HPLC (High Performance Liquid Chromatography) detection method for impurity FD-1 in vardenafil hydrochloride

Abstract

The invention relates to the field of drug detection and analysis, in particular to an HPLC (High Performance Liquid Chromatography) detection method for an impurity FD-1 in vardenafil hydrochloride. According to the method for detecting the impurity FD-1 in vardenafil hydrochloride, the high performance liquid chromatography is adopted, the peak shape of a chromatographic peak is good, tests of specificity, detection limit, quantitation limit, linear range, accuracy, precision, repeatability, durability, stability and the like are good, and the method is suitable for drug quality control of vardenafil hydrochloride.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, specifically relating to an HPLC method for the detection of impurity FD-1 in vardenafil hydrochloride. Background Technology

[0002] Vardenafil hydrochloride is a selective phosphodiesterase type 5 (PDE5) inhibitor primarily used to treat erectile dysfunction (ED) in men. Chemical name: 2-[2-ethoxy-5-(4-ethyl-piperazin-1-sulfonyl)phenyl]-5-methyl-7-propyl-3H-imidazol[5,1-f]-[1,2,4]triazine-4-one monohydrochloride trihydrate, molecular formula: C 23 H 32 N6O4S·HCl, molecular weight: 579.11, its chemical structural formula is:

[0003]

[0004] The synthetic route for vardenafil hydrochloride is shown below. Figure 1 Intermediate III (FD-1) reacts with N-ethylpiperazine to produce crude vardenafil hydrochloride.

[0005]

[0006] Figure 1 Synthetic route of vardenafil hydrochloride

[0007] According to the production process of vardenafil hydrochloride, FD-1 is the raw material for preparing crude FD. FD-1 contains a phenylsulfonyl chloride structure and is a commonly used sulfonating reagent with high reactivity, readily hydrolyzing to generate phenylsulfonic acid and hydrogen chloride. To ensure the safety of patient medication, strict control and accurate detection of the impurity FD-1 in vardenafil hydrochloride are required. Therefore, there is a need to develop a simple, sensitive, low-limit-of-detection, and reproducible method for detecting the impurity FD-1 in vardenafil hydrochloride. Summary of the Invention

[0008] The technical problem this invention aims to solve is to provide a method for detecting the impurity FD-1 in vardenafil hydrochloride. This method, by controlling and selecting a suitable diluent, can address issues such as poor stability and peak shape of FD-1. The method is simple to operate, highly versatile, and can be used for the quality control and evaluation of vardenafil hydrochloride raw materials.

[0009] To achieve the above-mentioned objectives, the present invention includes the following steps:

[0010] (1) Preparation of the test solution;

[0011] (2) Preparation of reference solution;

[0012] (3) Preparation of blank solution: The blank solution is selected from 0.05% hydrochloric acid acetonitrile, calculated by volume ratio;

[0013] (4) Determination method: Take the above blank solution, reference solution and test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the impurities according to the external standard method;

[0014] Furthermore, in step (1), the test solution is prepared by taking an appropriate amount of self-made vardenafil hydrochloride raw material, dissolving and diluting it with a diluent to prepare a solution of 0.5 mg / ml.

[0015] Furthermore, step (2) of preparing the reference solution involves taking an appropriate amount of FD-1 reference standard, dissolving and diluting it with a diluent to prepare a 0.5 μg / ml solution.

[0016] Furthermore, the chromatographic conditions for the high-performance liquid chromatography (HPLC) detection method in step (4) are selected from:

[0017] Chromatographic column: Waters ACQUITY UPLC BEH C18, column length 30–150 mm;

[0018] Column temperature: 20–60℃; flow rate: 0.2–0.6 ml / min; injection volume: 1–10 μl;

[0019] UV detector: 210–300 nm;

[0020] The diluent is selected from 0.05% to 0.10% acetonitrile hydrochloride, calculated by volume ratio;

[0021] Mobile phase A is selected from 0.05% to 0.15% phosphoric acid solution.

[0022] Mobile phase B is selected from acetonitrile;

[0023] Mobile phase A and mobile phase B are used for gradient elution at different volume ratios.

[0024] Furthermore, the gradient elution program is as follows: 0-1 min, 80% A, 20% B; 1-8 min, 80%-50% A, 20%-50% B; 8-9 min, 50% A, 50% B; 9-9.1 min, 50%-80% A, 50%-20% B; 9.1-13 min, 80% A, 20% B.

