A method for determining the content of pivalic acid in ciprefilate sodium by HPLC

Abstract

The application discloses a method for determining the content of valine acid in seviteronel sodium by HPLC, and points out that when the concentration of phosphoric acid in a diluent is in the range of 1% to 4%, seviteronel sodium will be precipitated, thereby solving the problem that seviteronel sodium is poor in stability and continues to degrade to produce valine acid during detection. The method comprises the following steps: preparing a test sample solution and a control sample solution; adopting a C18 chromatographic column, taking a phosphate buffer solution as mobile phase A and acetonitrile as mobile phase B to perform gradient elution; and calculating the content of valine acid by the peak area according to an external standard method. The method has the advantages of good separation effect, high sensitivity, short analysis time and simple operation, and is suitable for determining the content of valine acid in seviteronel sodium raw medicine and preparation, and can be used for drug quality control.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, specifically relating to an HPLC method for determining the content of tervastatin in cetirizine sodium. Background Technology

[0002] 2,2-Dimethylpropionic acid, chemical name: 2,2-dimethylpropionic acid, molecular formula: C5H 10 O2, chemical structural formula as follows:

[0003] Tervaline is a degradation impurity of cevelex sodium via high-temperature or hydrolysis pathways.

[0004] Pterovalic acid is readily soluble in organic solvents such as alcohols, ethers, and chloroform, and slightly soluble in water; its molecular weight is approximately 102.13; and its boiling point is approximately 164℃. Due to its relatively small molecular weight and moderate boiling point, it exhibits good peak elution under gas chromatography (GC) temperature programs. If necessary, it can be derivatized into methyl pterovalic acid to improve peak shape and sensitivity. Because its structure lacks p-π conjugation and contains only one carboxyl group, it exhibits no strong ultraviolet absorption and is generally not determined by high-performance liquid chromatography (HPLC).

[0005] However, because cevelexostat sodium degrades to pentovalinic acid at high temperatures (such as the GC injection temperature and GC temperature program), the amount of pentovalinic acid in the product cannot be accurately determined using GC. Furthermore, due to the poor thermal stability of cevelexostat sodium, high-temperature degradation can produce other impurity peaks and even baseline elevation, all of which can interfere with the specificity of pentovalinic acid determined by GC.

[0006] Pivalic acid is typically detected using polar gas chromatography columns, such as the DB-WAX with polyethylene glycol as the stationary phase, employing a direct injection temperature-programmed method. The analytical method discovered in this invention uses a conventional C18 HPLC column for the detection of pentivalic acid. Compared to gas chromatography methods, this method does not degrade cevelenostat sodium to produce pentivalic acid during detection. Because cevelenostat sodium has extremely poor solubility in acidic conditions, this invention uses phosphoric acid in a diluent to dissolve cevelenostat sodium in 50% acetonitrile for extraction. After this extraction, the diluent is added to precipitate the cevelenostat sodium, resulting in better sample solution stability and preventing the formation of additional pentivalic acid during analysis. Furthermore, pentivalic acid has a pKa value of approximately 5.03. Using a phosphoric acid-acetonitrile system at pH 3.0 and appropriately increasing the sample concentration can effectively enhance the HPLC response of pentivalic acid. The above two advantages solve the problems of poor stability of cevelexostat sodium and low response of tervastatin on HPLC, and increase the reliability of quality research on the determination of tervastatin in cevelexostat sodium in the pharmaceutical industry. Summary of the Invention

[0007] The technical problem to be solved: Gas chromatography (GC) itself causes cevelenoic acid sodium to degrade and produce pentovalinic acid, making it impossible to accurately determine the true content of pentovalinic acid in the product. Furthermore, pentovalinic acid has no strong UV absorption, and cevelenoic acid sodium will also hydrolyze and degrade to pentovalinic acid in solvents with a high water content over a long period, making it unsuitable to use conventional methods to dissolve cevelenoic acid sodium before HPLC determination. This invention uses 50% acetonitrile to dissolve cevelenoic acid sodium, rapidly extracts pentovalinic acid, then uses an acidic diluent to precipitate the cevelenoic acid sodium, and filters the filtrate to prevent its degradation and subsequent formation of pentovalinic acid. Simultaneously, based on the pKa of pentovalinic acid (approximately 5.03), a phosphoric acid-acetonitrile system at pH 3.0 is used, and the concentration of the test solution is appropriately increased, successfully solving the problems of poor stability of cevelenoic acid sodium and weak HPLC response of pentovalinic acid.

