Method for detecting and analyzing 1-phenyl-5-tetrazole-thione impurity in rosuvastatin calcium side chain

Abstract

The invention provides a method for detecting and analyzing a 1-phenyl-5-tetrazole-thione impurity in a rosuvastatin calcium side chain, which comprises the following steps: detecting a blank solution, a reference solution and a test solution by adopting high performance liquid chromatography to obtain a chromatogram, and confirming the content of 1-phenyl-5-tetrazole-thione according to the chromatogram. According to the method for detecting and analyzing the impurities of the 1-phenyl-5-tetrazole-thione, the impurities are well separated from adjacent peaks, the applicability is high, the accuracy is good, the specificity is high, the reproducibility and durability are good, the quantitation limit is 5.0040 mu g / mL, and the detection limit is 2.5020 mu g / mL. According to the method, the technical blank of 1-phenyl-5-tetrazole-thione trace impurity detection in the side chain of the rosuvastatin calcium is filled, the purity and the stability of the rosuvastatin calcium product can be better improved, safety guarantee is provided for strict control of the medicine quality, and the method has good application prospects and application values.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical analysis and more specifically relates to a method for detecting and analyzing 1-phenyl-5-mercaptotetrazolyl impurities in the side chain of rosuvastatin calcium. Background Art

[0002] Rosuvastatin calcium is a lipid-lowering drug widely used in clinical practice and belongs to the statin class. This class of drugs effectively lowers low-density lipoprotein cholesterol (LDL-C) levels in the blood by inhibiting HMG-CoA reductase, a key enzyme in cholesterol synthesis in the body, while also moderately increasing high-density lipoprotein cholesterol (HDL-C) levels. This is of great significance for the prevention and treatment of cardiovascular disease. However, as a chemically synthesized drug, rosuvastatin calcium inevitably introduces some impurities during the production process. These impurities may originate from residual raw materials, side reaction products, or degradation products during storage. Their presence not only affects the purity of the drug but also poses potential risks to human health. Therefore, comprehensive detection and strict control of impurities are key links in drug production.

[0003] 1-Phenyl-5-mercapto tetrazole is one of the important raw materials used in the side chain synthesis route of rosuvastatin calcium. It can be present in the final product as an impurity that is not fully converted during the subsequent reaction process, and its content is relatively high. 1-Phenyl-5-mercapto tetrazole has the characteristics of strong polarity and easy adsorption, which makes it face many challenges in trace analysis. Especially in complex drug matrices, its detection signal is easily disturbed, resulting in insufficient sensitivity and selectivity, which makes the trace detection of 1-Phenyl-5-mercapto tetrazole one of the difficulties in the field of pharmaceutical analysis. The detection of 1-Phenyl-5-mercapto tetrazole in the prior art is mainly concentrated on the determination of its purity after synthesis. For example, Chinese patent CN112062731B discloses a method for detecting the purity of 1-Phenyl-5-mercapto tetrazole by liquid chromatography. However, this method is applicable to the situation where the target compound content is relatively high, mainly detecting the purity of the target compound, and is not suitable for detection of impurities at trace levels. Furthermore, this method has limitations in terms of resolution, sensitivity, and resistance to matrix interference, making it difficult to meet the stringent monitoring requirements for trace impurities of 1-phenyl-5-mercaptotetrazole during the production of rosuvastatin calcium. Currently, there is a lack of sensitive and accurate methods for the analysis of trace amounts of 1-phenyl-5-mercaptotetrazole.

[0004] Currently, the main methods for drug impurity analysis include ultraviolet spectroscopy (UV), gas chromatography (GC), and high-performance liquid chromatography (HPLC). Among them, HPLC has become the mainstream analytical method due to its good separation effect, high sensitivity, and wide range of applications. However, for highly polar and easily adsorbed compounds such as 1-phenyl-5-mercaptotetrazole, traditional HPLC methods often have the following problems: insufficient separation between the target peak and adjacent impurity peaks, leading to biased results; filter membrane adsorption may occur during sample pretreatment, affecting the recovery rate; and slight changes in detection conditions may lead to decreased reproducibility and durability. In addition, existing analytical methods lack sufficient sensitivity and accuracy in trace detection, making it difficult to meet the needs of precise monitoring of 1-phenyl-5-mercaptotetrazole.