[0025] Furthermore, the chromatographic conditions are as follows: column temperature 28-32℃; flow rate 0.38-0.42 ml / min; UV detector detection wavelength 240-244 nm; initial mobile phase ratio 78:22-82:18; and phosphoric acid concentration 0.09%-0.11%.

[0026] Furthermore, the chromatographic column used was 2.1 mm × 50 mm, 1.7 μm, with a column temperature of 30 °C; the flow rate was 0.4 ml / min, the injection volume was 2 μl, the UV detector wavelength was 242 nm, mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile.

[0027] Beneficial effects of the present invention

[0028] 1. This invention provides for the first time an HPLC method for the detection of impurity FD-1 in vardenafil hydrochloride. The analytical method of this invention was determined by optimizing and screening the diluent and chromatographic conditions.

[0029] 2. This method is simple to operate, has strong versatility, good specificity, high sensitivity, low detection limit, good reproducibility, and accurate and reliable data. It can be used for the quality control and evaluation of FD-1 impurities in vardenafil hydrochloride raw material, meeting the needs of research and development and production. Attached Figure Description

[0030] Figure 1 Blank map;

[0031] Figure 2 Example 3: chromatogram of FD-1 reference solution;

[0032] Figure 3 Chromatogram of vardenafil hydrochloride test solution;

[0033] Figure 4 Chromatogram of vardenafil hydrochloride spiked test solution;

[0034] Figure 5 FD-1 linear relationship graph

[0035] Figure 6 Example 12 FD-1 reference solution chromatogram Detailed Implementation

[0036] The specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0037] Experimental apparatus: Waters Acquity UPLC with UV detector; diluent v / v: hydrochloric acid-acetonitrile (0.05:100)

[0038] Example 1: Preparation of vardenafil hydrochloride test solution, blank solution and reference solution

[0039] Blank solution: Same as diluent

[0040] Preparation of test solution: Take an appropriate amount of self-made vardenafil hydrochloride raw material, dissolve and dilute it with diluent to prepare a 0.5 mg / ml solution, which is used as the test solution.

[0041] Preparation of reference solution: Take an appropriate amount of FD-1 reference standard, dissolve and dilute it with diluent to prepare a 0.5 μg / ml solution, which is used as the reference solution.

[0042] Example 2 Chromatographic determination

[0043] Take the blank solution, reference solution, and test solution respectively, inject them into the high-performance liquid chromatograph, record the chromatograms, calculate the impurities according to the external standard method, and analyze them under the following chromatographic conditions:

[0044] Chromatographic column: Waters ACQUITY UPLC BEH C18, 2.1mm × 50mm, 1.7μm or other equivalent column;

[0045] Flow rate: 0.4 ml / min

[0046] Detection wavelength: 242nm

[0047] Column temperature: 30℃

[0048] Injection volume: 2 μl

[0049] Mobile phase A was selected from 0.1% phosphoric acid solution; mobile phase B was selected from nitrile; mobile phase A and mobile phase B were used for gradient elution at different volume ratios according to Table 1.

[0050] Table 1: Gradient elution procedure

[0051] Time, minutes 0 1 8 9 9.1 13 A％ 80 80 50 50 80 80 B％ 20 20 50 50 20 20

[0052] Example 3 Specificity Examination

[0053] The suitability of the system for preparing FD-1 with a limit of 0.1% is shown in the blank solution chromatogram. Figure 1 The chromatogram of the reference solution is shown below. Figure 2 The spectrum of the test solution is shown in the figure. Figure 3 The chromatogram of the spiked test solution is shown in [reference needed]. Figure 4 The results are shown in Table 2. The blank did not interfere with the detection of FD-1 in the sample.

[0054] Table 2: Specificity Results

[0055]

[0056] Example 4: Investigation of Limit of Detection and Limit of Quantification

[0057] The reference solution was serially diluted, and its signal-to-noise ratio was measured. A signal-to-noise ratio of approximately 10:1 was used as the limit of quantitation, and approximately 3:1 was used as the limit of detection. Two injections were performed at the limit of detection concentration, and six injections were performed at the limit of quantitation concentration. The results are shown in Table 3. This method can detect FD-1 at a level of 0.03% in vardenafil hydrochloride, and has high sensitivity.

