A method for detecting the content of all components of a compound tranexamic acid preparation and application thereof

Abstract

This invention discloses a method and application for detecting the total content of all components in a compound tranexamic acid preparation, belonging to the field of pharmaceutical analysis technology. The method employs high-performance liquid chromatography (HPLC) and includes the following steps: injecting a compound tranexamic acid test solution into an HPLC instrument, recording the chromatogram, and calculating the contents of tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate using the external standard method. This method can simultaneously detect five components in compound tranexamic acid tablets. Furthermore, the main peaks obtained by this method exhibit good peak symmetry, resolution, stability, and accuracy in content determination.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method and application for detecting the total content of all components in a compound tranexamic acid preparation. Background Technology

[0002] Compound tranexamic acid tablets are a complex preparation composed of tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate, and have the effect of whitening and fading spots. As a compound preparation, the multiple components have certain compatibility and ratio relationships, which effectively improves the activity of the drug while also improving the convenience of administration.

[0003] Currently, titration is the primary method used in pharmacopoeias of various countries to determine the content of tranexamic acid, vitamin C, glutathione, and L-cysteine. However, for a compound preparation, titration is not specific enough to accurately determine the content of each component. Therefore, finding a convenient, rapid, accurate, and efficient analytical method for determining the content of each component in compound preparations is of paramount importance.

[0004] However, the structures of each component in tranexamic acid compound tablets are as follows:

[0005]

[0006]

[0007] It can be seen that these five main components contain few conjugated double bonds in their structure, and mostly contain amino, carboxyl and hydroxyl groups. Their molecular polarity is relatively high and their retention is weak. It is difficult to retain and separate these five components using reversed-phase high-performance chromatography, which is a technical problem that needs to be solved in the current content testing of compound tranexamic acid tablets. Summary of the Invention

[0008] To address the issue that conventional techniques lack the ability to simultaneously detect the five components of compound tranexamic acid tablets using reversed-phase high-performance chromatography (RP-HPLC), this invention provides a method for detecting the total content of all components in compound tranexamic acid preparations. This method utilizes a conventional high-performance liquid chromatograph and a reversed-phase column to detect the five components in compound tranexamic acid tablets. Furthermore, the peak shape symmetry, resolution, stability, and content accuracy of each component obtained by this method are all excellent.

[0009] On one hand, this invention provides a method for detecting the total content of all components of a compound tranexamic acid preparation, using high-performance liquid chromatography (HPLC). The method includes the following steps: injecting a compound tranexamic acid test solution into an HPLC instrument, recording the chromatogram, and calculating the content of each component. The total components include tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate. The detection conditions for the HPLC method are as follows:

[0010] Stationary phase: A chromatographic column packed with octadecylsilane-bonded silica gel;

[0011] Mobile phase: Mobile phase A is a buffer solution containing sodium octane sulfonate ion-pairing reagent and phosphate; mobile phase B is methanol or acetonitrile; flow rate is 0.5-1 mL / min.

[0012] Detection wavelength: 200-230nm.

[0013] In the research and development process, in order to improve the retention of each component, the inventors explored and tried various methods and adopted a buffer solution containing phosphate and sodium octane sulfonate ions as mobile phase A, combined with an organic phase of methanol or acetonitrile, which can improve the retention of each component.

[0014] Understandably, the reagents such as methanol or acetonitrile mentioned above, according to conventional understanding in this field, generally refer to analytical grade reagents with a purity of 99.9% or higher. For the buffer solution of the mobile phase, the solvent is usually water, and the water used is generally purified water such as deionized water.

[0015] In some embodiments, the chromatographic column is an Agilent ZORBAX SB-C18 with dimensions of 3.0 mm × 150 mm and a diameter of 3.5 μm. Using this column in conjunction with the mobile phase system of this invention achieves good detection performance.