[0008] Technical solution: A method for determining the content of tervastatin in cetirizine sodium by HPLC, comprising the following steps: Step 1: Weigh the sodium cevelestat sample, add acetonitrile solution and sonicate to dissolve, dilute with solvent to 5 mg / mL, shake well and filter to obtain the test solution; Step 2: Weigh out the penvalerate reference standard, dissolve it in acetonitrile solution by sonication, and dilute it with solvent to prepare a reference standard solution with a penvalerate concentration of 10-100 μg / mL; Step 3: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, perform detection, record the chromatogram, and calculate the content of tervastatin by peak area according to the external standard method.

[0009] Furthermore, in steps 1 and 2, the concentration of the acetonitrile solution is 50% (v / v); the sonication time is 15 min, the frequency is 40 kHz; and the solvent is a phosphoric acid solution with a pH of 3.0 and a concentration of 1-4% (v / v).

[0010] Furthermore, the solvent is a phosphoric acid solution with a pH of 3.0 and a concentration of 3.5-4% (v / v).

[0011] Furthermore, in step 3, the chromatographic column used in the liquid chromatograph is a C18 column with octadecylsilane-bonded silica gel as the packing material; the volume of the reference solution and the test solution is 100 μL; the detection conditions are: gradient elution with phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B.

[0012] Furthermore, the gradient elution procedure is as follows:

[0013] Furthermore, the phosphoric acid solution has a pH of 3.0 and a concentration of 0.05-0.09% (v / v); the gradient elution flow rate is 1.0-1.4 mL / min.

[0014] Furthermore, the phosphoric acid solution has a pH of 3.0 and a concentration of 0.06-0.07% (v / v); the gradient elution flow rate is 1.2 mL / min.

[0015] Furthermore, this method has the following technical specifications: Tervaline showed a good linear relationship with peak area in the concentration range of 2.500-7.500 μg / mL, with a correlation coefficient r ≥ 0.995; the limit of detection was 1.0000 μg / mL, and the limit of quantitation was 2.500 μg / mL; the system precision RSD (n=6) < 1.0%; the repeatability range (n=6) was 0.0%, and the absolute difference in intermediate precision was 0.0%; the average recovery rate was 104.6%, and the RSD (n=9) of the recovery rate was 4.9%.

[0016] The method described above is applicable to the determination of tervastatin content in cevelex sodium raw material and preparations. Beneficial effects

[0017] The present invention provides a simple sample pretreatment method that does not require complex derivatization steps, thereby achieving the precipitation of cevelexta sodium and reducing interference from subsequent tervastatin quantification.

[0018] This invention employs optimized chromatographic conditions and a gradient elution program, enabling baseline separation of pentovalinic acid from cetylexadestat sodium and other impurities with a resolution greater than 1.5, ensuring the accuracy of the detection results. It also exhibits high detection sensitivity, with a detection limit of 1.0000 μg / mL and a quantitation limit of 2.500 μg / mL, meeting the requirements for detecting trace amounts of pentovalinic acid.

[0019] This invention features short analysis time, improved detection efficiency, high accuracy, good reproducibility, and a stable and reliable method. Attached Figure Description

[0020] Figure 1 This is an overlay image of the sample detected by the method in Comparative Example 1 and the spiked limit solution. Figure 2 This is a superimposed image of the sample detected by the method in Comparative Example 2 and the spiked limit solution. Figure 3 This is a superimposed image of the sample and the spiked limit solution detected by the method in Example 1; Figure 4 The spectrum of the spiked test solution detected by the method in Example 1 is shown below. Figure 5 The spectrum of the spiked test solution detected by the method in Example 2; Figure 6 The spectrum of the spiked test solution detected by the method in Example 3; Figure 7 The spectrum of the spiked test solution detected by the method in Example 4 is shown below. Figure 8 This is the spectrum of the blank solution for the specificity test; Figure 9 This is the chromatogram of the reference solution for the specificity test; Figure 10 Spectrum of the sample solution for specificity testing; Figure 11 Spectra of other impurity localization solutions (impurity A and XW1-A) for specificity testing; Figure 12 Linear relationship of pentanoic acid in methodological validation. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments: Example 1

