Method for detecting content of gluconate radicals in liquid preparation

Abstract

The invention relates to the field of medicine detection, in particular to a method for detecting the content of gluconate radicals in a liquid preparation. The detection method comprises the following steps: detecting the gluconate radical-containing liquid preparation dissolved by a sodium hydroxide solution through a high performance liquid chromatography, and the chromatographic conditions are as follows: a chromatographic column takes octadecyl bonded silica gel as a filler; the mobile phase comprises a mobile phase A and a mobile phase B; the mobile phase A is a mixed solution of a tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (85-95): (5-15), the mobile phase B is a mixed solution of a tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (30-50): (50-70), and the pH value of the tetrabutylammonium hydroxide solution is adjusted to 6.0-8.0 by adopting phosphoric acid; and gradient elution is carried out. According to the detection method, interference of impurities and other related substances in the liquid preparation can be avoided, the detection method is high in operability, easy and convenient to operate, high in sensitivity, good in repeatability and high in accuracy, the content of gluconate radicals in the liquid preparation medicine can be more accurately detected, and medicine quality control is facilitated.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical testing, specifically relating to a method for detecting gluconate content in liquid preparations. Background Technology

[0002] Gluconic acid is a sugar acid produced by the oxidation of the aldehyde group of glucose. It is a weak organic acid with five hydroxyl groups in its molecular formula, making it a pentahydroxyhexanoic acid. Gluconic acid is non-toxic and non-corrosive, and can form water-soluble complexes with metal ions such as sodium, zinc, and calcium through complexation.

[0003] Currently, many marketed drugs contain gluconate, such as calcium gluconate oral solution, zinc gluconate oral solution, calcium gluconate zinc oral solution, and calcium gluconate injection. During the research and development stage, there is a need to test the gluconate content of these drugs, which is beneficial to the quality control of the drugs.

[0004] On February 20, 2021, the Center for Drug Evaluation of the National Medical Products Administration published the "Technical Requirements for Pharmaceutical Research of the First Batch of Chemical Drugs Proposed Not to be Recommended as Reference Preparations" (Draft for Comments). The appendix, "Technical Requirements for Pharmaceutical Research of 14 Oral Preparations including Calcium Gluconate Tablets," includes technical requirements for pharmaceutical research of calcium gluconate zinc oral solution, stating that gluconate is a key quality attribute of calcium gluconate zinc oral solution and should be studied. A search revealed that currently, only the 2020 edition of the Chinese Pharmacopoeia (Part II) includes a quality standard for calcium gluconate oral solution, but this standard does not control gluconate. The supplementary version of the 2020 edition of the Chinese Pharmacopoeia (Part II) added a gluconate control item, recommending HPLC-UV using ion-pairing reagents. However, this method is extremely damaging to the chromatographic column, has very high detection costs, and exhibits significant impurity peaks that interfere with gluconate detection.

[0005] Furthermore, Chinese patent CN102565212B, published on July 11, 2012, and Chinese patent CN102590370B, published on July 18, 2012, disclose methods for determining gluconate using ion exchange chromatography equipped with a conductivity detector and ion exchange chromatography equipped with an electrochemical detector. Among these methods, ion exchange chromatography is highly susceptible to the quality of the eluent, requiring ultrapure water with secondary deionization for accurate determination. Gradient elution conditions are complex and the detection time is long.

[0006] Chinese patent CN115586285B, published on January 10, 2023, and Chinese patent CN117030877A, published on November 10, 2023, disclose the use of EDTA to treat test samples, which complexes with metal ions such as calcium and zinc in pharmaceuticals, promoting the dissociation of gluconate ions. However, after EDTA chelates metal ions, it forms large molecular chelates, and repeated injections can damage the chromatographic column life.

[0007] Chinese patent CN116297999B, published on June 23, 2023, discloses a method for detecting gluconate content in calcium zinc gluconate oral solution. The disclosed method is HPLC-UV method, but this method requires a special mixing strategy for the mobile phase and the mixing conditions are relatively harsh.

[0008] Therefore, there is an urgent need for a highly specific detection method that can more accurately detect the gluconate content in liquid pharmaceutical preparations. Summary of the Invention

[0009] To address the aforementioned technical problems, this application provides a method for detecting gluconate content in liquid preparations. This method is highly operable, simple to operate, highly sensitive, reproducible, and accurate. The method is simple to operate, requiring no special mixing strategy, and uses a conventional HPLC-UV detector. Furthermore, by treating the sample with sodium hydroxide instead of EDTA, gluconate is completely dissociated, minimizing damage to the chromatographic column and reducing detection costs. The gradient elution program includes five consecutive elution stages, ensuring good separation of the target analyte from adjacent impurity peaks during detection.