[0016] In some embodiments, the mobile phase A is a sodium 1-octanesulfonate-potassium dihydrogen phosphate buffer solution with a pH of 2.4 to 2.9, and the mobile phase B is methanol.

[0017] To achieve better separation between the components, this invention also screened the effect of different pH values ​​on the separation system. The results showed that each component was highly dependent on pH, with a mobile phase pH of 2.4–2.9 being ideal. Furthermore, the inventors discovered during the research and development process that the five main components were more stable in aqueous solution than in a 0.1% phosphoric acid aqueous solution; therefore, a 1-octanesulfonate-potassium dihydrogen phosphate buffer solution with a pH of 2.4–2.9 was selected as mobile phase A.

[0018] It is understandable that the pH value mentioned above will generally have an error of ±0.05 in actual operation. The pH value mentioned in this invention is the pH value measured by a pH meter during preparation, and the pH value is tested at a temperature of 25°C.

[0019] In some embodiments, the concentration of sodium 1-octanesulfonate in the mobile phase A is 1 ± 0.1 g / L, and the concentration of potassium dihydrogen phosphate is 50 ± 5 mM.

[0020] In some schemes, the mobile phase A is adjusted to pH 2.5 with phosphoric acid.

[0021] In some formulations, the mobile phase A is prepared as follows: 6.8 g of potassium dihydrogen phosphate and 1.0 g of sodium 1-octanesulfonate are weighed and placed in 1000 mL of water, dissolved and shaken well, and the pH is adjusted to 2.5 with phosphoric acid. The mixture is then filtered to obtain the mobile phase A.

[0022] In some protocols, elution is performed according to the following gradient elution procedure:

[0023]

[0024]

[0025] The gradient elution time changes at a constant rate from 3 min to 15 min.

[0026] The mobile phase ratios mentioned above refer to the percentage of each mobile phase volume in the total eluent volume. It can be understood that the gradient elution program described above means that at 0 min, the initial eluent consists of 95% mobile phase A and 5% mobile phase B; elution proceeds at a gradient of 5% mobile phase B within 0–3 min; at 15 min, the volume ratio of mobile phase A in the eluent decreases to 70%, and the volume ratio of mobile phase B increases to 30%; and elution proceeds at a constant gradient of 30% mobile phase B within 15–18 min.

[0027] Within 3–15 minutes, the conversion rate of mobile phase A from 95% to 70% is a uniform change. Correspondingly, the conversion rate of mobile phase B from 5% to 30% is also a uniform change.

[0028] In some schemes, the content of each component is calculated using the external standard method.

[0029] In some embodiments, the flow rate is 0.5-0.9 mL / min, preferably 0.7 mL / min.

[0030] In some schemes, the detection wavelength is 205-220nm, preferably 205-215nm, and more preferably 210nm.

[0031] Considering the spectral characteristics of tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate, the detection wavelengths are all terminal absorption. Therefore, in the above high-performance liquid chromatography method, the detection wavelength can be selected as 200-230 nm. However, considering factors such as the signal intensity of the target analyte and the stability of the detection baseline fluctuation, 205-220 nm can be selected, preferably 205-215 nm, and more preferably 210 nm.

[0032] In some schemes, the external standard method described above follows the conventional practice in the field, using standards and other reagents of different concentration gradients to establish a standard curve for quantitative detection.

[0033] In some schemes, the detection conditions also include column temperature, which is 35–45°C, for example, 40°C.

[0034] In some methods, the compound tranexamic acid test solution is an aqueous solution, and the injection volume is 5–10 μL, for example, 10 μL; wherein, in the compound tranexamic acid test solution, the concentration of tranexamic acid is 1.0 ± 0.2 mg / mL, the concentration of vitamin C is 0.54 ± 0.108 mg / mL, the concentration of glutathione is 0.068 ± 0.0136 mg / mL, the concentration of L-cysteine ​​is 0.1 ± 0.02 mg / mL, and the concentration of calcium pantothenate is 0.04 ± 0.008 mg / mL.

