HPLC-ELCD method for simultaneously detecting calcium gluconate, zinc gluconate and lysine hydrochloride and application

Abstract

The invention relates to the technical field of substance detection, and particularly discloses an HPLC-ELCD method for simultaneously detecting calcium gluconate, zinc gluconate and lysine hydrochloride and application. According to the HPLC-ELCD method for simultaneously detecting calcium gluconate, zinc gluconate and lysine hydrochloride, a cation exchange chromatographic column is adopted for isocratic elution of a sample to be detected, and a mobile phase is a methanesulfonic acid-oxalic acid solution; the mobile phase is a methanesulfonic acid-oxalic acid solution, the concentration of methanesulfonic acid is 0.002 mol / L to 0.004 mol / L, and the concentration of oxalic acid is 0.12 g / L to 0.18 g / L. The method disclosed by the invention is high in separation efficiency, good in selectivity, high in detection sensitivity and strong in operability.

Description

Technical Field

[0001] This invention relates to the field of substance detection technology, and more specifically, to an HPLC-ELCD method and its application for the simultaneous detection of calcium gluconate, zinc gluconate and lysine hydrochloride. Background Technology

[0002] Calcium gluconate and zinc oral solution can be used to treat problems caused by calcium and zinc deficiency, including osteoporosis, tetany, osteomalacia, rickets, calcium supplementation for pregnant and lactating women, postmenopausal women, growth retardation in children, loss of appetite, anorexia, recurrent oral ulcers, and acne.

[0003] Calcium gluconate and zinc gluconate oral solution is a compound preparation. Its active ingredients include calcium gluconate, zinc gluconate, and lysine hydrochloride. Current detection methods generally only allow for the individual determination of calcium gluconate, zinc gluconate, or lysine hydrochloride. Methods for determining calcium gluconate and zinc gluconate include titration and ion chromatography. Methods for determining lysine hydrochloride include nitrogen determination, high-performance liquid chromatography-evaporative light scattering (HPLC-ELSD), and ion chromatography. Among these methods, titration has a difficult endpoint determination and large errors; ELSD takes too long, requiring at least 2 hours to complete; HPLC-ELSD has relatively low sensitivity and requires a volatile mobile phase, prohibiting the use of non-volatile buffer salts and surfactants; and ion chromatography equipment is expensive and has high development costs.

[0004] To achieve efficient detection of all major components of calcium gluconate zinc oral solution, further research on detection methods is necessary. Summary of the Invention

[0005] One of the objectives of this invention is to provide an ideal method for the simultaneous detection of calcium gluconate, zinc gluconate, and lysine hydrochloride.

[0006] This invention provides an HPLC-ELCD method for the simultaneous detection of calcium gluconate, zinc gluconate, and lysine hydrochloride. The method employs a cation exchange column for isocratic elution of the sample, with a methanesulfonic acid-oxalic acid solution as the mobile phase. The methanesulfonic acid concentration in the mobile phase is 0.002 mol / L–0.004 mol / L, and the oxalic acid concentration is 0.12 g / L–0.18 g / L.

[0007] This invention reveals that when detecting samples containing calcium gluconate, zinc gluconate, and lysine hydrochloride simultaneously, high-performance liquid chromatography (HPLC) combined with a conventional conductivity detector (ELCD) using a cation exchange column under a specific mobile phase can effectively separate these three substances, enabling simultaneous detection of calcium gluconate, zinc gluconate, and lysine hydrochloride. This eliminates the need to design separate chromatographic detection conditions for each substance; a single analysis of the sample allows for simultaneous quantitative and qualitative assessment of all three substances, significantly reducing the time and cost of sample analysis.

[0008] Preferably, in the mobile phase of the HPLC-ELCD method of the present invention, the concentration of methanesulfonic acid is 0.003 mol / L and the concentration of oxalic acid is 0.15 g / L.

[0009] In the HPLC-ELCD method of the present invention, the chromatographic column is a Dionex IonPac™ SCS1.