[0005] In summary, there is currently no effective analytical method for the 1-phenyl-5-mercaptotetrazolyl impurity in the side chain of rosuvastatin calcium, which not only limits in-depth research on this impurity but also poses a risk to drug quality control. In this context, the development of a highly specific, sensitive, and stable detection method for rosuvastatin calcium would provide important technical support for the quality evaluation of rosuvastatin calcium. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the above existing problems, provide a method for detecting and analyzing 1-phenyl-5-mercaptotetrazolyl impurities in the side chain of rosuvastatin calcium, and promote the development of the field of rosuvastatin calcium quality control.

[0007] The first object of the present invention is to provide a method for detecting and analyzing 1-phenyl-5-mercaptotetrazolyl impurities in the side chain of rosuvastatin calcium.

[0008] The second object of the present invention is to provide an application of the above detection and analysis method.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] The present invention provides a method for detecting and analyzing 1-phenyl-5-mercaptotetrazole impurities in the side chain of rosuvastatin calcium. A blank solution, a reference solution, and a test solution are detected by high performance liquid chromatography to obtain a chromatogram, and the content of 1-phenyl-5-mercaptotetrazole is confirmed according to the chromatogram.

[0011] The chromatographic conditions of the HPLC are:

[0012] Column temperature: 50-55°C;

[0013] Mobile phase A: potassium dihydrogen phosphate solution, mobile phase B: methanol;

[0014] Injection volume: 1.5-2.5 μL;

[0015] Flow rate: 1.0-2.0 mL / min;

[0016] The mobile phase gradient elution program was as follows: within 0-8 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at (70-90): (10-30); within 8-15 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from (70-90): (10-30) to (30-50): (50-70); within 15-20 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at (30-50): (50-70); within 20-25 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from (30-90): (10-30) to (30-50): (50-70); -50:(50-70) linearly changes to (10-30):(70-90); within 25-32 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at (10-30):(70-90); within 32-34 minutes, the volume ratio of mobile phase A and mobile phase B changes linearly from (10-30):(70-90) to (70-90):(10-30); within 34-40 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at (70-90):(10-30).

[0017] Specifically, the rosuvastatin calcium side chain is rosuvastatin side chain intermediate 01, and the structural formula of the rosuvastatin side chain intermediate 01 is shown in formula (I):

[0018]

[0019] As an optional embodiment, the stationary phase of the chromatographic column of the high performance liquid chromatography analysis method is octadecyl bonded silica gel.

[0020] As an optional embodiment, the chromatographic column used in the high performance liquid chromatography analysis method is Agilent ZORBAX Eclipse XDB-C18, with specifications of 150×4.6 mm and 5 μm.

[0021] As an optional embodiment, the detector of the high performance liquid chromatography analysis method is a diode array detector with a detection wavelength of 220-260 nm.

[0022] Specifically, the detection wavelength is 240 nm.

[0023] As an optional embodiment, the blank solution is methanol.

[0024] As an optional embodiment, the reference solution and the test solution are prepared using methanol as the solvent.

[0025] As an optional embodiment, the concentration of the test solution is 5-15 mg / mL.

[0026] Preferably, the concentration of the test solution is 10 mg / mL.

[0027] As an optional embodiment, the test sample is rosuvastatin side chain intermediate 01.

[0028] As an alternative embodiment, the concentration of the control solution is 5-15 μg / mL.

[0029] As an optional embodiment, the concentration of the reference solution is 10 μg / mL.

[0030] Specifically, the reference substance is 1-phenyl-5-mercaptotetrazole.

[0031] Preferably, in the chromatographic conditions of the high performance liquid chromatography analysis method, the column temperature is 52°C.

[0032] Preferably, in the chromatographic conditions of the high performance liquid chromatography analysis method, the injection volume is 2 μL.

[0033] Preferably, in the chromatographic conditions of the high performance liquid chromatography analysis method,

[0034] The mobile phase gradient elution program was as follows: within 0-8 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 80:20; within 8-15 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 80:20 to 40:60; within 15-20 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 40:60; within 20-25 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 40:60 to 20:80; within 25-32 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 20:80; within 32-34 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 20:80 to 80:20; within 34-40 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 80:20.

[0035] The application of the above-mentioned detection and analysis method in the quality control of rosuvastatin calcium or its preparations should also be within the scope of protection of the present invention.

[0036] The present invention has the following beneficial effects:

[0037] The present invention provides a method for quantitatively analyzing 1-phenyl-5-mercaptotetrazolyl impurities in the side chain of rosuvastatin calcium. The method utilizes high-performance liquid chromatography (HPLC) and explores parameters such as a gradient elution program, column temperature, sample concentration, and injection volume to achieve trace detection of 1-phenyl-5-mercaptotetrazolyl impurities. The method for detecting and analyzing 1-phenyl-5-mercaptotetrazolyl impurities of the present invention achieves good separation of the impurity from adjacent peaks, is highly applicable, accurate, and specific, and also exhibits excellent reproducibility and durability. The limit of quantification is 5.0040 μg / mL and the limit of detection is 2.5020 μg / mL.