[0035] On the other hand, the present invention also provides the application of the above-mentioned detection method in the quality control of compound tranexamic acid preparations.

[0036] In some formulations, the compound tranexamic acid preparation is in the form of tablets.

[0037] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0038] The reagents and raw materials used in this invention are all commercially available.

[0039] The positive and progressive effects of this invention are as follows:

[0040] This invention provides a method for detecting the total content of all components in a compound tranexamic acid preparation. This method solves the problem that titration has poor specificity and cannot accurately determine the content of each component in a compound tranexamic acid preparation. It can use reversed-phase high-performance liquid chromatography to detect the content of all components in compound tranexamic acid tablets, solving the problem that highly polar small molecule compounds are poorly retained in reversed-phase chromatography. Moreover, it does not require high-end equipment; a common liquid chromatograph with a cost-effective chromatographic column can be used to complete the detection.

[0041] Meanwhile, the main peaks of each component obtained by this detection method have good peak shape symmetry, separation, stability and content accuracy, and can simultaneously determine the content of five components. Attached Figure Description

[0042] Figure 1 This is a superimposed diagram showing the retention and separation of each component in the phosphoric acid aqueous solution-acetonitrile system.

[0043] Figure 2This is a superimposed image showing the retention and separation of each component in a system using 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 0.1% phosphoric acid aqueous solution and acetonitrile.

[0044] Figure 3 The retention and separation spectra of each component in the system using 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 20 mM potassium dihydrogen phosphate solution and methanol are shown.

[0045] Figure 4 The retention and separation spectra of each component in the system consisting of 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 20 mM potassium dihydrogen phosphate solution - 0.1% triethylamine (pH adjusted to 2.5 with phosphoric acid) and methanol are shown.

[0046] Figure 5 The retention and separation spectra of each component in the methanol system using 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 50 mM potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) are shown.

[0047] Figure 6 The retention and separation spectra of each component in the methanol system using 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 50 mM potassium dihydrogen phosphate solution (pH adjusted to 2.3 with phosphoric acid) are shown.

[0048] Figure 7 This is a superimposed image showing the retention and separation of each component in a methanol system using a 1 g / L ion-pairing reagent (sodium 1-octanesulfonate) - 50 mM potassium dihydrogen phosphate solution (pH adjusted to 2.4–2.9 with phosphoric acid).

[0049] Figure 8 The retention and separation overlays of each component were obtained using a 1 g / L ion-pairing reagent (sodium 1-octanesulfonate)-50 mM potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid) and methanol system at a flow rate of 0.5–0.9 ml / min.

[0050] Figure 9 The retention and separation overlays of each component were obtained using a 1 g / L ion-pairing reagent (sodium 1-octanesulfonate)-50 mM potassium dihydrogen phosphate solution (pH adjusted to 2.5 with phosphoric acid), methanol system, and column temperatures of 35℃, 40℃, and 45℃.

[0051] Figure 10 Overlay diagrams showing the retention and separation of components in specific experiments and mixed standard solutions. Detailed Implementation

[0052] The present invention is further illustrated below by way of embodiments, but these embodiments are not intended to limit the invention to their scope. Experimental methods in the following embodiments, unless otherwise specified, were performed according to conventional methods and conditions, or as selected in the product instructions.

[0053] Example 1

[0054] This embodiment explores and optimizes the method for determining the content of five components in compound tranexamic acid preparations: tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate.

[0055] I. Reagents and Instruments

[0056] 1. Reference standard

[0057] 1.1 Source of reference standard

[0058] The content of each raw material was standardized and assigned by two people using the reference standards from the China National Institutes for Food and Drug Control (Table 1) and then used as working reference standards (as secondary reference standards).