[0058] Table 3: Results of Limit of Detection and Limit of Quantitation

[0059]

[0060]

[0061] Example 5: Examination of Linear Relationships

[0062] Using the FD-1 impurity limit concentration as 100%, six points were selected within the LOQ–150% level range. Determination was performed under the above chromatographic conditions, and regression curves were plotted against the corresponding peak areas to calculate the regression equation and correlation coefficient. The results are shown in Table 4, and the linear relationship graph for FD-1 is shown below. Figure 5 FD-1 exhibits good linearity in the range of 0.1550–0.7749 μg / ml.

[0063] Table 4: Linearity Results

[0064]

[0065] Example 6 Accuracy Examination

[0066] The recovery rates of FD-1 in vardenafil hydrochloride at the limit levels of 50% (0.05%), 80% (0.08%), 100% (0.10%), and 120% (0.12%) were investigated. Each concentration level was tested in triplicate. The results are shown in Table 5. The recovery rates were all between 70% and 130%, and the RSD was less than 20.0%, indicating that the data obtained by this method are accurate and reliable.

[0067] Table 5: Accuracy Results

[0068]

[0069]

[0070] Example 7: Sample Injection Precision Study

[0071] The reference solution was injected six times consecutively, and the chromatograms were recorded. The results are shown in Table 6. The RSD of the FD-1 peak area after six consecutive injections was less than 10%, indicating that the method has good injection precision.

[0072] Table 6: Injection Precision Results

[0073] Impurity Name 1# 2# 3# 4# 5# 6# RSD (%) FD-1 8231 8585 8493 8475 8446 8474 1.4

[0074] Example 8 Repeatability Test

[0075] Six parallel solutions of the test sample and the spiked test sample were prepared, with FD-1 added to the spiked test sample at a level of 0.1%. The results are shown in Table 7. The RSD of each impurity content in the six background test samples and the spiked test sample solutions was less than 20%. The recoveries of each impurity in the six spiked test samples were all between 70% and 130%, with RSDs all less than 20.0%, indicating that the method has good repeatability.

[0076] Table 7: Repeatability Results

[0077]

[0078] Example 9 Durability Test

[0079] Under normal conditions, different wavelengths, flow rates, column temperatures, and initial mobile phase ratios were tested. The varying parameters are shown in Table 8. After the instrument system stabilized, the test solution and reference solution were tested. Only one parameter was changed each time, and the system suitability (theoretical plate number and peak area RSD) and the ratio of the test sample content to the normal results were statistically analyzed under each parameter variation condition. The results of the method robustness test are shown in Table 9. When the chromatographic conditions were slightly varied, the RSD of the peak area of ​​the reference solution (6 needles) was less than 10%, the theoretical plate number calculated based on the impurity FD-1 peak was not less than 5000, and the impurity FD-1 was not detected in the test solution.

[0080] Table 8: Durability Variation Parameters

[0081] Changing parameters Change 1 Normal level Change 2 wavelength nm 240 242 244 Flow rate (ml / min) 0.38 0.40 0.42 Column temperature ℃ 28 30 32 Initial flow phase ratio 78:22 80:20 82:18 Phosphoric acid concentration 0.09％ 0.10％ 0.11％

[0082] Table 9: Durability Results

[0083]

[0084] Example 10 Stability Study

[0085] Samples were injected according to the test method requirements. The reference solution, background test solution, and spiked test solution (0.1%) were tested at different time points (at least 48 hours). The results are shown in Table 10. The reference solution was stable after 50 hours at room temperature; the background test solution and the spiked test solution were stable after 48 hours at room temperature.

[0086] Table 10: Stability Results

[0087]

[0088]

[0089] Example 11 Test Sample Determination

[0090] Samples were injected according to the test method requirements, and three batches of vardenafil hydrochloride test solutions were tested. The results are shown in Table 11. FD-1 was not detected in any of them.

[0091] Table 11: Test Results of Typical Samples

[0092] Test sample FD-1 Test sample 1 Not detected Test sample 2 Not detected Test sample 3 Not detected

[0093] Example 12 Methodology Development - Diluent Selection

[0094] The chromatographic conditions were the same as in Example 2. The effects of different diluents on the recovery rate and peak shape of FD-1 were investigated. Considering that FD-1 contains acyl chloride structures, common substances such as water, alcohol, and alkali should be avoided in the diluent. Acetonitrile is a commonly used inert diluent. Common production methods for acetonitrile include propylene ammoxidation by-product method, acetic acid ammoxidation method, acetylene ammoxidation method, or ethanol ammoxidation method. Considering that different manufacturers and different batches of acetonitrile may have different levels of alkaline substances such as ammonia remaining, the tailing factor and theoretical plate number of the acid concentration in the diluent were investigated. The results are shown in Table 12.