[0022] A method for determining the content of pentovalinic acid in cetirizine sodium by HPLC: Prepare the solvent-phosphoric acid solution: Take 40 mL of phosphoric acid, add 1000 mL of water and shake well, then adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution; Test solution: Weigh approximately 50 mg of cevelexat sodium accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Example 2

[0023] A method for determining the content of pentovalinic acid in cetirizine sodium by HPLC: Prepare the solvent-phosphoric acid solution: Take 30 mL of phosphoric acid, add 1000 mL of water and shake well, then adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution; Test solution: Weigh approximately 50 mg of cevelexat sodium accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Example 3

[0024] A method for determining the content of pentovalinic acid in cetirizine sodium by HPLC: Prepare the solvent-phosphoric acid solution: Take 20 mL of phosphoric acid, add 1000 mL of water and shake well, then adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution; Test solution: Weigh approximately 50 mg of cevelexat sodium accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Example 4

[0025] A method for determining the content of pentovalinic acid in cetirizine sodium by HPLC: Prepare the solvent-phosphoric acid solution: Take 10 mL of phosphoric acid, add 1000 mL of water and shake well, then adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution; Test solution: Weigh approximately 50 mg of cevelexat sodium accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Comparative Example 1

[0026] The difference between this comparative example and Example 1 is that mobile phase A and acetonitrile are used as solvents, as detailed below: Test solution: Weigh approximately 50 mg of cevelexat sodium accurately and place it in a 10 mL volumetric flask. Mix mobile phase A and acetonitrile in a 1:1 volume ratio to prepare a diluent. Dilute to the mark, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of pentvalerate reference standard accurately and place it in a 200 mL volumetric flask. Mix mobile phase A and acetonitrile in a 1:1 volume ratio as a diluent, dilute to the mark, and shake well. Accurately measure 1 mL of the solution and place it in a 10 mL volumetric flask. Dilute to the mark with solvent and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Comparative Example 2

[0027] The difference between this comparative example and Example 1 is that the concentration of phosphoric acid in the solvent is reduced, as detailed below: Prepare the solvent-phosphoric acid solution: Take 1 mL of phosphoric acid, add 1000 mL of water and shake well, then adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution; Test solution: Weigh approximately 50 mg of cevelexat sodium accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with solvent, shake well, filter, and collect the filtrate. Reference solution: Weigh approximately 10 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, shake well, accurately measure 1 mL, place it in a 10 mL volumetric flask, dilute to the mark with solvent, and shake well. Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (Welch Xtimate C18, 4.6 × 250 mm, 5 μm or equivalent column); mobile phase A was phosphoric acid solution (0.7 mL phosphoric acid added to 1000 mL water, pH adjusted to 3.0 with sodium hydroxide), and mobile phase B was acetonitrile, with gradient elution performed according to the table below; flow rate was 1.2 mL / min; column temperature was 25 °C; detection wavelength was 210 nm; injection volume was 100 μL. The elution conditions are as follows: Results Analysis

[0028] The results of Comparative Example 1 are as follows Figure 1 As shown, the peak area of ​​tervastatin in the spiked sample was inconsistent with that in the tervastatin solution. It is suspected that this is because the solution of cevelexostat sodium is alkaline, while tervastatin is acidic. The pH of the solution in the spiked solution is inconsistent with that of the control sample, resulting in a strong matrix effect and a large difference in area. Therefore, it is not possible to accurately quantify tervastatin while dissolving the sample (cevelexostat sodium). In Comparative Example 2, based on the research and analysis of Comparative Example 1, and considering the characteristics of cevelenoic acid sodium (extremely low solubility in acidic solutions), only a very small amount of acetonitrile was used to dissolve the sample. Tervalerate was then extracted using ultrasound while the sample was completely dissolved. After complete extraction, an acidic diluent was added to precipitate cevelenoic acid sodium. Tervalerate acid still exhibited excellent solubility under these conditions. After filtration, tervalerate acid was added to the filtrate, while cevelenoic acid sodium was discarded with the precipitate. Injection revealed that, at the same concentration level, the peak area of ​​tervalerate acid in the spiked solution and the reference solution was essentially the same. Figure 2 However, the chromatographic peaks did not overlap, suggesting that the amount of added phosphoric acid was insufficient and did not form a strong buffering capacity, resulting in incomplete precipitation of cevelexat sodium. A small amount of matrix interference remained, causing a slight difference in pH between the spiked test solution and the reference solution, which further led to the incomplete overlap of chromatographic peaks, characterized by inconsistent retention times.