[0059] Table 1. Reference standards for 5 components from the China National Institutes for Food and Drug Control

[0060]

[0061]

[0062] 1.2 Preparation of reference solution

[0063] 1.2.1 Preparation of single-item control standards

[0064] Take approximately 50 mg of L-cysteine, 50 mg of glutathione, 50 mg of calcium pantothenate, 50 mg of vitamin C, and 50 mg of tranexamic acid, respectively, accurately weigh them, and place them in 20 ml volumetric flasks. Dilute with diluent and bring to the mark, shake well, and use them as reference solutions for each single product.

[0065] 1.2.2 Preparation of Mixed Control Standards

[0066] Accurately weigh approximately 108 mg of L-cysteine, approximately 68 mg of glutathione, and 40 mg of calcium pantothenate, place them in the same 100 ml volumetric flask, dilute with diluent and bring to volume, shake well, and prepare two parallel portions as mixed stock solutions, labeled Stock-1 and Stock-2 respectively.

[0067] Accurately weigh approximately 50 mg of tranexamic acid and approximately 27 mg of vitamin C, place them in the same 50 ml volumetric flask, dilute and dissolve them with diluent, accurately transfer 5 ml of the above mixed stock solution into the above 50 ml volumetric flask, dilute to the mark with diluent, shake well, and prepare two parallel aliquots as the reference solution, labeled as STD-1 and STD-2 respectively.

[0068] 2. Test sample

[0069] 2.1 Source of test sample

[0070] The test sample was tranexamic acid compound tablets, which were developed and produced by Shanghai Yaotan Pharmaceutical Research and Development Co., Ltd.

[0071] 2.2 Preparation of test solution

[0072] Take 10 tranexamic acid compound tablets and grind them into a fine powder. Weigh approximately 1150 mg of the ground powder accurately into a 100 ml volumetric flask, add an appropriate amount of diluent, sonicate for 2 min, remove and cool, dilute with diluent and bring to volume, shake well, let stand, centrifuge at 5000 rpm for 5 minutes, take the supernatant and filter (filter membrane: aqueous syringe filter, 0.45 μm), discard 2 ml of initial filtrate, take the filtrate for analysis, prepare two parallel aliquots, labeled SPL1-1 and SPL1-2 respectively, as test solution 1, for the determination of calcium pantothenate content.

[0073] Take 5 ml of the above test solution 1 into a volumetric flask and dilute to the mark. Shake well and prepare two parallel portions, labeled SPL2-1 and SPL2-2 respectively, as test solution 2, for the determination of the contents of other components.

[0074] 3. Instruments

[0075] The high-performance liquid chromatograph used in the method of this invention is a Thermo Fisher UltiMate 3000.

[0076] II. Chromatographic Conditions

[0077] The chromatograms of compound tranexamic acid preparations (tablets) were examined under different chromatographic conditions to explore and optimize the detection conditions.

[0078] 1. Phosphoric acid aqueous solution-acetonitrile system

[0079] Column: Agilent ZORBAX Eclipse Plus C18, 4.6mm × 250mm, 5μm.

[0080] Mobile phase A: 0.1% aqueous phosphoric acid solution.

[0081] Mobile phase B: Acetonitrile.

[0082] Flow rate: 1.0 mL / min.

[0083] Column temperature: 40℃.

[0084] Injection volume: 5 μL.

[0085] Detection wavelength: 210nm.

[0086] The gradient elution procedure is as follows:

[0087] Table 2. Gradient elution program

[0088] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0 95 5 3 95 5 15 5 95 15.1 95 5 20 95 5

[0089] Take the above-mentioned single-item reference solutions and inject them separately. The resulting chromatograms are shown below. Figure 1 As shown in the figure, L-cysteine, vitamin C, tranexamic acid, and glutathione are poorly retained in this system.

[0090] 2. Sodium octane sulfonate-phosphate-acetonitrile system

[0091] Column: Waters Symmetry C18, 4.6 mm × 250 mm, 5 μm.

[0092] Mobile phase A: 1 g / L sodium 1-octanesulfonate - 0.1% phosphoric acid aqueous solution.

