A method for detecting lactic acid content by liquid chromatography

Abstract

The invention relates to the technical field of pharmaceutical analysis, and provides a method for detecting lactic acid content by liquid chromatography, wherein octadecylsilane bonded silica gel is used as a filler, the detection wavelength is 200-220 nm, the column temperature is 15-25° C., the flow rate is 0.4-0.6 ml / min, and the injection volume is 5-30 μl. The method of the invention has strong specificity, good separation, high stability, and high accuracy, and solves the problems of poor separation and inaccurate detection of adjacent chromatographic peaks in lactic acid and famotidine injection in the existing method.

Description

Technical Field

[0001] The invention relates to the technical field of medical analysis, and in particular to a method for detecting lactic acid content by using liquid chromatography. Background Art

[0002] Famotidine injection is indicated for upper gastrointestinal bleeding caused by peptic ulcer, gastric and duodenal mucosal erosion and bleeding caused by various reasons other than tumors and esophageal and gastric fundus varices. Its main ingredient is famotidine, a histamine H2 receptor blocker that has a significant inhibitory effect on gastric acid secretion and can also inhibit pepsin secretion.

[0003] Lactic acid is an excipient for famotidine injection, which acts as a pH regulator and can also improve drug solubility. It also plays an important role in the safety, stability and other properties of the preparation. Therefore, it is necessary to pay attention to the relationship between the stability trend of famotidine injection and the lactic acid content in it to ensure product quality.

[0004] At present, the lactic acid determination method reported in the literature is mainly gas chromatography. For example, patent No. CN202310979796.9 discloses a method for detecting lactic acid content, which includes: (1) preparing a test solution: dissolving the test sample in a first solvent to obtain a test solution. (2) preparing a test solution: mixing the test solution with periodic acid to obtain a test solution, wherein the molar amount of periodic acid added is greater than the molar amount of lactic acid in the test sample. (3) using gas chromatography to detect the test solution to obtain the lactic acid content in the test sample. However, gas chromatography has limitations in determining lactic acid. Lactic acid has poor thermal stability and is easily decomposed in a high-temperature vaporization chamber. Therefore, lactic acid needs to be derivatized into esters before determination. The operation is relatively complicated and there may be matrix interference, resulting in low accuracy of the test results. Summary of the invention

[0005] Therefore, in view of the above, the present invention provides a method for detecting lactic acid content by liquid chromatography, which solves the problems of poor separation of lactic acid and adjacent chromatographic peaks in famotidine injection and inaccurate detection in the existing method.

[0006] To achieve the above object, the present invention is achieved through the following technical solutions:

[0007] A method for detecting lactic acid content by liquid chromatography, using octadecylsilane bonded silica gel as filler, with a detection wavelength of 200-220nm, a column temperature of 15-25°C, a flow rate of 0.4-0.6ml / min, and an injection volume of 5-30μl.

[0008] Further improvements are: the detection wavelength is 210 nm, the column temperature is 15° C., the flow rate is 0.5 ml / min, and the injection volume is 20 μl.

[0009] A further improvement is: using water as diluent, 0.1% formic acid solution as mobile phase A, and methanol as mobile phase B for gradient elution.

[0010] A further improvement is: based on the mobile phase volume ratio of 100%, the elution gradient of the mobile phase is:

[0011] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 25 100 0 25.1 80 20 35 80 20 35.1 100 0 50 100 0

[0012] A further improvement is that the method for detecting lactic acid content by liquid chromatography comprises the following steps:

[0013] (1) Prepare reference solution;

[0014] (2) Prepare the test solution;

[0015] (3) Take the reference solution and the test solution and inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the lactic acid content using the external standard method.

[0016] A further improvement is that the preparation method of the reference solution is: take an appropriate amount of sodium lactate reference substance, use water as a diluent, dilute and dissolve it to a concentration of 0.3 mg / ml.

[0017] A further improvement is that the test solution is prepared by taking an appropriate amount of famotidine injection, diluting it 10 times with water as a diluent.