[0029] Figure 3 As shown in Example 1, increasing the concentration of the solvent phosphoric acid caused sodium cevelestat to precipitate. After filtration and injection, it was found that at the same concentration level, the peak areas of tervastatin in the spiked solution and the reference solution were comparable, and the retention times of the chromatographic peaks were essentially the same. Furthermore, the greater the amount of phosphoric acid added to the diluent, the more complete the precipitation of sodium cevelestat. Figure 4-7 As shown, the smaller the other chromatographic peaks in the chromatogram, the better. Methodological verification of the method in Example 1 above.

[0030] 1. Specificity test Experimental process Blank solution: Accurately measure 1 mL of 50% acetonitrile, place it in a 10 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0031] Diluent: Phosphoric acid solution (add 40 mL of phosphoric acid to 1000 mL of water, and adjust the pH to 3.0 with 10 mol / L sodium hydroxide solution).

[0032] Reference stock solution ①: Weigh 12.45 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, and shake well.

[0033] Reference solution ①: Accurately measure 1 mL of the reference stock solution ① and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0034] Reference stock solution ②: Weigh 10.73 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add 50% acetonitrile, sonicate for 15 min to dissolve and dilute to the mark, and shake well.

[0035] Reference solution ②: Accurately measure 1 mL of reference stock solution ② and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0036] Test solution: Weigh 52.12 mg of this product accurately, place it in a 10 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with diluent, shake well, filter, and take the filtrate.

[0037] Other impurity mixed standard solution: Accurately weigh approximately 6 mg of impurity A reference standard and approximately 2 mg of XW1-A reference standard, place them in a 50 mL volumetric flask, add 1 mL of 50% acetonitrile, sonicate for 15 min to dissolve, dilute to the mark with diluent, and shake well. Accurately measure 2.5 mL, place it in a 20 mL volumetric flask, dilute to the mark with diluent, and shake well.

[0038] Accurately measure the above solutions and inject them into the liquid chromatograph, then record the chromatograms.

[0039] Acceptable standards In a blank solution, there should be no interference at the elution position of tervastatin.

[0040] There should be no other peaks in the test solution that interfere with the detection of pentylamino acid.

[0041] Impurities A and XW1-A should not interfere with the detection of tervastatin.

[0042] Experimental results like Figure 8 , Figure 9 As shown, in the blank solution, there is no interference at the peak position of tervastatin.

[0043] like Figure 10 As shown, tervastatin was not detected in the test solution, and no other components interfered with the detection of tervastatin.

[0044] like Figure 11 As shown, neither impurity A nor XW1-A interferes with the detection of tervastatin.

[0045] Experimental conclusions The specificity of the method for detecting pentovalinic acid in cevelex sodium meets the requirements.

[0046] 2. Precision System precision Experimental process Reference solution: Take the reference solution ① from section 1.1.

[0047] Accurately measure the reference solution, inject it into the liquid chromatograph, inject it six times consecutively, and record the chromatogram.

[0048] Acceptable standards The RSD value of the retention time of the tervastatin peak in six consecutive reference solutions should not exceed 5.0%.

[0049] The RSD value of the peak area of ​​tervastatin in the reference solution for six consecutive tests should not exceed 5.0%.

[0050] Experimental results Table 1 System precision results

[0051] Experimental conclusions In six consecutive reference solutions, the RSD of the peak area of ​​pentavolic acid was 0.9%, and the RSD of the retention time was 0.1%, both less than 5.0% (see Table 1). The precision of the method for detecting pentavolic acid in cetirizine sodium meets the requirements.

[0052] 3. Repeatability Experimental process Reference solution: Take reference solution ① and reference solution ② from section 1.1.

[0053] Spiked test solution: Accurately weigh approximately 50 mg of this product and place it in a 10 mL volumetric flask. Add 1 mL of the reference stock solution to dissolve the sample, sonicate for 15 min, dilute to the mark with diluent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions.

[0054] The sample weights of the spiked test solution were: 49.47 mg, 45.76 mg, 48.42 mg, 46.15 mg, 48.95 mg, and 47.92 mg.

[0055] Accurately measure the reference solution and 6 spiked test solutions, inject them separately into the liquid chromatograph, and record the chromatograms.