[0093] Mobile phase B: Acetonitrile.

[0094] Flow rate: 1.0 mL / min.

[0095] Column temperature: 40℃.

[0096] Injection volume: 5 μL.

[0097] Detection wavelength: 210nm

[0098] The gradient elution procedure is as follows:

[0099] Table 3. Gradient elution program

[0100] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0 95 5 3 95 5 15 70 30 15.1 95 5 20 95 5

[0101] Take the above-mentioned single-item reference solutions and inject them separately. The resulting chromatograms are shown below. Figure 2 As shown in the figure, the components in this system are retained and enhanced and can be separated from each other, but the peak shapes are poor and the introduced ion-pairing reagents cause fluctuations in the system.

[0102] 3. Sodium octane sulfonate-20mM potassium dihydrogen phosphate-methanol system

[0103] Column: Waters Symmetry C18, 4.6 mm × 250 mm, 5 μm.

[0104] Mobile phase A: 1 g / L sodium 1-octanesulfonate-20 mM potassium dihydrogen phosphate aqueous solution.

[0105] Mobile phase B: Methanol.

[0106] Flow rate: 1.0 mL / min.

[0107] Column temperature: 40℃.

[0108] Injection volume: 5 μL.

[0109] Detection wavelength: 210nm.

[0110] The gradient elution procedure is as follows:

[0111] Table 4. Gradient elution program

[0112]

[0113]

[0114] Take the above mixed reference solution, inject it for detection, and the resulting chromatogram is shown below. Figure 3 As shown in the figure, vitamin C and glutathione cannot be separated in this system, and the separation degree and peak shape of each peak do not meet the requirements.

[0115] 4. Sodium octane sulfonate - 20mM potassium dihydrogen phosphate - 0.1% triethylamine - methanol system (pH 2.5)

[0116] Column: Agilent ZORBAX SB-C18, 3.0 mm × 150 mm, 3.5 μm.

[0117] Mobile phase A: 1 g / L sodium 1-octanesulfonate - 20 mM potassium dihydrogen phosphate aqueous solution - 0.1% triethylamine (adjusted to pH 2.5 with phosphoric acid).

[0118] Mobile phase B: Methanol.

[0119] Flow rate: 1.0 mL / min.

[0120] Column temperature: 40℃.

[0121] Injection volume: 5 μL.

[0122] Detection wavelength: 210nm.

[0123] The gradient elution procedure is as follows:

[0124] Table 5. Gradient elution program

[0125] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0 95 5 3 95 5 15 70 30 15.1 95 5 20 95 5

[0126] Two injections were performed under the above conditions. The first injection used a mixed reference standard of the above five components, but tranexamic acid was not detected in the chromatogram. The second injection used a high-concentration (2.5 mg / ml) tranexamic acid standard solution alone. The chromatogram is shown below. Figure 4 As shown in the figure (the first injection is below the baseline, and the second injection is above the baseline), it can be seen from the figure that, under this system, vitamin C and L-cysteine ​​were not separated after the mixed reference sample was injected, and tranexamic acid had a weak response.

[0127] 5. Sodium octane sulfonate - 50mM potassium dihydrogen phosphate - phosphoric acid - methanol system (pH 2.5)

[0128] Column: Agilent ZORBAX SB-C18, 3.0 mm × 150 mm, 3.5 μm.

[0129] Mobile phase A: 1 g / L sodium 1-octanesulfonate - 50 mM potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.5 with phosphoric acid).

[0130] Mobile phase B: Methanol.

[0131] Flow rate: 0.7 mL / min.

[0132] Column temperature: 40℃.

[0133] Injection volume: 10 μL.

[0134] Detection wavelength: 210nm.