[0095] Table 12: Evaluation of Diluent Selection

[0096] diluent Tail Factor Theoretical number of plates 0.05% Acetonitrile Hydrochloride 1.1 51699 0.10% Acetonitrile Hydrochloride 1.0 49932

[0097] Conclusion: Adding different concentrations of hydrochloric acid to acetonitrile did not significantly affect the tailing factor and theoretical plate number of FD-1 between the two diluents. Therefore, 0.05% hydrochloric acid-acetonitrile was chosen as the diluent. (See details...) Figure 6 .

[0098] Example 13 Methodology Development - Gradient Selection

[0099] The chromatographic conditions were the same as in Example 2, and the effects of different gradients on peak shape and separation were investigated. Mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile. The peak shape of FD-1 under different gradients was investigated, and the results are shown in Table 13.

[0100] Table 13: Gradient Examination

[0101]

[0102]

[0103] Conclusion: Different mobile phases resulted in different peak shapes for FD-1. Using a 0.1% phosphoric acid solution / acetonitrile system with a Waters ACQUITY UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 μm) improved the peak shape of FD-1, resulting in a tailing factor close to 1.0 and a relatively high theoretical plate number.

[0104] Through the above Examples 1 to 10, the specificity, limit of detection and limit of quantitation, linearity, accuracy, injection precision, repeatability, robustness and stability of the method were investigated. All tests met the requirements, proving that the method is suitable for the detection and analysis of FD-1 in vardenafil hydrochloride.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. An HPLC method for detecting impurity FD-1 in vardenafil hydrochloride, characterized in that, Includes the following steps: (1) Preparation of the test solution; (2) Preparation of reference solution; (3) Preparation of blank solution; (4) Determination method: Take the above blank solution, reference solution and test solution respectively, inject them into the high performance liquid chromatograph, record the chromatogram, and calculate the impurities according to the external standard method.

2. The HPLC detection method according to claim 1, characterized in that, The preparation of the test sample in step (1) involves dissolving and diluting an appropriate amount of self-made vardenafil hydrochloride raw material with a diluent to prepare a solution of 0.5 mg / ml.

3. The HPLC detection method according to claim 1, characterized in that, The preparation of the reference solution in step (2) involves taking an appropriate amount of FD-1 reference standard, dissolving and diluting it with a diluent to prepare a solution of 0.5 μg / ml.

4. The HPLC detection method according to claim 1, characterized in that, The blank solution in step (3) is prepared by selecting 0.05% hydrochloric acid acetonitrile, calculated by volume ratio.

5. The HPLC detection method according to claim 1, characterized in that, The chromatographic conditions for high performance liquid chromatography in step (4) are selected from: Chromatographic column: Waters ACQUITY UPLC BEH C18, column length 30–150 mm; Column temperature: 20–60℃; flow rate: 0.2–0.6 ml / min; injection volume: 1–10 μl; UV detector: 210–300 nm; The diluent was selected from 0.05% acetonitrile hydrochloride, calculated by volume ratio; Mobile phase A is selected from 0.05% to 0.15% phosphoric acid solution. Mobile phase B is selected from acetonitrile; Mobile phase A and mobile phase B are used for gradient elution at different volume ratios.

6. The HPLC detection method according to claim 5, characterized in that, The gradient elution procedure is as follows: 。 7. The HPLC detection method according to claim 5, characterized in that, The chromatographic conditions were as follows: column temperature 28-32℃; flow rate 0.38-0.42 ml / min; UV detector wavelength 240-244 nm; initial mobile phase ratio 78:22-82:18; and phosphoric acid concentration 0.09%-0.11%.

8. The HPLC detection method according to claim 5, characterized in that, The chromatographic column used was 2.1 mm × 50 mm, 1.7 μm, with a column temperature of 30 °C; the flow rate was 0.4 ml / min, and the injection volume was 2 μl; the UV detector was used at a wavelength of 242 nm; mobile phase A was 0.1% phosphoric acid solution, and mobile phase B was acetonitrile.

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