[0056] Acceptable standards are shown in Table 2: Table 2 Repeatability Standards

[0057] Experimental results Table 3 Repeatability Results

[0058] Experimental conclusions The content values ​​of the six parallel test solutions were all 0.13%, with an average of 0.13% and a range of 0.0%, which is less than 0.1% (see Table 3 for details). The repeatability of the method for detecting pentovalinic acid in cevelex sodium meets the requirements.

[0059] 4. Intermediate precision Experimental process Reference stock solution ①: Weigh 12.470 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add diluent, sonicate to dissolve and dilute to the mark, and shake well.

[0060] Reference solution ①: Accurately measure 1 mL of the reference stock solution ① and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0061] Reference solution ②: Weigh 13.070 mg of tervastatin reference standard accurately, place it in a 200 mL volumetric flask, add diluent, sonicate to dissolve and dilute to the mark, and shake well.

[0062] Reference solution ②: Accurately measure 1 mL of reference stock solution ② and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0063] Spiked test solution: Accurately weigh approximately 50 mg of this product and place it in a 10 mL volumetric flask. Add 1 mL of the reference stock solution to dissolve the sample, sonicate for 15 min, dilute to the mark with diluent, shake well, filter, and collect the filtrate. Prepare 6 parallel solutions.

[0064] The sample weights of the spiked test solution were: 49.93 mg, 50.32 mg, 50.27 mg, 50.04 mg, 50.25 mg, and 50.16 mg.

[0065] Accurately measure the reference solution and 6 spiked test solutions, inject them separately into the liquid chromatograph, and record the chromatograms.

[0066] Acceptable standards For the second experimenter, the 6 parallel spiking test solutions should meet the following conditions, as detailed in Table 4: Table 4 Precision Standards

[0067] For the average of the results from the two experimenters, tervastatin should meet the following conditions, as detailed in Table 5: Table 5 Precision (Second Person) Standard

[0068] Experimental results Table 6 Intermediate Precision (Second Person) Results

[0069] Table 7 Intermediate Precision Results

[0070] Experimental conclusions The content of the six parallel test solutions obtained by the second experimenter was 0.12%, with an average of 0.12% and a range of 0.0%, which is less than 0.1% (see Table 6). The RD value of the average content measured by the two experimenters was 0.0% (see Table 7). The intermediate precision of the method for detecting pentovalinic acid in cetirizine sodium meets the requirements.

[0071] 5. Limit of Quantification Experimental process Reference solution: Take the reference solution ① from section 1.1.

[0072] Limit of Quantification Solution: Accurately measure 5 mL of the reference solution and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0073] Accurately measure the solution to the limit of quantitation and inject it into the liquid chromatograph. Inject the solution continuously for 6 injections and record the chromatogram.

[0074] Acceptable standards In six consecutive injections of the limit of quantitation solution, the peak height signal-to-noise ratio of tervastatin should be no less than 10.

[0075] In six consecutive injections of the limit of quantitation solution, the RSD value of the peak area of ​​tervastatin should not exceed 10.0%.

[0076] Report the concentration of pentylamino acid in the solution at the limit of quantitation.

[0077] Experimental results Table 8 Results of Limit of Quantification

[0078] Experimental conclusions In six consecutive injections of the limit-of-quantitation (LOQ) solution, the peak height signal-to-noise ratio of pentovalinic acid was the lowest at 12 and greater than 10; the RSD value of the peak area was 1.5% and less than 10.0% (see Table 8 for details); the concentration of the LOQ solution was 2.500 μg / mL. The LOQ of the method for detecting pentovalinic acid in cetylpenicillin sodium meets the requirements.

[0079] 6. Detection limit Experimental process Limit of Quantification Solution: Take the limit of quantification solution from section 3.1.

[0080] Detection limit solution: Accurately measure 4 mL of the quantitation limit solution and place it in a 10 mL volumetric flask. Dilute to the mark with diluent and shake well.

[0081] Accurately measure the solution at the detection limit, inject it into the liquid chromatograph, and record the chromatogram.

[0082] Acceptable standards In the detection limit solution, the peak height signal-to-noise ratio of the pentvalic acid peak should be no less than 3.

[0083] The report indicates the detection limit for the concentration of pentylcholine in the solution.