[0135] The gradient elution procedure is as follows:

[0136] Table 6. Gradient elution program

[0137] Time (min) Mobile phase A (% v / v) Mobile phase B (% v / v) 0 95 5 3 95 5 15 70 30 18 70 30 18.1 95 5 25 95 5

[0138] Take the above mixed reference solution, inject it for detection, and the resulting chromatogram is shown below. Figure 5 As shown in the figure, the system exhibits good baseline, good retention of each component, and good separation and peak shape among the peaks.

[0139] 6. Sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.3)

[0140] Mobile phase A: 1 g / L sodium 1-octanesulfonate - 50 mM potassium dihydrogen phosphate aqueous solution (pH adjusted to 2.3 with phosphoric acid).

[0141] Mobile phase B: Methanol.

[0142] The remaining chromatographic conditions were the same as those in item 5 above for the sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5).

[0143] Take the above mixed reference solution, inject it for detection, and the resulting chromatogram is shown below. Figure 6 As shown in the figure, glutathione and calcium pantothenate cannot be well separated under pH 2.3 conditions.

[0144] 7. Adjust the pH of the system

[0145] Referring to the conditions of the sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5) in item 5 above, the pH of mobile phase A was adjusted to 2.4, 2.5, 2.7 and 2.9 only with phosphoric acid, and the chromatograms were recorded after each injection.

[0146] Chromatogram as shown Figure 7 As shown in the figure, under pH conditions of 2.4–2.9, each component can be well retained, and the separation and peak shape between each peak are good.

[0147] 8. System flow rate adjustment

[0148] Referring to the conditions of the sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5) in item 5 above, only the flow rate was adjusted to 0.5 mL / min, 0.7 mL / min, 0.8 mL / min and 0.9 mL / min, and the chromatograms were recorded after each injection.

[0149] Chromatogram as shown Figure 8 As shown in the figure, under the condition of 0.5 to 0.9 mL / min, each component can be well retained, and the separation and peak shape between each peak are good.

[0150] 9. Column temperature adjustment

[0151] Referring to the conditions of the sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5) in item 5 above, only the column temperature was adjusted to 35℃, 40℃ and 45℃, and the chromatograms were recorded after each injection.

[0152] Chromatogram as shown Figure 9 As shown in the figure, when the column temperature is between 35 and 45°C, each component can be well retained, and the separation and peak shape between each peak are good.

[0153] Example 2

[0154] This embodiment performs methodological verification on the detection method obtained from the screening in Example 1.

[0155] The detection conditions used were in accordance with the “sodium octane sulfonate-50mM potassium dihydrogen phosphate-phosphoric acid-methanol system (pH 2.5)” under item 5 of Example 1.

[0156] 1. Specificity and System Applicability

[0157] 1.1 Blank solvent and blank excipient solution

[0158] Use purified water as the diluent as the blank solution. Weigh out the blank excipients (excluding the active ingredient) of the compound tranexamic acid tablets and place them in a volumetric flask. Dissolve, dilute, and bring to volume, then shake well and filter to obtain the blank excipient solution. Inject the blank solution and the blank excipient solution separately into a high-performance liquid chromatograph for detection. The results are as follows: Figure 10 As shown in the figure, there is no interference between the blank solution and the blank excipient solution at the peaks of the five components.

[0159] 1.2 Resolution

[0160] The above-mentioned STD-1 was injected into a high-performance liquid chromatograph for detection. The resolutions between L-cysteine ​​and vitamin C, glutathione and L-cysteine, calcium pantothenate and glutathione, and tranexamic acid and calcium pantothenate were 8.8, 11.5, 6.7 and 34.8, respectively.

[0161] The results show that the proposed method has good specificity.

[0162] 1.3 System Applicability

[0163] First, inject STD-1 five times consecutively, and STD-2 twice consecutively to start testing the sample. Inject STD-1 every 12 injections, and end the experiment with an injection of STD-1.