[0038] The present invention utilizes conventional HPLC equipment and reagents, is simple and easy to operate, and is highly practical, making it widely applicable to the quality control process in the actual production of rosuvastatin calcium. The present invention fills a technological gap in the detection of trace impurities of 1-phenyl-5-mercaptotetrazole in the side chain of rosuvastatin calcium, can better improve the purity and stability of rosuvastatin calcium products, and provides a safety guarantee for strict control of drug quality. The invention has great application prospects and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the HPLC chromatogram of the blank solution of Example 1.

[0040] Figure 2 1 is the HPLC chromatogram of the control solution of Example 1.

[0041] Figure 3 1 is the HPLC chromatogram of the test solution of Example 1.

[0042] Figure 4 HPLC chromatogram of the detection limit solution of Example 7.

[0043] Figure 5 The figure is the HPLC chromatogram of the quantitative limit solution of Example 7.

[0044] Figure 6 This is the HPLC chromatogram of the L-0.08 linear solution of Example 8.

[0045] Figure 7 This is the HPLC chromatogram of the L-0.1 linear solution of Example 8.

[0046] Figure 8 This is the HPLC chromatogram of the L-0.12 linear solution of Example 8.

[0047] Figure 9 This is the HPLC chromatogram of the L-0.15 linear solution of Example 8.

[0048] Figure 10The test results of linearity and range detection of Example 8 (Figure A is the linearity graph; Figure B is the residual graph).

[0049] Figure 11 This is the HPLC chromatogram of the 0.05% spiked solution of Example 9.

[0050] Figure 12 This is the HPLC chromatogram of the 0.1% spiked solution of Example 9.

[0051] Figure 13 This is the HPLC chromatogram of the 0.15% spiked solution of Example 9.

[0052] Figure 14 The figure is a high performance liquid chromatogram of the centrifuged test solution in Example 10.

[0053] Figure 15 This is a high performance liquid chromatogram of the test solution filtered in Example 10 (0 mL discarded).

[0054] Figure 16 This is the HPLC chromatogram of the filtered test solution (2 mL discarded) in Example 10.

[0055] Figure 17 This is the HPLC chromatogram of the filtered test solution (4 mL discarded) in Example 10.

[0056] Figure 18 The HPLC chromatograms of the gradient elution procedures with different mobile phases in Comparative Example 1 are shown.

[0057] Figure 19 The high performance liquid chromatograms of comparative example 4 were processed with different injection volumes.

[0058] Figure 20 The figure is the superimposed HPLC chromatogram of the test solution and the reference solution of Comparative Example 5.

[0059] Figure 21 The figure is the HPLC chromatogram of the test solution of Comparative Example 5.

[0060] Figure 22 The figure is the superimposed HPLC chromatogram of the test sample spiked solution and the reference solution in Comparative Example 5. DETAILED DESCRIPTION

[0061] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0062] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0063] In the following examples, the instruments used are: 1 / 100,000 analytical balance, high performance liquid chromatography, pH meter; the reagents used are: methanol (HPLC), potassium dihydrogen phosphate (AR), phosphoric acid (AR).

[0064] 1-Phenyl-5-mercaptotetrazolyl, CAS No. 86-93-1, structural formula:

[0065] Rosuvastatin side chain intermediate 01, the structural formula is:

[0066]

[0067] Example 1 Detection and Analysis Method of 1-Phenyl-5-Mercaptotetrazolyl in the Side Chain of Rosuvastatin Calcium

[0068] 1. Solution preparation

[0069] Reference substance: 1-phenyl-5-mercaptotetrazolyl (denoted as RM06501);

[0070] Test sample: rosuvastatin side chain intermediate 01 (denoted as STCA01);

[0071] Diluent / blank solution: methanol;

[0072] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0073] Reference solution stock solution: weigh 50 mg of the reference substance into a 100 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, shake well;

[0074] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute it to the scale, then shake well.

[0075] The test solution was prepared in multiple replicates and analyzed by high performance liquid chromatography.

[0076] 2. Chromatographic conditions

[0077] The chromatographic conditions are shown in Table 1.

[0078] Table 1 Chromatographic conditions parameters

[0079]

[0080]

[0081] 3. Experimental Operation

[0082] After the baseline is balanced, according to the chromatographic conditions of this example, the control solution and the test solution are injected according to the injection sequence in Table 2, and the chromatogram is recorded.