[0084] Experimental results Table 9 Detection Limit Results

[0085] Experimental conclusions In the solution with the detection limit, the peak height signal-to-noise ratio of pentavolic acid is 6, which is greater than 3 (see Table 9 for details); the detection limit concentration is 1.0000 μg / mL. The detection limit of the pentavolic acid detection method in cetylpenicillin sodium meets the requirements.

[0086] 7. Linear Experimental process Reference stock solution: Take the reference stock solution ① from section 1.1.

[0087] Prepare linear solutions according to Table 10. Accurately measure each linear solution, inject it into the liquid chromatograph, and record the chromatogram.

[0088] Table 10 Preparation of linear solutions

[0089] Acceptable standards The linear correlation coefficient r should be no less than 0.990.

[0090] Y 100% The deviation of the axis intercept should not exceed 10.0%.

[0091] Report the sum of squared residuals.

[0092] Experimental results Table 11 Linearity Results

[0093] Experimental conclusions The linear graph of pentylamino acid is as follows Figure 12 As shown, the correlation coefficient r is 0.998, which is greater than 0.990; Y 100% The axial intercept deviation is 2.3%, which is less than 10.0%; the sum of squared residuals is 0.00, as detailed in Table 11. The linearity of the method for detecting pentovalinic acid in cevelex sodium meets the requirements.

[0094] 8. Accuracy Experimental process Reference solution: Take the reference solution ① from section 1.1.

[0095] Reference stock solution: Take the reference stock solution ① from section 1.1.

[0096] Prepare solutions of various accuracy levels according to Table 12.

[0097] Table 12 Preparation of Accuracy Solutions

[0098] Accurately measure the reference solution, control sample, and various accuracy solutions, inject them into the liquid chromatograph, and record the chromatogram.

[0099] Acceptable standards The individual recovery rates should be between 80.0% and 120.0%.

[0100] The average recovery rate should be between 85.0% and 115.0%.

[0101] The RSD value (n=9) for the recovery rate at each level should not exceed 10.0%.

[0102] Experimental results Table 13 Experimental results of accuracy

[0103] Experimental conclusions For pentavolic acid, the individual recoveries ranged from 99.5% to 112.6%, with an average recovery of 104.6% and an RSD (n=9) of 4.9%, all meeting the requirements (see Table 13 for details). The accuracy of the method for detecting pentavolic acid in cevelex sodium met the requirements.

[0104] 9. Scope The concentration range of pentovalinic acid that can be accurately determined by analytical methods and meets the requirements of precision and linearity is 2.500 μg / mL to 7.500 μg / mL.

[0105] Filter membrane adsorption Experimental process Spiked test solution: Take the first portion of the spiked test solution under section 2.1.1.

[0106] Control sample: Take the unfiltered spiked test solution and centrifuge at 8000 rpm for 10 min.

[0107] Each subsequent filtrate: Take the unfiltered spiked test solution, pass it through a PTFE filter membrane, and discard 1 mL, 3 mL, and 5 mL respectively.

[0108] Precisely measure and control the sample and each subsequent filtrate, inject them into the liquid chromatograph, and record the chromatogram.

[0109] Acceptable standards If the ratio of the peak area of ​​pentovalinic acid in the filtrate to that in the control sample is between 0.9 and 1.1, the filter membrane is considered to be non-adsorbent.

[0110] Experimental results Table 14 Filter membrane adsorption results

[0111] Experimental conclusions The ratio of the peak area of ​​pentvalerate in each filtrate to that in the control sample was 1.0, ranging from 0.9 to 1.1, as detailed in Table 14. The membrane adsorption of pentvalerate in cetirizine sodium met the requirements.

[0112] 10. Solution stability Experimental process Reference solution: Take the reference solution ② from section 1.1.

[0113] Spiked test solution: Take the second portion of the spiked test solution under section 2.1.1.

[0114] Accurately measure the reference solution and the spiked test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0115] Acceptable standards For both the reference solution and the test solution, if the peak area of ​​pentvallic acid at each time point is between 0.8 and 1.2, and the peak area of ​​pentvallic acid in the control sample is between 0.8 and 1.2, the solution is considered stable.