[0018] By adopting the above technical solution, the beneficial effects of the present invention are:

[0019] The liquid chromatography method for detecting lactic acid content of the present invention has strong specificity, good separation, high stability, high precision and high accuracy, and has practical significance for the quality control of famotidine injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the specificity spectrum of the reference solution in Example 1 of the present application;

[0021] Figure 2 It is the specificity spectrum of the test solution in Example 1 of the present application;

[0022] Figure 3 This is a standard curve diagram of Example 1 of the present application. DETAILED DESCRIPTION

[0023] The following will describe the implementation methods of the present invention in detail in conjunction with specific embodiments, so that the implementation process of how the present invention applies technical means to solve technical problems and achieve technical effects can be fully understood and implemented accordingly.

[0024] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the reagents and products used are all commercially available. The sources, trade names and components of the reagents used are indicated when they first appear.

[0025] Chromatographic column: Waters Atlantis T3 C18 (4.6 mm×250 mm, 5 μm).

[0026] Detector: UV; Detection wavelength: 210nm; Flow rate: 0.5ml / min; Column temperature: 15℃; Injection volume: 20μl; Diluent: water; Mobile phase A: 0.1% formic acid solution; Mobile phase B: methanol. Use gradient mobile phase for elution. Based on the mobile phase volume ratio of 100%, the mobile phase gradient is set as follows:

[0027]

[0028]

[0029] Example 1

[0030] The method for detecting lactic acid content by liquid chromatography comprises the following steps:

[0031] (1) Solution preparation

[0032] Blank solution: water.

[0033] Blank excipient solution: Accurately measure 2 ml of blank excipient, place in a 20 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0034] Reference solution: Take 7.5 mg of sodium lactate reference solution, weigh accurately, place in a 20 ml volumetric flask, add water to dissolve and dilute to scale, shake well, and prepare two in parallel.

[0035] Test solution: Accurately measure 2 ml of famotidine injection, place it in a 20 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0036] (2) Take the reference solution and the test solution and inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the lactic acid content by the external standard method.

[0037] Example 2

[0038] The detection method of Example 1 was validated from several aspects, including specificity, system applicability, solution stability, linearity and range, accuracy, and repeatability, as described in detail below.

[0039] (1) Exclusivity

[0040] The specificity results are shown in Table 1.

[0041] Table 1 Specificity results

[0042] Solution name Component name Retention time (minutes) blank solution / / Blank excipient solution / / Reference solution lactic acid 13.670 Test solution lactic acid 13.620

[0043] From the results in Table 1, it can be seen that the blank solution and blank auxiliary material solution have no interference with lactic acid detection.

[0044] (2) System applicability

[0045] The system suitability results are shown in Table 2.

[0046] Table 2 System suitability results

[0047]

[0048] As can be seen from Table 2, the RSD of the peak area of ​​reference solution 1# measured five times continuously was 0.57%, and the recovery rate of the response factor of the two reference solutions was 100.4%, which was in line with the regulations, indicating that the method had good specificity and system applicability.

[0049] (3) Solution stability test

[0050] The stability results of the reference solution are shown in Table 3.

[0051] Table 3 Stability results of reference solution

[0052] Time (h) Main peak area of ​​reference solution 0 549.282 15 541.029 22 540.511 32 541.328 39 538.989 48 538.387 RSD / % 0.73

[0053] The stability results of the test solution are shown in Table 4.

[0054] Table 4 Stability results of test solution

[0055] Time (h) Main peak area of ​​test solution 0 553.354 1 555.828 14 548.111 24 547.330 37 546.293 42 547.495 51 547.585 RSD / % 0.67

[0056] It can be seen from Tables 3 and 4 that, under room temperature conditions, the reference solution is stable after 48 hours, and the test solution is stable after 51 hours.

[0057] (4) Linearity and range

[0058] Blank solution: water.

[0059] Linear stock solution: Take 18.7 mg of sodium lactate reference substance, weigh accurately, place in a 25 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0060] Linear solution 1: Accurately measure 2 ml of linear stock solution, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0061] Linear solution 2: Accurately measure 2 ml of linear stock solution, place it in a 5 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0062] Linear solution 3: Accurately measure 2.5 ml of linear stock solution, place it in a 5 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0063] Linear solution 4: Accurately measure 3 ml of linear stock solution, place it in a 5 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0064] Linear solution 5: Accurately measure 7.5 ml of the linear stock solution, place it in a 10 ml volumetric flask, add water to dilute to the scale, shake well, and the solution is ready.