[0010] Therefore, this application provides a method for detecting gluconate content in liquid preparations, comprising: detecting gluconate-containing liquid preparation test solutions treated with sodium hydroxide solution by high performance liquid chromatography (HPLC), wherein the detection conditions of the HPLC include:

[0011] The chromatographic column is packed with octadecyl-bonded silica gel.

[0012] The mobile phase comprises mobile phase A and mobile phase B; mobile phase A is a mixed solution of tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (85-95):(5-15), and mobile phase B is a mixed solution of tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (30-50):(50-70), wherein the tetrabutylammonium hydroxide solution is adjusted to a pH of 6.0 to 8.0 with phosphoric acid;

[0013] Gradient elution procedures include:

[0014] .

[0016] In some embodiments, the gradient elution process includes:

[0017] .

[0019] In some embodiments, the gradient elution process includes:

[0020] .

[0022] In some embodiments, the concentration of tetrabutylammonium hydroxide in the mobile phase is from 0.1% (v / v) to 0.3% (v / v); preferably, the concentration of the mobile phase is 0.2% (v / v).

[0023] In some embodiments, the tetrabutylammonium hydroxide is adjusted to a pH of 7.4-7.8, preferably 7.6, using phosphoric acid.

[0024] In some embodiments, the volume ratio of the mobile phase A tetrabutylammonium hydroxide solution to acetonitrile is more preferably 90:10; and / or the volume ratio of the mobile phase B tetrabutylammonium hydroxide solution to acetonitrile is 40:60.

[0025] In some embodiments, the chromatographic column has dimensions of 250 × 4.6 mm and a packing particle size of 5 μm. Preferably, the chromatographic column is an Agilent ZORBAX Eclipse XDB-C18 or Ultimate Plus C18.

[0026] In some embodiments, the column temperature of the chromatographic column is 25 to 35°C.

[0027] In some embodiments, the column temperature of the chromatographic column is 30°C.

[0028] In some embodiments, the flow rate of the mobile phase is 0.8 to 1.2 mL / min.

[0029] In some embodiments, the flow rate of the mobile phase is 1 mL / min.

[0030] In some embodiments, the chromatographic conditions further include an injection volume of 15 μL to 25 μL.

[0031] In some embodiments, the chromatographic conditions further include an injection volume of 20 μL.

[0032] In some embodiments, the detection wavelength of the chromatographic conditions is from 208 nm to 212 nm.

[0033] In some embodiments, the detection wavelength is 210 nm.

[0034] In some embodiments, the detection method includes preparing a test solution and preparing a reference solution.

[0035] In some embodiments, the test solution preparation step includes: accurately measuring 1 ml of the sample to be tested, placing it in a 100 ml volumetric flask, adding 1 ml of 1 mol / L sodium hydroxide solution, diluting with water to the mark, sonicating for 5 min, and shaking well.

[0036] In some embodiments, the preparation of the reference solution includes: accurately weighing approximately 12 mg of sodium gluconate reference standard, placing it in a 20 ml volumetric flask, adding an appropriate amount of water to dissolve it, adding 0.2 ml of 1 mol / L sodium hydroxide solution, diluting with water to the mark, sonicating for 5 min, and shaking well.

[0037] In some embodiments, the detection method includes: accurately measuring the test solution and the reference solution, injecting them separately into a liquid chromatograph, calculating the peak area using the external standard method, and multiplying the result by 0.8946.

[0038] In some embodiments, the liquid preparation in the detection method is selected from calcium gluconate oral solution, zinc gluconate oral solution, calcium gluconate and zinc oral solution, and calcium gluconate injection; preferably, calcium gluconate oral solution, zinc gluconate oral solution, and calcium gluconate and zinc oral solution.

[0039] The embodiments of this application achieve the following beneficial effects:

[0040] The method for detecting gluconate content in liquid formulations in this application does not use EDTA to chelate metal ions in the test sample. Since EDTA forms a large molecular chelate after chelating with metal ions, multiple injections will damage the chromatographic column life.

[0041] The method for detecting gluconate content in liquid preparations disclosed in this application involves treating the sample with sodium hydroxide instead of EDTA, which allows for complete dissociation of gluconate, minimizes damage to the chromatographic column, and reduces detection costs.