[0083] Table 2 Injection sequence

[0084] Sample name Number of injection needles blank solution 1 Control solution 1 Test solution 1 1 Test solution 2 1 Test solution 3 1 Test solution 4 1 Test solution 5 1

[0085] 4. Calculation

[0086] According to the chromatogram results, the content of 1-phenyl-5-mercaptotetrazole was calculated according to the following formula.

[0087]

[0088] Where:

[0089] A i is the peak area of the related substance measured in the test solution;

[0090] A S is the average value of the peak area of RM06501 measured in the control solution;

[0091] W T is the sample weight of the test sample, mg;

[0092] W S The mass of RM06501 weighed to prepare the control solution, mg;

[0093] P S The residual amount of RM06501 weighed to prepare the control solution (%);

[0094] D T is the dilution multiple of the test sample;

[0095] D S is the dilution factor of the control solution.

[0096] 5. Test Results

[0097] The HPLC chromatograms of blank solution, control solution and test solution are as follows: Figure 1 、 Figure 2 and Figure 3 The specific detection results are shown in Table 3. The results show that the detection limit of 1-phenyl-5-mercaptotetrazole is ≤1000ppm, which can achieve trace detection of 1-phenyl-5-mercaptotetrazole.

[0098] Table 3 Test results

[0099]

[0100] Example 2 System suitability test

[0101] 1. Solution preparation

[0102] Reference substance: 1-phenyl-5-mercaptotetrazolyl (RM06501);

[0103] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0104] Diluent / blank solution: methanol;

[0105] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0106] Reference solution stock solution: Weigh RM06501 sample into a 100 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of diluent, and shake well;

[0107] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, then shake well.

[0108] 2. Experimental Operation

[0109] After the system is equilibrated, inject one or two injections of blank solution and one injection of the test solution. Perform the assay according to the chromatographic conditions of Example 1 and record each chromatogram. Report the theoretical plate number of the STCA01 main peak in the chromatogram of the test solution.

[0110] 3. Experimental Results

[0111] The system suitability results are shown in Table 4. The results show that the theoretical plate number of the test sample STCA01 is 411140, which is much larger than the standard 5000, and the system suitability meets the requirements.

[0112] Table 4 System suitability results

[0113]

[0114] Example 3 Specificity Test

[0115] 1. Solution preparation

[0116] Reference substance: 1-phenyl-5-mercaptotetrazolyl (RM06501);

[0117] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0118] Diluent / blank solution: methanol;

[0119] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0120] Reference solution stock solution: Weigh RM06501 sample into a 100 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of diluent, and shake well;

[0121] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, then shake well.

[0122] Test sample spiked solution: Take 0.5 mL of the test sample solution, accurately weigh it, and place it in a 50 mL volumetric flask. Then transfer 1.0 mL of the control stock solution to the above volumetric flask, dissolve it in an appropriate amount of diluent and dilute it to the scale, shake well, and filter it through a 0.45 μm filter membrane.

[0123] 3. Experimental Operation

[0124] According to the assay method, inject one sample each of the blank solution, test solution, control solution, and test-spiked solution. Perform the assay according to the chromatographic conditions of Example 1 and record the corresponding chromatograms. Report the retention time, peak area, and resolution of RM06501 from adjacent chromatographic peaks in the blank solution, test solution, control solution, and test-spiked solution.

[0125] 4. Experimental Results

[0126] The specificity results are shown in Table 5. The results show that the blank solution has no interference at the peak of 1-phenyl-5-mercaptotetrazole. The above results show that the liquid chromatography method of the present invention has good specificity and is suitable for the detection of 1-phenyl-5-mercaptotetrazole.

[0127] Table 5 Specificity results

[0128]

[0129] Example 4 Detection limit and quantification limit test

[0130] 1. Solution preparation

[0131] Reference substance: 1-phenyl-5-mercaptotetrazolyl (RM06501);

[0132] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0133] Diluent / blank solution: methanol;

[0134] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0135] Reference solution stock solution: Weigh RM06501 sample into a 100 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of diluent, and shake well;

[0136] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, then shake well.

[0137] L-1.0: Pipette 1 mL of the test solution into a 100 mL volumetric flask and dilute to the mark with methanol.

[0138] Quantitation limit solution: Pipette 0.5 mL of control stock solution and 2.5 mL of L-1.0 into a 50 mL volumetric flask and dilute to the mark with methanol.