[0164] The relative standard deviations of the peak areas of five consecutive STD-1 injections were 0.2% for vitamin C, 0.2% for L-cysteine, 0.1% for glutathione, 0.5% for calcium pantothenate, and 0.3% for tranexamic acid. The recoveries of STD-2 relative to the response of STD-1 were 99.3% for vitamin C, 99.2% for L-cysteine, 99.3% for glutathione, 99.4% for calcium pantothenate, and 99.4% for tranexamic acid. The recoveries of intermittently injected STD-1 relative to the average response of five consecutive STD-1 injections were 100.7%–101.6% for vitamin C, 100.1%–100.9% for L-cysteine, 100.6%–101.5% for glutathione, 99.9%–101.4% for calcium pantothenate, and 100.3%–101.2% for tranexamic acid.

[0165] The results show that the method of the present invention has good system applicability.

[0166] 2. Linearity and Range

[0167] According to the range of 50%-150% of the labeled content (specifically selecting five concentration points: 50%, 80%, 100%, 120%, and 150%), a series of standard linear solutions with vitamin C concentrations of 0.27–0.81 mg / ml, L-cysteine ​​concentrations of 0.054–0.162 mg / ml, glutathione concentrations of 0.034–0.102 mg / ml, calcium pantothenate concentrations of 0.02–0.06 mg / ml, and tranexamic acid concentrations of 0.5–1.5 mg / ml were prepared and injected into a high-performance liquid chromatograph for detection.

[0168] Standard curves for each component were plotted based on the obtained chromatograms. The linear correlation coefficients for each component were as follows: vitamin C 0.9996, L-cysteine ​​0.9999, glutathione 1.0000, calcium pantothenate 1.0000, and tranexamic acid 1.0000.

[0169] The results show that the standard curve of the detection method of the present invention has a high correlation coefficient and good linearity.

[0170] 3. Accuracy

[0171] A series of accuracy solutions were prepared at 80%, 100%, and 120% of the labeled concentration levels, with three replicates prepared for each concentration level. The corresponding blank excipients and mixed reference solutions were added to each accuracy solution. The corresponding concentrations at each level were: Vitamin C 0.432 mg / ml, 0.540 mg / ml, 0.648 mg / ml; L-cysteine ​​0.08 mg / ml, 0.10 mg / ml, 0.12 mg / ml; glutathione 0.0544 mg / ml, 0.068 mg / ml, 0.0816 mg / ml; calcium pantothenate 0.032 mg / ml, 0.04 mg / ml, 0.048 mg / ml; and tranexamic acid 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml. These solutions were injected into a high-performance liquid chromatograph (HPLC) for detection, and the recovery rate was calculated based on the obtained chromatograms.

[0172] The average recovery rates of vitamin C in the nine accuracy solutions were 100.5% (RSD 0.9%), L-cysteine ​​99.7% (RSD 0.3%), glutathione 100.9% (RSD 0.4%), calcium pantothenate 100.9% (RSD 0.2%), and tranexamic acid 99.7% (RSD 0.6%).

[0173] The results show that the method of the present invention has good accuracy, and the linear range that can be accurately detected is 0.432-0.648 mg / ml for vitamin C, 0.08-0.12 mg / ml for L-cysteine, 0.0544-0.0816 mg / ml for glutathione, 0.032-0.048 mg / ml for calcium pantothenate, and 0.8-1.2 mg / ml for tranexamic acid.

[0174] 4. Repeatability

[0175] Following the preparation method of the test sample in Example 1, six parallel samples were prepared and injected into a high-performance liquid chromatograph for detection. The content of each component and the relative standard deviation of the content were calculated based on the obtained chromatograms.

[0176] The results showed that the average labeled amount of vitamin C was 98.9% with an RSD of 0.4%, the average labeled amount of L-cysteine ​​was 95.9% with an RSD of 0.3%, the average labeled amount of glutathione was 91.3% with an RSD of 0.4%, the average labeled amount of calcium pantothenate was 95.2% with an RSD of 0.4%, and the average labeled amount of tranexamic acid was 96.8% with an RSD of 0.3%.

[0177] The results show that the method of the present invention has good repeatability.