[0010] In the HPLC-ELCD method of this invention, the column temperature is 30±5℃, the flow rate is 1±0.2ml / min, and the injection volume is 5~25μl.

[0011] The method of this invention uses a conventional high-performance liquid chromatography column and is equipped with a conductivity detector. Compared with traditional titration or high-performance liquid chromatography detection methods, it is convenient and accurate. Compared with ion chromatography, the detection equipment is cheaper and easier to operate.

[0012] This invention also provides the application of the above-described HPLC-ELCD method in the detection of calcium zinc gluconate oral solution.

[0013] The present invention also provides a method for simultaneously detecting calcium gluconate, zinc gluconate and lysine hydrochloride in calcium gluconate zinc oral solution, wherein the calcium gluconate zinc oral solution sample is detected by the above-mentioned HPLC-ELCD method.

[0014] In the method of the present invention, the sample is diluted with water before testing.

[0015] The method of the present invention further includes a step of quantifying calcium gluconate, zinc gluconate and / or lysine hydrochloride by means of an external standard method after detecting calcium gluconate, zinc gluconate and / or lysine hydrochloride in the sample.

[0016] Those skilled in the art can calculate the content of each target component in the sample to be tested based on the peak area of ​​each target component (calcium gluconate, zinc gluconate, and lysine hydrochloride) using the conventional external standard method.

[0017] If the sample to be tested is a pharmaceutical preparation with a defined specification, the formulas for calculating the external standard method for each target component are as follows: ; M 对 The sample weight of the target component in the reference solution, in mg; V 对 : The volume of the target component diluted in the reference solution, in ml; A 对 Peak area of ​​the target component in the reference solution; A 供 Peak area of ​​the target component in the test solution; V 供 The dilution factor of the test solution; C: Target component specification concentration (mg / ml).

[0018] The present invention also provides the application of the above method in the quality monitoring of calcium gluconate zinc oral solution.

[0019] In the application of this invention, the quality monitoring refers to the qualitative and / or quantitative monitoring of the active ingredients in the calcium gluconate zinc oral solution; the active ingredients include calcium gluconate, zinc gluconate and / or lysine hydrochloride.

[0020] The active ingredients in calcium gluconate and zinc gluconate oral solution include calcium gluconate, zinc gluconate, and lysine hydrochloride. Detecting their content is helpful in judging product quality and process stability. The method of this invention can efficiently detect multiple active ingredients simultaneously, which is beneficial for the industrial production of calcium gluconate and zinc gluconate oral solution.

[0021] The beneficial effects of this invention are at least as follows: The high-performance liquid chromatography method of this invention has high sensitivity, high separation efficiency, and good selectivity; the sample pretreatment method is simple, using a one-step preparation method, which consumes little solvent and has high detection efficiency; and the method has strong applicability, which can simultaneously and effectively determine the contents of calcium gluconate, zinc gluconate, and lysine hydrochloride. Attached Figure Description

[0022] Figure 1 This is the spectrum of specificity detection in Example 1. Detailed Implementation

[0023] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0024] 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 or prepared according to conventional methods in the art.

[0025] Example 1

[0026] This embodiment provides a method for simultaneously detecting calcium gluconate, zinc gluconate, and lysine hydrochloride in an oral solution of calcium gluconate and zinc gluconate, as detailed below: 1. Instruments and chromatographic conditions

[0027] Shimadzu LC-20AT high performance liquid chromatograph, quaternary pump, conductivity detector, autosampler.

[0028] Cation exchange column: Dionex IonPac™ SCS1 column (4.0 mm × 250 mm).

[0029] The mobile phase was a methanesulfonic acid-oxalic acid solution (weigh 0.15 g of oxalic acid, dissolve it in an appropriate amount of water, add 3 mL of 1 mol / L methanesulfonic acid, and dilute to 1000 mL with water, then filter).

[0030] Elution method: isocratic elution.