[0139] Detection limit solution: Pipette 0.5 mL of the control stock solution and 2.5 mL of L-1.0 into a 100 mL volumetric flask and dilute to the mark with methanol.

[0140] 2. Experimental Operation

[0141] After baseline equilibrium, inject one injection of the blank solution, and three injections of the quantification limit solution and the detection limit solution. Perform the assay according to the chromatographic conditions of Example 1 and record the chromatograms. Report the peak areas, RSDs of the peak areas, and signal-to-noise ratio (S / N) of RM06501 and STCA01 in the quantification limit solution; report the peak areas and signal-to-noise ratio (S / N) of RM06501 and STCA01 in the detection limit solution. Calculate the quantification limit and detection limit concentrations of RM06501 and the percentage of these concentrations relative to the test solution concentration.

[0142] 3. Experimental Results

[0143] The HPLC chromatograms of the detection limit solution and the quantification limit solution are shown as follows: Figure 4 and Figure 5 The detection limit and quantification limit results are shown in Table 6. The results show that:

[0144] 1. The signal-to-noise ratio of the detection limit solution RM06501 peak for three consecutive injections is greater than 18.3 (≥3);

[0145] 2. The signal-to-noise ratio of the detection limit solution STCA01 peak for three consecutive injections is greater than 9.1 (≥3);

[0146] 3. The signal-to-noise ratio of the RM06501 peak of the quantitative limit solution for three consecutive injections is greater than 39.7 (≥10); the RSD value of the RM06501 peak of the quantitative limit solution for three consecutive injections is 4.79 (≤10.0%);

[0147] 4. The signal-to-noise ratio of the STCA01 peak of the quantitative limit solution for three consecutive injections is greater than 24.3 (≥10); the RSD value of the STCA01 peak of the quantitative limit solution for three consecutive injections is 8.71 (≤10.0%);

[0148] 5. The detection limit of RM06501 is 2.5020μg / mL, the detection limit of STCA01 is 2.5235μg / mL, the quantification limit of RM06501 is 5.0040μg / mL, and the quantification limit of STCA01 is 5.0470μg / mL, which meet the detection requirements.

[0149] Table 6 Detection limit and quantification limit results

[0150]

[0151]

[0152] Example 5 Linearity and range detection

[0153] 1. Solution preparation

[0154] Reference substance: 1-phenyl-5-mercaptotetrazolyl (RM06501);

[0155] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0156] Diluent / blank solution: methanol;

[0157] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0158] Reference solution stock solution: Weigh RM06501 sample into a 100 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of diluent, and shake well;

[0159] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, then shake well.

[0160] L-1.0: Pipette 1 mL of the test solution into a 100 mL volumetric flask and dilute to the mark with methanol.

[0161] Quantitation limit solution: Pipette 0.5 mL of control stock solution and 2.5 mL of L-1.0 into a 50 mL volumetric flask and dilute to the mark with methanol.

[0162] Detection limit solution: Pipette 0.5 mL of the control stock solution and 2.5 mL of L-1.0 into a 100 mL volumetric flask and dilute to the mark with methanol.

[0163] Linear solution: Accurately pipette different volumes of control stock solution as shown in Table 7 into volumetric flasks of different volumes, dilute to the scale with solvent, and shake well to obtain the linear solution.

[0164] Table 7 Preparation scheme of linear solutions of different concentrations

[0165]

[0166] 2. Experimental Operation

[0167] After the system suitability is qualified, take a blank solution injection, take the linear solution and the quantitative limit solution at each concentration level, and inject once at each concentration from low concentration to high concentration. Measure according to the chromatographic conditions of Example 1 and record the chromatogram.

[0168] A univariate linear regression was performed on the concentration of RM06501 using the average peak area value. The linear equation, linear correlation coefficient r, residual sum of squares, Y-axis intercept, absolute value of the Y-axis intercept at 0.1% concentration ratio, and 95% confidence interval of the Y-axis intercept of RM06501 were reported respectively, and a linear graph was drawn.

[0169] 3. Experimental Results

[0170] The HPLC results of linear solutions with different concentrations are as follows: Figure 6 、 Figure 7 、 Figure 8 and Figure 9 As shown, the linear graph and residual graph are Figure 10 As shown in Figures A and B of the present invention, the linear results are shown in Table 8. The results show that the linear correlation coefficient is 0.9939, and the absolute value of the Y-axis intercept and the 0.1% concentration response value is 1.2%, indicating that the liquid chromatography method of the present invention has a high degree of linearity between peak area and concentration, and the detection result of RM06501 is accurate.