[0042] The method for detecting gluconate content in liquid formulations disclosed in this application includes a gradient elution procedure comprising five consecutive elution stages. By optimizing the high-performance liquid chromatography detection method and avoiding interference from other substances, the target analyte can be well separated from adjacent impurity peaks during detection, thus enabling accurate detection of gluconate content in the formulation.

[0043] The method for detecting gluconate content in liquid preparations provided in this application makes the quality testing indicators of drugs containing gluconate more comprehensive, which is conducive to better control of the quality of liquid preparations. Attached Figure Description

[0044] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0045] Figure 1 The HPLC chromatogram of the test solution in Example 1;

[0046] Figure 2 The HPLC chromatogram of the reference solution in Example 1;

[0047] Figure 3 This is the HPLC chromatogram of the blank solution in Example 1;

[0048] Figure 4 The HPLC chromatogram of the 20% linear solution is shown in the linearity test.

[0049] Figure 5 The HPLC chromatogram of the 50% linear solution is shown in the linearity test.

[0050] Figure 6 The HPLC chromatogram of the 80% linear solution is shown in the linearity test.

[0051] Figure 7 The HPLC chromatogram of the 100% linear solution is shown in the linearity test.

[0052] Figure 8 The HPLC chromatogram of the 160% linear solution is shown in the linearity test.

[0053] Figure 9 This is the HPLC chromatogram of the negative solution from the recovery test.

[0054] Figure 10 The HPLC chromatogram of the 80% spiked test solution for the recovery test;

[0055] Figure 11 The HPLC chromatogram of the 100% spiked test solution is shown in the recovery test.

[0056] Figure 12 HPLC chromatogram of the 120% spiked test solution for recovery test;

[0057] Figure 13 HPLC chromatogram of the test sample solution under standard conditions for durability testing;

[0058] Figure 14 HPLC chromatogram of the test solution at a column temperature of 25°C for durability testing;

[0059] Figure 15HPLC chromatogram of the test solution at a column temperature of 35℃ for durability testing;

[0060] Figure 16 HPLC chromatogram of the test solution with a robust mobile phase pH of 7.4;

[0061] Figure 17 HPLC chromatogram of the test solution with a robust mobile phase at pH 7.8;

[0062] Figure 18 Replace the column with an Ultimate Plus C18 (4.6*250mm, 5μm) for durability; HPLC chromatogram of the test solution.

[0063] Figure 19 Replace the HPLC chromatogram of the test solution in the Shimadzu LC-2050C instrument for durability purposes;

[0064] Figure 20 The HPLC chromatogram of the blank solution in Comparative Example 1;

[0065] Figure 21 The HPLC chromatogram of the reference solution in Comparative Example 1;

[0066] Figure 22 The image shows the HPLC chromatogram of the test solution in Comparative Example 2. Detailed Implementation

[0067] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0068] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Unless otherwise specified, all quantitative analysis experiments in the following examples were performed in triplicate, and the results were averaged.

[0069] This application uses calcium gluconate oral solution as an example to illustrate the method for detecting gluconate in liquid preparations of this application.

[0070] The instruments and reagents used in the embodiments of this application are as follows:

[0071] 1. Instruments:

[0072] Aglient 1260II high performance liquid chromatograph.

[0073] Electronic balances, Mettler Toledo XSR105 and XPR2 / A.

[0074] pH meter, Mettler Toledo S220-K.

[0075] 2. Chromatographic column: C18 (Aglient ZORBAX Eclipse XDB-C18, 4.6mm×250mm, 5μm)

[0076] 3. Reagents:

[0077] Test sample: Calcium gluconate oral solution, which is self-made by our company, batch number 24080501, specification 10ml: 100mg, the ingredients are calcium gluconate 100mg (equivalent to calcium 9mg), and the excipients are lactic acid, calcium hydroxide and flavoring.

[0078] Sodium gluconate reference standard: Brand is China National Institutes for Food and Drug Control, chemical reference standard, content is 99.8%.

[0079] Tetrabutylammonium hydroxide: Brand: Anaiji, Purity: Analytical grade.

[0080] Acetonitrile: Brand: Fisher; Purity: chromatographic grade.

[0081] Phosphoric acid: Brand: Tianjin Fuyu; Purity: Analytical grade.