[0031] The flow rate was 1.0 ml per minute, with an unsuppressed conductivity detector; the column temperature was 30 °C; and the injection volume was 10 μl.

[0032] 2. Experimental Procedure

[0033] The calcium gluconate and zinc oral solution involved in this invention is in the specification of 10ml, and each 10ml of the solution contains 600mg of calcium gluconate, 30mg of zinc gluconate, 100mg of lysine hydrochloride, 10mg of sodium benzoate, 4mg of aspartame, 8mg of acesulfame potassium, 90mg of lactic acid, and 2.5mg of peach flavoring.

[0034] Blank solvent: water.

[0035] Test solution: Accurately measure 1 ml of calcium gluconate and zinc oral solution, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution; this is equivalent to a solution containing approximately 1.2 mg of calcium gluconate, 0.06 mg of zinc gluconate, and 0.2 mg of lysine hydrochloride per 1 ml of test solution.

[0036] Reference solution: Accurately weigh appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve and dilute with water to prepare a mixed solution containing 1.2 mg of calcium gluconate, 0.06 mg of zinc gluconate, and 0.2 mg of lysine hydrochloride per 1 ml.

[0037] Blank excipient stock solution: Weigh 10 mg sodium benzoate, 4 mg aspartame, 8 mg acesulfame potassium, 90 mg lactic acid, and 2.5 mg peach flavoring into a 10 ml volumetric flask, dissolve and dilute with water to the mark, and shake well; Blank excipient solution: Accurately measure 1 ml of blank excipient stock solution, place it in a 50 ml volumetric flask, dilute with water to the mark, and shake well.

[0038] Accurately measure the amounts of each of the above solutions and inject them into the liquid chromatograph, then record the chromatograms. Calculate the content of each main component using the peak area based on the external standard method.

[0039] Typical specificity result graphs are shown below Figure 1 .result: Figure 1 The blank solvent and blank excipient did not produce peaks at the calcium gluconate, zinc gluconate and lysine hydrochloride levels, meaning they did not interfere with the detection of these three components.

[0040] Example 2

[0041] This embodiment verifies the linearity, limit of quantitation and limit of detection, solution stability, precision, robustness and accuracy of the detection method of Embodiment 1 of the present invention.

[0042] 1. Linear range test: Preparation of linear solution: Take appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve them in water, and quantitatively dilute them to prepare a mixed solution containing approximately 0.12 mg of calcium gluconate, 0.006 mg of zinc gluconate, and 0.02 mg of lysine hydrochloride per 1 ml as the linear L1; Take appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve them in water, and quantitatively dilute them to prepare a mixed solution containing approximately 0.6 mg of calcium gluconate, 0.03 mg of zinc gluconate, and 0.1 mg of lysine hydrochloride per 1 ml as linear L2; Take appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve them in water, and quantitatively dilute them to prepare a mixed solution containing approximately 1.2 mg of calcium gluconate, 0.06 mg of zinc gluconate, and 0.2 mg of lysine hydrochloride per 1 ml as linear L3; Take appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve them in water, and quantitatively dilute them to prepare a mixed solution containing approximately 1.8 mg of calcium gluconate, 0.09 mg of zinc gluconate, and 0.3 mg of lysine hydrochloride per 1 ml as linear L4; Take appropriate amounts of calcium gluconate, zinc gluconate, and lysine hydrochloride reference standards, dissolve them in water, and quantitatively dilute them to prepare a mixed solution containing approximately 2.4 mg of calcium gluconate, 0.12 mg of zinc gluconate, and 0.4 mg of lysine hydrochloride per 1 ml as the linear L5. The test results are shown in Tables 1-3.