[0171] Table 8 Linearity results

[0172]

[0173] Example 6 Accuracy Test

[0174] 1. Solution preparation

[0175] Reference substance: 1-phenyl-5-mercaptotetrazolyl (RM06501);

[0176] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0177] Diluent / blank solution: methanol;

[0178] Reference solution stock solution: Weigh RM06501 sample into a 100 mL volumetric flask, dissolve and dilute to the mark with an appropriate amount of diluent, and shake well;

[0179] Control solution: Pipette 1 mL of control solution stock solution into a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute to the mark, then shake well.

[0180] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0181] Accuracy solution: Accurately weigh 0.5 mL of the test sample and place it in a 50 mL volumetric flask. Prepare nine replicates. According to the scheme shown in Table 9, pipette spiked solutions of different concentrations into each of the nine volumes, dissolve the solution to the mark, and dilute to the mark. Shake well and filter through a 0.45 μm filter. Prepare three replicates for each concentration.

[0182] Table 9 Preparation scheme of spiked solutions at different concentrations

[0183] Concentration level 0.05% spike solution 0.1% spike solution 0.15% spike solution Control stock solution (mL) 0.5 1 1.5 Volumetric flask (mL) 50 50 50

[0184] 2. Experimental Operation

[0185] After the system suitability is confirmed, inject one injection of the blank solution, one injection of the test solution, and one injection of each accuracy solution at each concentration level. Perform the measurement according to the chromatographic conditions of Example 1 and record the chromatogram. Calculate and report the single value of the RM06501 recovery for each of the nine samples, the 95% confidence interval of the average recovery (n = 9), and the RSD of the recovery (n = 9).

[0186] The residual amount and recovery rate of RM06501 in the test solution before and after spike addition were calculated according to the following formula:

[0187]

[0188] Theoretical amount = C S ×V

[0189]

[0190] Where:

[0191] C Sis the concentration of RM06501 stock solution added to the test sample spike solution, mg / mL;

[0192] V is the volume of RM06501 stock solution added to each test sample spike solution, mL;

[0193] C S+T is the residual amount of RM06501 measured in the spiked solution of each test sample;

[0194] C i is the residual amount of RM06501 measured in the test solution;

[0195] is the average value of the RM06501 residue measured in the test solution;

[0196] A i is the peak area of RM06501 in the test solution;

[0197] A S is the average peak area of RM06501 in three reference solutions;

[0198] A S+T is the peak area of RM06501 in the spiked solution of each test sample;

[0199] W T is the amount of the test sample in the test solution, mg;

[0200] W S is the sample amount of RM06501 in the control solution, mg;

[0201] W S+T is the amount of test sample in the spiked solution of each test sample, mg;

[0202] P S is the content of RM06501 in the control solution (%);

[0203] D T is the dilution factor of the test solution;

[0204] D S is the dilution multiple of the control solution;

[0205] D S+T is the dilution factor of the test sample in the spiked solution.

[0206] 3. Experimental Results

[0207] The HPLC chromatograms of 0.05% spiked solution, 0.1% spiked solution, and 0.15% spiked solution are shown in Figure 1. Figure 11 、 Figure 12 and Figure 13As shown in Table 10, the accuracy results show that the spike recovery rate is 95.76~109.88% (80%~120%); the RSD value of the spike recovery rate is 4.2 (≤10.0%), indicating that the liquid chromatography method of the present invention has good accuracy and is suitable for RM06501 detection.

[0208] Table 10 Accuracy results

[0209]

[0210]

[0211] Example 7 Durability Test

[0212] 1. Solution preparation

[0213] Test sample: Rosuvastatin side chain intermediate 01 (STCA01);

[0214] Test solution: Take 0.5 mL of the test sample, accurately weigh it, and place it in a 50 mL volumetric flask. Dissolve it with an appropriate amount of diluent and dilute it to the mark. Shake well and filter it through a 0.45 μm filter membrane.

[0215] Centrifuge the test solution: Centrifuge the test solution at 10,000 rpm for 5 minutes and take the supernatant.

[0216] Filter the test solution: Filter the test solution with a 0.45 μm filter membrane, discard 0 mL, 2 mL, and 4 mL of the filtered liquid respectively, and use the remaining filtrate as the filtered test solution.

[0217] 2. Experimental Operation

[0218] After the system is qualified, take one injection of the filtered test solution and one injection of the centrifuged test solution, and measure according to the chromatographic conditions of Example 1, record the chromatogram, and calculate the ratio of the residual amount of RM06501 in the filtered and centrifuged test solution.