[0178] 5. Stability.

[0179] STD-1 and the test solution (prepared as described in Example 1) were placed at 4°C and injected into a high-performance liquid chromatograph at intervals of 4, 8, 14, 24, and 28 hours for detection. The relative standard deviations of the peak areas of STD-1 and the test solution at each time point were calculated based on the obtained chromatograms.

[0180] The results showed that the relative standard deviations of the peak areas at each time point for STD-1 were 0.6% for vitamin C, 0.3% for L-cysteine, 0.6% for glutathione, 0.6% for calcium pantothenate, and 0.6% for tranexamic acid; the relative standard deviations of the peak areas at each time point for the test solution were 0.8% for vitamin C, 0.7% for L-cysteine, 0.8% for glutathione, 0.8% for calcium pantothenate, and 0.6% for tranexamic acid.

[0181] The results showed that the reference and test solutions were stable for at least 28 hours at 4°C.

[0182] The verification results of this embodiment show that the method of the present invention has good system applicability, specificity, linearity and range, accuracy, repeatability and stability.

Claims

1. A method for detecting the total content of a complex tranexamic acid preparation, characterized by, The detection was performed using high-performance liquid chromatography (HPLC), including the following steps: The compound tranexamic acid test solution was injected into the HPLC instrument, the chromatogram was recorded, and the content of each component was calculated. The total components included tranexamic acid, vitamin C, glutathione, L-cysteine, and calcium pantothenate. The detection conditions for HPLC were as follows: Stationary phase: A chromatographic column packed with octadecylsilane-bonded silica gel; Mobile phase: Mobile phase A is a 1-octanesulfonate sodium-potassium dihydrogen phosphate buffer solution with pH 2.4~2.9, mobile phase B is methanol, and the flow rate is 0.5-1 mL / min. In mobile phase A, the concentration of 1-octanesulfonate sodium is 1±0.1 g / L, and the concentration of potassium dihydrogen phosphate is 50±5 mM. Elution was performed according to the following gradient elution procedure: ； The gradient elution time changes from 3 min to 15 min with a uniform rate of change in the mobile phase; Column temperature: 35~45℃; Detection wavelength: 205~220nm.

2. The detection method according to claim 1, characterized in that, The chromatographic column was an Agilent ZORBAX SB-C18, with dimensions of 3.0 mm × 150 mm and a diameter of 3.5 μm.

3. The method of claim 1, wherein, The mobile phase A was adjusted to pH 2.5 with phosphoric acid.

4. The method of claim 1, wherein The detection conditions meet the following criteria: the content of each component is calculated using the external standard method.

5. The method of claim 1, wherein The detection conditions meet the following conditions: the flow rate is 0.5-0.9 mL / min.

6. The detection method according to claim 5, characterized in that, The detection conditions meet the following condition: the flow rate is 0.7 mL / min.

7. The method of claim 1, wherein, The detection conditions meet the following conditions: the detection wavelength is 205~215nm.

8. The method of claim 1, wherein, The detection conditions meet the following conditions: the detection wavelength is 210nm.

9. The method of claim 1, wherein, The column temperature is 40°C.

10. The detection method according to claim 1, characterized in that, The compound tranexamic acid test solution is an aqueous solution, and the injection volume is 5~10 μL; wherein, in the compound tranexamic acid test solution, the concentration of tranexamic acid is 1.0±0.2 mg / mL, the concentration of vitamin C is 0.54±0.108 mg / mL, the concentration of glutathione is 0.068±0.0136 mg / mL, the concentration of L-cysteine ​​is 0.1±0.02 mg / mL, and the concentration of calcium pantothenate is 0.04±0.008 mg / mL.

11. The detection method according to claim 10, characterized in that, The injection volume of the compound tranexamic acid test solution was 10 μL.

12. The application of the detection method according to any one of claims 1-11 in the quality detection of compound tranexamic acid preparations.

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