[0082] The solutions used in the embodiments of this application are prepared as follows:

[0083] 1. Test solution

[0084] Specific steps: Accurately measure 1 ml of the sample to be tested, place it in a 100 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, dilute with water to the mark, sonicate for 5 min, shake well, and the test solution is obtained.

[0085] 2. Reference solution

[0086] Specific steps: Weigh 12.33 mg of sodium gluconate reference standard accurately, place it in a 20 ml volumetric flask, add an appropriate amount of water to dissolve it, add 0.2 ml of 1 mol / L sodium hydroxide solution, dilute with water to the mark, sonicate for 5 min, and shake well to obtain the reference standard solution.

[0087] 3. Blank solution

[0088] Specific steps: Prepare a 0.01 mol / L sodium hydroxide solution as a blank solution.

[0089] Example 1: Detection of gluconate content in samples

[0090] The prepared test solutions, reference solutions, and blank solutions were analyzed by high-performance liquid chromatography.

[0091] The chromatographic conditions are as follows:

[0092] Column: C18 (Agilent ZORBAX Eclipse XDB-C18, 4.6mm × 250mm, 5μm);

[0093] Mobile phase A: 0.2% (v / v) tetrabutylammonium hydroxide solution (pH adjusted to 7.6 with phosphoric acid) to acetonitrile in a volume ratio of 90:10;

[0094] Mobile phase B: 0.2% (v / v) tetrabutylammonium hydroxide solution (pH adjusted to 7.6 with phosphoric acid) to acetonitrile in a volume ratio of 40:60;

[0095] Flow rate: 1.0 mL / min;

[0096] Column temperature: 30℃;

[0097] Detection wavelength: 210nm;

[0098] Injection volume: 20 μL;

[0099] The elution procedure is shown in Table 1 below:

[0100] Washout stage Washing time (min) Mobile phase A (%) Mobile phase B (%) Phase 1 (Isocratic Washout) 10 100 0 Second stage (gradient elution) 2 100→0 0→100 Phase 3 (Isocratic Washout) 8 0 100 Phase 4 (gradient elution) 5 0→100 100→0 Phase 5 (Isocratic Washing) 25 100 0

[0101] The HPLC detection results of the test solution and the reference solution in this embodiment are shown in Table 2 and Table 3, respectively.

[0102] Calculation of gluconate content: The gluconate content is calculated by peak area using the external standard method and multiplied by 0.8946.

[0103] Calculation formula:

[0104]

[0105] in:

[0106] W 对 : Sample weight of the reference standard, mg;

[0107] A 对 : Peak area of ​​gluconate in the chromatogram of the reference solution;

[0108] A 样 : The peak area of ​​gluconate in the chromatogram of the test solution;

[0109] V 样 : Sampling volume of the test sample;

[0110] 0.8946: Conversion factor between gluconate and sodium gluconate.

[0111] Table 2: HPLC chromatographic peak results of the test solution

[0112]

[0113] Table 3: HPLC chromatographic peak results of the reference solution

[0114]

[0115] Based on Table 2 above and Figure 1 The HPLC chromatogram of the test sample solution shows that the retention times of the impurity peak and the glucate peak are 4.748 min and 3.923 min, respectively. The glucate peak and the adjacent impurity peak have been completely separated.

[0116] Based on Table 3 and Figure 2 The HPLC chromatogram of the reference solution shows that the retention time of the glucate peak is 3.918 min, the theoretical plate number is 5163, the tailing factor is 1.057, the overall peak shape is good, and the baseline is stable.

[0117] in accordance with Figure 3 The HPLC chromatogram of the blank solution shows that the blank solution does not interfere with the detection of glucate ions, indicating that this method has good specificity.

[0118] Example 2 Linearity Test

[0119] Accurately weigh 59.99 mg of sodium gluconate reference standard, place it in a 25 ml volumetric flask, dissolve and dilute with water to the mark, shake well, and use as a linear stock solution (2.395 mg / ml).

[0120] Sodium gluconate reference solutions with gluconate concentrations of 0.120 mg / ml, 0.299 mg / ml, 0.479 mg / ml, 0.599 mg / ml, and 0.958 mg / ml were prepared using a stock solution. Solutions of different concentrations were injected sequentially from lowest to highest, with 20 μl injected at each concentration, and the peak area was recorded. A linear regression was performed with peak area on the ordinate and injection concentration (mg / ml) on the abscissa, yielding the regression equation y = 610.8134x - 1.9308, R0. 2 =1.0000. Experimental results show that gluconate exhibits good linearity within the concentration range of 0.120-0.958 mg / ml. Representative chromatograms are shown below. Figures 4-8 .