[0219] 3. Experimental Results

[0220] The HPLC chromatogram of the centrifuged test solution is as follows Figure 14 As shown, the HPLC chromatograms of the filtered test solution are as follows: Figure 15 、 Figure 16 and Figure 17 The durability results are shown in Table 11. The results show that the ratio of the residual amount of the test solution RM06501 after filtration and after centrifugation is between 0.9 and 1.1, which meets the requirements.

[0221] Table 11 Durability results

[0222]

[0223] Mobile phase gradient in the detection method of Comparative Example 1

[0224] 1. Test methods

[0225] High performance liquid chromatography detection was performed according to the method of Reference Example 1 (including solution preparation, chromatographic conditions and experimental operation), which differed from the chromatographic conditions of the embodiment in that the mobile phase gradient elution procedure was replaced.

[0226] The mobile phase gradient elution program of this comparative example is specifically shown in Table 12, and the high performance liquid chromatography test was carried out using the mobile phase gradient elution programs shown in sequence numbers 0-5.

[0227] Table 12 Liquid chromatography mobile phase gradient

[0228]

[0229]

[0230] 2. Test results

[0231] The test results are shown in Table 13 and Figure 18 As shown, the results show that in the initial gradient investigation, it can be seen that the lower the initial proportion of phase B, the later the RM06501 peak time, and the higher the proportion of phase B, the earlier the RM06501 peak time. From the data in Table 13, it can be seen that the best separation is when the initial proportion of phase B is 25, 27, and 57. However, considering that when the phase B is 57, the target peak is too weakly retained and almost unretained, it may peak at the same time as the air peak when other instruments are used, and be deducted as a blank, so the condition of an initial proportion of phase B of 57 is not considered.

[0232] Table 13 HPLC results of different mobile phase gradient elution programs

[0233]

[0234]

[0235] Comparative Example 2 Investigation of Column Temperature

[0236] After investigating the initial proportion of phase B in comparative experiments, considering that the column temperature will affect the selectivity of the chromatographic column on the chromatographic peak, the initial gradient of phase B was selected as 20% (method No. 2) and 25% (method No. 3) under the condition that the retention time peak is after 5 minutes, and column temperature investigation was carried out respectively.

[0237] 1. Test methods

[0238] The chromatographic conditions were similar to those of Method 2 and Method 3 in Comparative Example 1, except that the column temperature was changed. The column temperature parameters were adjusted according to Table 14, and then the HPLC test was performed.

[0239] Table 14 Column temperature parameters

[0240] Mobile phase B initial gradient 25% 20℃ 30℃ 50℃ 55℃ Mobile phase B initial gradient 20% 50℃ 50℃ 50℃ ——

[0241] 2. Test results

[0242] The test results are shown in Table 15. The results show that the separation between RM06501 and adjacent impurity peaks is optimal when the initial gradient of phase B is at a column temperature of 55°C. However, considering that low temperature can extend the service life of the chromatographic column, the column temperature is determined to be 52°C and the initial gradient of phase B is 20%.

[0243] Table 15 HPLC results of different column temperature parameters

[0244]

[0245] Comparative Example 3 Test sample concentration investigation

[0246] 1. Test methods

[0247] High performance liquid chromatography was performed according to the method of Example 1 (including solution preparation, chromatographic conditions and experimental procedures), except that: the concentration of the test sample was changed.

[0248] The concentration of the test solution before adjustment is 25 mg / mL: take 2.5 mL (about 2.5 g) of the test solution into a 100 mL volumetric flask, shake well, and filter;

[0249] The adjusted test sample concentration is 10 mg / mL. Take 0.5 mL of the test sample, accurately weigh it, place it in a 50 mL volumetric flask, dissolve it with an appropriate amount of diluent and dilute it to the scale, shake well, and filter it through a 0.45 μm filter membrane.

[0250] 2. Experimental Results

[0251] The results are shown in Table 16. The peak height of the 10 mg / mL test sample concentration was still 3000, indicating that the sample concentration before adjustment may have been overloaded. Therefore, the test sample concentration was determined to be 10 mg / mL. Since the test sample was a reaction solution, 0.5 mL of the test sample was accurately weighed and placed in a 50 mL volumetric flask. Dissolve and dilute to the mark with an appropriate amount of diluent, shake well, and filter through a 0.45 μm filter.

[0252] Table 16 HPLC results of samples with different concentrations

[0253]

[0254] Comparative Example 4: Injection Volume Investigation

[0255] 1. Test methods

[0256] High performance liquid chromatography (HPLC) was performed according to the method of Example 1 (including solution preparation, chromatographic conditions, and experimental procedures), with the difference from the chromatographic conditions of Example 1 being that the injection volume was changed. The injection volume of Example 1 was 2 μL, while the injection volume of this comparative example was 5 μL.