[0043] Table 1. Methodological validation of calcium gluconate content - linearity results

[0044] Table 2. Methodological validation of zinc gluconate content - linear results

[0045] Table 3. Methodological validation of lysine hydrochloride content - linear results

[0046] Conclusion: Within the limit concentration range of 0.1231–2.462 mg / ml, the peak area of ​​calcium gluconate showed a linear relationship with concentration, y = 8.6254 × 10⁻⁶. 5 X +4.8189×10 3 The correlation coefficient r = 0.9999 > 0.999, and the sum of squared residuals is 2982459075; Within the limit concentration range of 0.0062–0.1232 μg / ml, the peak area of ​​zinc gluconate showed a linear relationship with concentration, y = 6.8485 × 10⁻⁶. 5 X +5.8211×10 1 The correlation coefficient r = 0.9999 > 0.999, and the sum of squared residuals is 519936; Within the limit concentration range of 0.0207–0.4147 mg / ml, the peak area of ​​lysine hydrochloride showed a linear relationship with concentration, y = 1.3960 × 10⁻⁶. 6 X + 2.1598 × 10 3 The correlation coefficient r = 0.9999 > 0.999, and the sum of squared residuals is 579522596.

[0047] 2. Limit of Quantitation and Limit of Detection Tests: Reference standard mixed stock solution: Take appropriate amounts of calcium gluconate, zinc gluconate and lysine hydrochloride reference standards, dissolve and dilute with water to prepare a solution containing approximately 1 mg each of calcium gluconate, zinc gluconate and lysine hydrochloride per 1 ml. Take an appropriate amount of the mixed stock solution of reference standards and dilute it stepwise with water until the S / N value is about 10 to obtain the limit of quantitation solution; dilute it stepwise with water until the S / N value is about 3 to obtain the limit of detection solution.

[0048] The test results are shown in Table 4.

[0049] Table 4 Results of Limit of Quantitation and Limit of Detection

[0050] Conclusion: The limit of quantification for calcium gluconate was 0.0053 mg / ml, and the RSD of the peak area of ​​the limit of quantification for 6 consecutive injections was 6.8% (not greater than 10%), with the S / N also greater than 10. The limit of detection was 0.0031 mg / ml, and the RSD of the peak area of ​​the limit of detection for 3 consecutive injections was 5.3% (not greater than 10%), with the S / N also greater than 3. The limit of quantification for zinc gluconate is 0.0062 mg / ml, and the RSD of the peak area of ​​the limit of quantification for 6 consecutive injections is 3.5% (not greater than 10%), and the S / N is also greater than 10. The limit of detection is 0.0035 mg / ml, and the RSD of the peak area of ​​the limit of detection for 3 consecutive injections is 3.1% (not greater than 10%), and the S / N is also greater than 3. The limit of quantification for lysine hydrochloride was 0.0045 mg / ml, with an RSD of 4.6% (not greater than 10%) for the peak area of ​​the limit of quantification for six consecutive injections, and the S / N ratio was also greater than 10 for all injections. The limit of detection was 0.0021 mg / ml, with an RSD of 8.7% (not greater than 10%) for the peak area of ​​the limit of detection for three consecutive injections, and the S / N ratio was also greater than 3 for all injections.

[0051] 3. Stability testing: The reference solution and the test solution from Example 1 were placed at room temperature for 40 hours, and the peak areas at different time points were measured. The results are shown in Tables 5 and 6.

[0052] Table 5. Stability results of the reference solution

[0053] Table 6. Stability results of the test solution

[0054] Conclusion: After being placed at room temperature for 40 hours, the peak areas at different time points and the peak area at 0 h all had RSD values ​​within the range of 2.0%, which indicates that the reference solution and the test solution were stable within 40 hours of being placed at room temperature.

[0055] 4. Precision test: Following the method for preparing the test solution in Example 1, six replicates were prepared and tested. The test results (the ratio of the tested content to the theoretical content) are shown in Table 7.

[0056] Table 7 Precision Results

[0057] Conclusion: The contents of calcium gluconate, zinc gluconate, and lysine hydrochloride in the six test solutions were all between 98.0% and 102.0%, and the RSD values ​​of the six test solutions were all less than 2.0%, indicating that the method of the present invention has good precision.