[0121] Table 4: Results of the linear experiment

[0122]

[0123] Example 3 Recovery Test

[0124] Negative solution: Prepare a solution that does not contain calcium gluconate according to the prescription dosage of calcium gluconate oral solution.

[0125] Transfer 0.1 ml of the negative solution to a 10 ml volumetric flask, add 0.1 ml of sodium hydroxide (1 mol / L), dilute to the mark with water, sonicate for 5 min, and shake well. Inject the sample for detection. No chromatographic peak appeared at the retention time of the target peak, indicating that the negative solution does not interfere with the detection of the gluconate peak, demonstrating the good specificity of this method. See the chromatogram below. Figure 9 .

[0126] Preparation of spiked recovery solutions: Accurately weigh approximately 4.8 mg, 6.0 mg, and 7.2 mg of sodium gluconate reference standard, respectively, and place them in separate 10 mL volumetric flasks. Add 0.1 mL of negative control solution, dissolve in water, add 0.1 mL of sodium hydroxide (1 mol / L), and dilute to the mark with water. Sonicate for 5 min, shake well, and prepare triplicate for each solution as 80%, 100%, and 120% recovery solutions. The experimental results show that the average recovery rate of gluconate was 99.7%, with an RSD of 0.50%, indicating good accuracy of the method. Representative chromatograms are shown below. Figures 10-12 .

[0127] Table 5: Results of Recovery Test

[0128]

[0129] Example 4 Solution Stability Test

[0130] The reference solution and the test solution were placed at room temperature and measured at 0, 2, 4, 8, 12, 24 and 48 h. The RSD value (n=7) of the peak area of ​​the reference solution within 48 h was 0.36%, and the mean detection amount of the test solution was 54.8 mg / ml, which was 100.0% of the labeled amount, with an RSD value (n=7) of 0.38%, indicating that the reference solution and the test solution were stable within 48 h at room temperature.

[0131] Table 6: Solution stability results

[0132]

[0133] Example 5 Durability Test

[0134] The chromatographic conditions for the gluconate detection method were varied according to the standard conditions and Table 7 (tables showing different column temperatures, pH values, column types, and instruments). The standard conditions were: column temperature 30℃, mobile phase buffer pH 7.6, Agilent ZORBAX Eclipse XDB-C18 column, and a 1260II HPLC system. Results showed that the system suitability met the requirements with minor variations in these conditions. The resolution between the gluconate peak and adjacent peaks in the test solution was excellent, and the RSD was 0.50%, indicating good robustness of the method. Representative chromatograms are attached. Figures 13-19 .

[0135] Table 7: Durability Results

[0136]

[0137] Comparative Example 1

[0138] The difference between this comparative example and the determination method of the test solution and reference standard in Example 1 is that this comparative example uses isocratic elution, which is different from the initial ratio of tetrabutylammonium hydroxide solution to acetonitrile in the mobile phase of Example 1. In this comparative example, the volume ratio of tetrabutylammonium hydroxide solution to acetonitrile in the mobile phase is 70:30. Other chromatographic conditions are the same as in Example 1.

[0139] The HPLC results of the blank solution and the reference solution of this comparative example are shown in Table 8.

[0140] Table 8: HPLC chromatographic peak results of the test solution

[0141]

[0142] The HPLC chromatograms of the blank solution and the reference solution for this comparative example are shown below. Figure 20 and Figure 21 According to Table 8 and Figures 20-21 The HPLC chromatograms of the blank solution and the reference solution show that the retention time of the gluconate peak is 1.682 min. The blank solution has an inverted peak at the same retention time, which interferes with the detection of the gluconate peak.

[0143] By using the method in Comparative Example 1, which involves changing the volume ratio of tetrabutylammonium hydroxide solution to acetonitrile in the mobile phase, the gluconate peak could not be separated from the blank solvent peak, interfering with the detection.

[0144] Comparative Example 2

[0145] The difference between this comparative example and the test solution and reference method in Example 1 is that the gradient elution procedure is different from that in Example 1. Specifically, this comparative example uses the initial ratio of Example 1 as the mobile phase for isocratic elution.

[0146] The elution procedure is shown in Table 9 below:

[0147] Washout stage Washing time (min) Mobile phase A (%) Mobile phase B (%) Isocratic elution 30 100 0

[0148] The HPLC detection results of the test solution of the comparative example are shown in Table 10.