[0257] 2. Test results

[0258] The test results are shown in Table 17 and Figure 19 As shown, the results show that the peak shape of STCA01 with an injection volume of 2 μL is sharper and more symmetrical than that with an injection volume of 5 μL, and the injection volume is determined to be 2 μL.

[0259] Table 17 HPLC results of different injection volumes

[0260] Injection volume Peak height of STCA 2μL 1986.53 5μL 3333.14

[0261] Comparative Example 5

[0262] High performance liquid chromatography (HPLC) was performed using the method of Reference Example 1 (including solution preparation, chromatographic conditions, and experimental procedures), which differs from Example 1 in that the chromatographic conditions are shown in Table 18. The chromatographic conditions of this comparative example were set with reference to the prior art method for detecting the purity of 1-phenyl-5-mercaptotetrazole (Chinese Patent CN112062731B).

[0263] Table 18 Chromatographic conditions

[0264]

[0265] 2. Test results

[0266] The test results are as follows Figure 20 、 Figure 21 and Figure 22 As shown, the results show that the main peak of the reference solution bifurcates into three peaks, the peak shape of the sample impurity peak is abnormal compared to the main peak, and the target impurity peak after the test sample is spiked is inconsistent with the reference. This shows that the existing method of using liquid chromatography to detect the purity of 1-phenyl-5-mercaptotetrazole is not suitable for the trace detection of 1-phenyl-5-mercaptotetrazole.

[0267] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for detecting and analyzing 1-phenyl-5-mercaptotetrazolyl impurities in the side chain of rosuvastatin calcium, characterized in that: The blank solution, the reference solution and the test solution were detected by high performance liquid chromatography to obtain a chromatogram, and the content of 1-phenyl-5-mercaptotetrazole was confirmed according to the chromatogram; The chromatographic conditions of the HPLC are: Column temperature: 50-55°C; Mobile phase A: potassium dihydrogen phosphate solution, mobile phase B: methanol; Injection volume: 1.5-2.5 μL; Flow rate: 1.0-2.0 mL / min; The mobile phase gradient elution program was as follows: within 0-8 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at (70-90): (10-30); within 8-15 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from (70-90): (10-30) to (30-50): (50-70); within 15-20 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at (30-50): (50-70); within 20-25 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from (30-90): (10-30) to (30-50): (50-70); -50:(50-70) linearly changes to (10-30):(70-90); within 25-32 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at (10-30):(70-90); within 32-34 minutes, the volume ratio of mobile phase A and mobile phase B changes linearly from (10-30):(70-90) to (70-90):(10-30); within 34-40 minutes, the volume ratio of mobile phase A and mobile phase B remains unchanged at (70-90):(10-30).

2. The detection and analysis method according to claim 1, characterized in that: The rosuvastatin calcium side chain is rosuvastatin side chain intermediate 01, and the structural formula of the rosuvastatin side chain intermediate 01 is shown in formula (I):

3. The detection and analysis method according to claim 1, characterized in that: The stationary phase of the chromatographic column of the high performance liquid chromatography analysis method is octadecyl bonded silica gel.

4. The detection and analysis method according to claim 1, characterized in that: The detector of the high performance liquid chromatography analysis method is a diode array detector, and the detection wavelength is 220-260 nm.

5. The detection and analysis method according to claim 1, characterized in that: The blank solution is methanol.

6. The detection and analysis method according to claim 1, characterized in that: The concentration of the test solution is 5-15 mg / mL.

7. The detection and analysis method according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography analysis method include a column temperature of 52°C.

8. The detection and analysis method according to claim 1, characterized in that: In the chromatographic conditions of the high performance liquid chromatography analysis method, the injection volume is 2 μL.

9. The detection and analysis method according to claim 1, characterized in that: In the chromatographic conditions of the high performance liquid chromatography analysis method, The mobile phase gradient elution program was as follows: within 0-8 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 80:20; within 8-15 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 80:20 to 40:60; within 15-20 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 40:60; within 20-25 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 40:60 to 20:80; within 25-32 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 20:80; within 32-34 minutes, the volume ratio of mobile phase A and mobile phase B was linearly changed from 20:80 to 80:20; within 34-40 minutes, the volume ratio of mobile phase A and mobile phase B was maintained at 80:

20.

10. Use of the detection and analysis method according to any one of claims 1 to 9 in the quality control of rosuvastatin calcium or its preparations.

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