[0065] The linear relationship of lactic acid is shown in Table 5.

[0066] Table 5 Lactic acid linear relationship

[0067]

[0068] It can be seen from Table 5 that the linear relationship of lactic acid is good using the detection method provided by the present invention.

[0069] (5) Accuracy

[0070] Blank solution: water.

[0071] Reference solution: Take 7.5 mg of sodium lactate reference substance, weigh accurately, place in a 20 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0072] Blank auxiliary material solution: Accurately measure 2 ml of blank auxiliary material, place it in a 20 ml volumetric flask, add water to dilute to the scale, shake well, and obtain.

[0073] Stock solution of reference substance: Take 37.4 mg of sodium lactate reference substance, weigh accurately, place in a 50 ml volumetric flask, add water to dissolve and dilute to the scale, shake well, and obtain.

[0074] 80% spiked test solution: Accurately measure 4 ml of reference stock solution and 1 ml of blank auxiliary material, place in a 10 ml volumetric flask, add water to dilute to the mark, shake well, and prepare 3 copies in parallel.

[0075] 100% spiked test solution: Accurately measure 5 ml of reference stock solution and 1 ml of blank auxiliary material, place in a 10 ml volumetric flask, add water to dilute to the mark, shake well, and prepare 3 copies in parallel.

[0076] 120% spiked test solution: Accurately measure 6 ml of reference stock solution and 1 ml of blank auxiliary material, place in a 10 ml volumetric flask, add water to dilute to the mark, shake well, and prepare 3 copies in parallel.

[0077] The accuracy test results are shown in Table 6.

[0078] Table 6 Accuracy test results

[0079]

[0080]

[0081] As can be seen from the table, the average recovery rate of lactic acid (n=9) is 98.4%, and the RSD is 0.27%. This method has high accuracy.

[0082] (6) Repeatability

[0083] Six test sample solutions were prepared in parallel, and the lactic acid content was calculated using the external standard method. The test results are shown in Table 7.

[0084] Table 7 Repeatability results

[0085]

[0086] As shown in Table 7, the average content is 3.05 mg / ml and the RSD is 0.16%, indicating that the detection method provided by the present invention has good precision.

[0087] In summary, the method for detecting the lactic acid content in famotidine injection established in the present invention has strong specificity, good separation, high stability, high precision and high accuracy, and can be used as a method for detecting the lactic acid content in famotidine injection.

[0088] The above records are only embodiments of the present invention. Any modification or change made by a person familiar with the present technology using the present invention shall fall within the patent scope claimed by the present invention and shall not be limited to those disclosed in the embodiments.

Claims

1. A method for detecting lactic acid content by liquid chromatography, characterized in that: Octadecylsilane bonded silica gel is used as filler, the detection wavelength is 200-220 nm, the column temperature is 15-25° C., the flow rate is 0.4-0.6 ml / min, and the injection volume is 5-30 μl.

2. The method for detecting lactic acid content by liquid chromatography according to claim 1, characterized in that: The detection wavelength was 210 nm, the column temperature was 15° C., the flow rate was 0.5 ml / min, and the injection volume was 20 μl.

3. The method for detecting lactic acid content by liquid chromatography according to claim 1, characterized in that: Water was used as the diluent, 0.1% formic acid solution was used as the mobile phase A, and methanol was used as the mobile phase B for gradient elution.

4. The method for detecting lactic acid content by liquid chromatography according to claim 3, characterized in that: Taking the mobile phase volume ratio as 100%, the elution gradient of the mobile phase is:

5. The method for detecting lactic acid content by liquid chromatography according to claim 1, characterized in that: The following steps are involved: (1) Prepare reference solution; (2) Prepare the test solution; (3) Take the reference solution and the test solution and inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the lactic acid content using the external standard method.

6. The method for detecting lactic acid content by liquid chromatography according to claim 5, characterized in that: The preparation method of the reference solution is as follows: take an appropriate amount of sodium lactate reference substance, use water as a diluent, and dilute and dissolve it to a concentration of 0.3 mg / ml.

7. The method for detecting lactic acid content by liquid chromatography according to claim 5, characterized in that: The preparation method of the test solution is: take an appropriate amount of famotidine injection, use water as a diluent, and dilute it 10 times to obtain the solution.

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