[0058] 5. Accuracy Test: Reference stock solution: Take appropriate amounts of calcium gluconate, zinc gluconate and lysine hydrochloride reference standards, dissolve and dilute with water to prepare a solution containing approximately 12 mg of calcium gluconate, 0.6 mg of zinc gluconate and 2 mg of lysine hydrochloride per 1 ml.

[0059] 80% accuracy solution: Take 1 ml of the blank excipient stock solution from Example 1, place it in a 50 ml volumetric flask, accurately add 4 ml of the reference stock solution, dilute with water to the mark and shake well to obtain the solution; prepare 3 parallel portions. 100% accuracy solution: Take 1 ml of the blank excipient stock solution from Example 1, place it in a 50 ml volumetric flask, accurately add 5 ml of the reference stock solution, dilute with water to the mark and shake well to obtain the solution; prepare 3 parallel portions. 120% accuracy solution: Take 1 ml of the blank excipient stock solution from Example 1, place it in a 50 ml volumetric flask, accurately add 6 ml of the reference stock solution, dilute with water to the mark, and shake well. Prepare 3 parallel solutions. The test results are shown in Tables 8 to 10.

[0060] Table 8 Accuracy Results - Calcium Gluconate

[0061] Table 9 Accuracy Results - Zinc Gluconate

[0062] Table 10 Accuracy Results - Lysine Hydrochloride

[0063] Conclusion: The recovery rates of the product at three concentrations in nine samples were all within the range of 98.0% to 102.0% as determined by this method. Furthermore, the RSD of the recovery rates of calcium gluconate, zinc gluconate, and lysine hydrochloride in the nine samples were all less than 2.0%, indicating that the method of this invention has good accuracy.

[0064] 6. Durability test: The column temperature, flow rate, and concentration of methanesulfonic acid or oxalic acid in Example 1 were changed separately, and the test solution in Example 1 was tested. The test results (the ratio of the test content to the theoretical content) are shown in Table 11.

[0065] Table 11 Content Method Validation - Robustness Results - Test Solution

[0066] Conclusion: By varying the column temperature by ±5℃, flow rate by ±0.2ml / min, methanesulfonic acid concentration by 0.003±0.001mol / L, and oxalic acid concentration by 0.15±0.03g / L, the RSDs of calcium gluconate, zinc gluconate, and lysine hydrochloride were 0.8%, 0.5%, and 0.8%, respectively, all not exceeding 2%; thus, the method of the present invention has good robustness.

[0067] Comparative Example 1

[0068] This comparative example provides a chromatographic detection method using a Shim-pack IC-C4 column (150 mm × 4.6 mm I.D., 7 μm), a mobile phase of 2.5 mM oxalic acid aqueous solution, a flow rate of 1 mL / min, a column temperature of 40℃, and an injection volume of 25 μL; isocratic elution is employed.

[0069] The recovery rate of the method in Comparative Example 1 was tested using three 100% accuracy solutions from the accuracy test section of Example 2. The results are shown in Table 12. The test results show that the recovery rate of calcium gluconate determined using the method in Comparative Example 1 is not between 98.0% and 102.0%, which is low. This indicates that the detection result of calcium gluconate in Comparative Example 1 is lower and the accuracy is worse than that of the Example.

[0070] Table 12 Comparison of detection results between Comparative Example 1 and Example 1

[0071] Comparative Example 2

[0072] This comparative example provides a detection method that is basically the same as that in Example 1, except that the chromatographic column is replaced with a Shim-pack IC-C4, 150 mm × 4.6 mm ID, 7 μm.

[0073] The recovery rate of the method in Comparative Example 2 was tested using three 100% accuracy solutions from the accuracy test section of Example 2. The results are shown in Table 13. The test results show that the recovery rate of calcium gluconate determined using the method in Comparative Example 2 is not between 98.0% and 102.0%, which is low. This indicates that the detection result of calcium gluconate in Comparative Example 2 is low, and the accuracy is worse than that of the Example.

