glucuronic acid in calcium gluconate raw material, characterized in that, Specifically, the following steps are included: 1) Preparation of the test solution of the active pharmaceutical ingredient Take calcium gluconate raw material, add solvent to dissolve it, and prepare test solution; 2) Preparation of reference solution Dissolve D-glucuronic acid reference standard in a solvent to prepare a reference standard solution. 3) Accurately measure the test solution and the reference solution separately, inject them into the ion chromatograph, record the chromatograms, and calculate the content of glucuronic acid in the test solution by peak area using the external standard method.

2. The method according to claim 1, characterized in that, The solvent is purified water.

3. The method according to claim 1, characterized in that, The concentration of the test sample solution is 10 mg / ml.

4. The method according to claim 1, characterized in that, The concentration of the reference solution was 5 μg / ml.

5. The method according to claim 4, characterized in that, The method for preparing the reference solution is as follows: Take an appropriate amount of D-glucuronic acid reference standard, accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing about 50 μg per 1 ml, as the reference standard stock solution; accurately transfer 1 mL of the reference standard stock solution into a 10 ml volumetric flask, dilute it with water to the mark, and prepare a solution containing about 5 μg of glucuronic acid per 1 ml.

6. The method according to claim 1, characterized in that, The detection conditions for the ion chromatography are as follows: Detector: Conductivity detector Detection method: Suppressed conductivity detection Column: Dionex IonPac TM AS11-HC RFIC TM (4*250mm) Analytical; Eluent concentration: 9 mmol / L sodium hydroxide aqueous solution; Elution type: isocratic elution; Flow rate: 1.0 ml / min; Injection volume: 20 μl.

7. The method according to claim 1, characterized in that, This detection method can quantitatively detect as little as 0.002% of glucuronic acid.

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