[0116] Experimental results Table 15 Stability results of reference solutions

[0117] Table 16 Stability results of the test solution

[0118] Experimental conclusions For the reference solution, at room temperature (10–30°C), the peak area ratio of pentavolic acid measured at 77 h compared to 0 h was 1.0; under refrigeration (2–8°C), the peak area ratio of pentavolic acid measured at 76 h compared to 0 h was also 1.0, both within the range of 0.8–1.2 (see Table 15). For the test solution, at room temperature (10–30°C), the peak area ratio of pentavolic acid measured at 75 h compared to 0 h was 1.0; under refrigeration (2–8°C), the peak area ratio of pentavolic acid measured at 74 h compared to 0 h was also 1.0, both within the range of 0.8–1.2 (see Table 16). The stability of the solution for the pentavolic acid detection method in cetirizine sodium meets the requirements.

[0119] 11. Durability Experimental process Reference solution: Take reference solution ① and reference solution ② from section 1.1.

[0120] Spiked test solution: Take the 5th portion of the spiked test solution under section 2.1.1.

[0121] The chromatographic conditions were changed as follows: Table 17 Chromatographic conditions and parameters

[0122] Acceptable standards For the sequences after changing the chromatographic parameters: The absolute difference between the measured pentovalin content and the initial conditions should meet the standards in the table below.

[0123] Table 18 Criteria for Absolute Differences

[0124] Experimental results Table 19 Durability Results

[0125] Experimental conclusions For each sequence after changing the chromatographic parameters, the absolute difference in the measured content values ​​compared with the initial conditions was all within 0.00% (see Table 19). The robustness of the method for detecting pentovalinic acid in cevelex sodium meets the requirements.

[0126] The specificity, precision, limit of quantitation, limit of detection, linearity, accuracy, range, membrane adsorption, solution stability, and robustness of this analytical method were all verified to meet the requirements. For the reference solution, it was stable for 77 hours at room temperature (10–30°C) and 76 hours under refrigeration (2–8°C). For the test solution, it was stable for 75 hours at room temperature (10–30°C) and 74 hours under refrigeration (2–8°C). This analytical method is suitable for the determination of pentovalinic acid in cevelex sodium.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for determining the content of tervastatin in cetirizine sodium by HPLC, characterized in that, Includes the following steps: Step 1: Weigh the sodium cevelestat sample, dissolve it in acetonitrile solution by sonication, dilute it to 5 mg / mL with 1-4% (v / v) phosphoric acid solution, shake well, and filter to obtain the test solution; Step 2: Weigh the penvalerate reference standard, dissolve it in acetonitrile solution by sonication, and dilute it with 1-4% (v / v) phosphoric acid solution to prepare a reference solution with a penvalerate concentration of 10-100 μg / mL; Step 3: Accurately pipette the reference solution and the test solution, inject them into the liquid chromatograph, perform detection, record the chromatogram, and calculate the content of tertival acid by peak area according to the external standard method.

2. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 1, characterized in that, In steps 1 and 2, the concentration of the acetonitrile solution is 50% (v / v); the sonication time is 15 min and the frequency is 40 kHz; the pH value of the phosphoric acid solution is 3.0 and the concentration is 3.5-4% (v / v).

3. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 1, characterized in that, In step 3, the chromatographic column used in the liquid chromatograph is a C18 column with octadecylsilane-bonded silica gel as the packing material; the volume of the reference solution and the test solution is 100 μL; the detection conditions are: gradient elution with phosphoric acid solution as mobile phase A and acetonitrile as mobile phase B.

4. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 3, characterized in that, The gradient elution procedure is as follows: 。 5. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 3, characterized in that: The phosphoric acid solution has a pH of 3.0 and a concentration of 0.05-0.09% (v / v); the gradient elution flow rate is 1.0-1.4 mL / min.

6. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 5, characterized in that, The phosphoric acid solution has a pH of 3.0 and a concentration of 0.06-0.07% (v / v); the gradient elution flow rate is 1.2 mL / min.

7. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 1, characterized in that, This method has the following technical specifications: Tervaline showed a good linear relationship with peak area in the concentration range of 2.500-7.500 μg / mL, with a correlation coefficient r ≥ 0.995; the limit of detection was 1.0000 μg / mL, and the limit of quantitation was 2.500 μg / mL; the system precision RSD (n=6) < 1.0%; the repeatability range (n=6) was 0.0%, and the absolute difference in intermediate precision was 0.0%; the average recovery rate was 104.6%, and the RSD (n=9) of the recovery rate was 4.9%.

8. The method for determining the content of tervastatin in cetirizine sodium by HPLC according to claim 1, characterized in that, This method is applicable to the determination of tervastatin content in cevelex sodium raw material and preparations.

Images