[0074] Table 13 Comparison of detection results between Comparative Example 2 and Example 1

[0075] Comparative Example 3

[0076] This comparative example provides a detection method that is basically the same as that in Example 1, except that the mobile phase is changed to a 2.5 mM oxalic acid aqueous solution.

[0077] The recovery rate of the method in Comparative Example 3 was tested using three 100% accuracy solutions from the accuracy test section of Example 2. The results are shown in Table 14. The test results show that the recovery rates of calcium gluconate and zinc gluconate determined using the method in Comparative Example 3 were not between 98.0% and 102.0%, which is low. This indicates that the detection results of calcium gluconate and zinc gluconate in Comparative Example 3 are likely to be low, and the accuracy is worse than that of the Example.

[0078] Table 14 Comparison of detection results between Comparative Example 3 and Example 1

[0079] Comparative Example 4

[0080] This comparative example provides a detection method that is basically the same as that in Example 1, except that the mobile phase is changed to a methanesulfonic acid-pyridine dicarboxylic acid solution (weigh 0.12 g of pyridine dicarboxylic acid, add an appropriate amount of water to dissolve it, then add 3 mL of 1 mol / L methanesulfonic acid, and dilute to 1000 mL with water, then filter).

[0081] The recovery rate of the method in Comparative Example 3 was tested using three 100% accuracy solutions from the accuracy test section of Example 2. The results are shown in Table 15. The test results show that the recovery rate of zinc gluconate determined using the method in Comparative Example 4 is not between 98.0% and 102.0%, which is low. This indicates that the detection result of zinc gluconate in Comparative Example 4 is low, and the accuracy is worse than that of the Example.

[0082] Table 15 Comparison of detection results between Comparative Example 4 and Example 1

[0083] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An HPLC-ELCD method for the simultaneous detection of calcium gluconate, zinc gluconate, and lysine hydrochloride, characterized in that, The sample was eluted isocratically using a cation exchange column, with a methanesulfonic acid-oxalic acid solution as the mobile phase. The concentration of methanesulfonic acid in the methanesulfonic acid-oxalic acid solution was 0.002 mol / L-0.004 mol / L, and the concentration of oxalic acid was 0.12 g / L-0.18 g / L.

2. The HPLC-ELCD method according to claim 1, characterized in that, In the mobile phase, the concentration of methanesulfonic acid is 0.003 mol / L and the concentration of oxalic acid is 0.15 g / L.

3. The HPLC-ELCD method according to claim 1 or 2, characterized in that, The chromatographic column is a Dionex IonPac™ SCS1.

4. The HPLC-ELCD method according to any one of claims 1-3, characterized in that, The column temperature was 30±5℃, the flow rate was 1±0.2ml / min, and the injection volume was 5-25μl.

5. The application of the HPLC-ELCD method according to any one of claims 1-4 in the detection of calcium gluconate zinc oral solution.

6. A method for simultaneously detecting calcium gluconate, zinc gluconate, and lysine hydrochloride in an oral solution of calcium gluconate and zinc gluconate, characterized in that, The calcium gluconate zinc oral solution sample was detected using the HPLC-ELCD method according to any one of claims 1-4.

7. The method according to claim 6, characterized in that, The sample was diluted with water before testing.

8. The method according to claim 6 or 7, characterized in that, The method further includes the step of quantifying calcium gluconate, zinc gluconate and / or lysine hydrochloride by external standard method after detecting calcium gluconate, zinc gluconate and / or lysine hydrochloride in the sample.

9. The application of the method according to any one of claims 6-8 in the quality control of calcium gluconate zinc oral solution.

10. The application according to claim 9, characterized in that, The quality monitoring refers to the qualitative and / or quantitative monitoring of the active ingredients in the calcium gluconate zinc oral solution; the active ingredients include calcium gluconate, zinc gluconate and / or lysine hydrochloride.

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