[0149] Table 10: HPLC chromatographic peak results of the test solution

[0150]

[0151] The HPLC chromatogram results of the test solution for this comparative example are shown below. Figure 22 According to Table 10 and Figure 22 The HPLC chromatogram of the test solution shows that the retention times of the gluconate peak and the impurity peak are 2.384 min and 2.945 min, respectively. The gluconate peak and the adjacent impurity peak are well separated, indicating that this initial ratio can effectively separate them. However, the baseline after gluconate shows a significant increase, which is due to the elution of impurities remaining in the column from the previous injection.

[0152] Using the method in Comparative Example 2, which involves changing the elution program to isocratic elution, impurities that cannot be eluted in the current test solution will remain in the next test, interfering with the detection.

[0153] In summary, the detection method optimized in this application is highly specific and sensitive, and can accurately detect the content of gluconate, making it suitable for determining the content of gluconate in preparations containing gluconate.

[0154] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for detecting gluconate content in a liquid preparation, characterized in that, It includes: The test solution of the liquid preparation containing gluconate, which has been dissolved in sodium hydroxide solution, is analyzed by high performance liquid chromatography (HPLC). The detection conditions of the HPLC include: The chromatographic column is packed with octadecyl-bonded silica gel. The mobile phase comprises mobile phase A and mobile phase B; mobile phase A is a mixed solution of tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (85-95):(5-15), and mobile phase B is a mixed solution of tetrabutylammonium hydroxide solution and acetonitrile in a volume ratio of (30-50):(50-70), wherein the tetrabutylammonium hydroxide solution is adjusted to a pH of 6.0 to 8.0 with phosphoric acid; Gradient elution procedures include:

2. The detection method according to claim 1, characterized in that, The gradient elution procedure includes:

3. The detection method according to claim 1, characterized in that, The concentration of tetrabutylammonium hydroxide in the mobile phase is from 0.1% (v / v) to 0.3% (v / v); Preferably, the concentration of the mobile phase is 0.2% (v / v).

4. The detection method according to claim 1, characterized in that, The tetrabutylammonium hydroxide is prepared by adjusting the pH value to 7.4-7.8 with phosphoric acid, preferably 7.

6.

5. The detection method according to any one of claims 1-4, characterized in that, The volume ratio of the mobile phase A, tetrabutylammonium hydroxide solution, to acetonitrile is 90:10; and / or The volume ratio of the mobile phase B tetrabutylammonium hydroxide solution to acetonitrile is 40:

60.

6. The detection method according to claim 1, characterized in that, The chromatographic column has dimensions of 250 mm × 4.6 mm and a packing particle size of 5 μm. Preferably, the chromatographic column is an Agilent ZORBAX Eclipse XDB-C18 or Ultimate Plus C18.

7. The detection method according to claim 1, characterized in that, The column temperature of the chromatographic column is 25 to 35°C, preferably 30°C; and / or The flow rate of the mobile phase is 0.8 to 1.2 mL / min, preferably 1 mL / min.

8. The detection method according to claim 1, characterized in that, The injection volume for the chromatographic conditions is 15 μL to 25 μL, preferably 20 μL; and / or The detection wavelength for the chromatographic conditions is from 208 nm to 212 nm, preferably 210 nm.

9. The detection method according to any one of claims 1-8, characterized in that, It includes: The preparation of the test solution includes: accurately measuring 1 ml of the sample to be tested, placing it in a 100 ml volumetric flask, adding 1 ml of 1 mol / L sodium hydroxide solution, diluting with water to the mark, sonicating for 5 min, and shaking well; It also includes the preparation of a reference solution, wherein the preparation of the reference solution includes: taking about 12 mg of sodium gluconate reference standard, accurately weighing it, placing it in a 20 ml volumetric flask, adding an appropriate amount of water to dissolve it, adding 0.2 ml of 1 mol / L sodium hydroxide solution, diluting it with water to the mark, sonicating for 5 min, and shaking well; Preferably, the method further includes the steps of: accurately measuring the test solution and the reference solution, injecting them separately into the liquid chromatograph, calculating the peak area using the external standard method, and multiplying the result by 0.8946.

10. The detection method according to claim 1, characterized in that: The liquid preparation is selected from calcium gluconate oral solution, zinc gluconate oral solution, calcium gluconate zinc oral solution, and calcium gluconate injection.

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