Separation and determination method for related impurities of besifloxacin and timolol eye drops

Abstract

The present application discloses a method for the separation and determination of related impurities in besifloxacin and timolol maleate eye drops, using HPLC method: Test solution: Dissolve and dilute the test sample with water; Control solution: Dissolve and dilute timolol maleate with water; System suitability solution: Take besifloxacin and timolol maleate eye drops and their related impurities, mix and dilute with water; Blank excipient solution: Take sodium chloride, disodium hydrogen phosphate heptahydrate, citric acid, benzalkonium chloride and bimatoprost, dilute with water; Retention time determination: Inject the blank excipient solution and the system suitability solution for analysis to determine the retention time; Separation and detection of target substances: Inject the test solution and the control solution, and calculate the content of related impurities in the test solution by the external standard method of the main component; The stationary phase is octadecylsilyl silica gel, mobile phase A is sodium octanesulfonate buffer solution, and mobile phase B is methanol. The method of the present application has strong specificity, high sensitivity, good repeatability and durability.

Description

Technical Field

[0001] The present application relates to the field of high performance liquid chromatography analysis, and particularly to a method for separating and determining related impurities of besifloxacin and timolol maleate eye drops. Background Art

[0002] Besifloxacin and timolol maleate eye drops are a compound preparation composed of besifloxacin and timolol maleate. Besifloxacin is a synthetic prostamide and a prostaglandin structural analogue with intraocular pressure (IOP) - lowering activity. Besifloxacin selectively mimics the action of prostamide and reduces IOP by increasing the outflow of aqueous humor through two pathways: the trabecular meshwork and the uveoscleral pathway. Timolol is a non - selective β - adrenergic receptor blocker that can reduce IOP in both patients with high IOP and normal individuals. The exact mechanism of timolol in reducing IOP is not yet clear. Tonography and aqueous humor fluorophotometry studies suggest that its IOP - lowering effect is related to reducing aqueous humor production. Timolol has no obvious endogenous sympathomimetic activity and local anesthetic effect, and has no direct inhibitory effect on the myocardium.

[0003] Generally speaking, compound drugs introduce a variety of impurities, and there are large differences in the properties of impurities, so it is necessary to control the introduced impurities. At present, the quality standards of besifloxacin and timolol maleate eye drops are not included in the United States Pharmacopeia, the Japanese Pharmacopeia, the import drug registration standards of the National Medical Products Administration, etc. In view of this, it is of great significance to develop a highly specific, efficient and accurate analytical method for quality analysis of besifloxacin and timolol maleate eye drops. Summary of the Invention

[0004] To achieve precise analytical control of the impurity content in besifloxacin and timolol maleate eye drops, the present application provides a method for separating and determining related impurities of besifloxacin and timolol maleate eye drops.

[0005] A method for separating and determining related impurities of besifloxacin and timolol maleate eye drops, using the HPLC method, includes the following steps:

[0006] Prepare the test solution: Dissolve and dilute the test sample with water to obtain it;

[0007] Prepare the control solution: Dissolve and dilute timolol maleate with water to obtain it;

[0008] Prepare the system suitability solution: Take besifloxacin and timolol maleate eye drops and their related impurities, mix them and dilute with water to obtain it;

[0009] Prepare the blank excipient solution: Take sodium chloride, disodium hydrogen phosphate heptahydrate, citric acid, benzalkonium chloride and besifloxacin, dissolve and dilute with water to obtain it;

[0010] Retention time determination: Inject the blank excipient solution and the system suitability solution for analysis to determine the retention times of the related impurities in the besifloxacin and timolol maleate eye drops;

[0011] Separation and detection of the target substance: Inject the test solution and the reference solution for detection, and calculate the content of the related impurities in the test solution by the external standard method based on the main component;

[0012] In the HPLC, octadecylsilane chemically bonded silica gel is used as the stationary phase, sodium octanesulfonate buffer solution is used as mobile phase A, and methanol is used as mobile phase B for gradient elution.

[0013] The main components of the besifloxacin and timolol maleate eye drops in this application are besifloxacin and timolol maleate, and their molecular structures are shown below. Based on this, the reference substance used in the analysis method of this application is timolol maleate, which is beneficial to improving the reliability and accuracy of the analysis results.

[0014] Besifloxacin

[0015]

[0016] Timolol maleate

[0017] Preferably, the related impurities include:

[0018] Impurity B: 3-(tert-butylamino)-2-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-1-propanol maleate;

[0019] Impurity C: N-(tert-butyl)-2,3-bis((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-1-propanamine maleate;

[0020] Impurity D: 4-morpholino-1,2,5-thiadiazol-3-ol;

[0021] Impurity E: (S,Z)-4-((1-(tert-butylamino)-3-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)propan-2-yl)oxy)-4-oxobut-2-enoic acid;

[0022] Impurity F: 4-(4-chloro-1,2,5-thiadiazol-3-yl)morpholine;

[0023] Impurity G: 4-morpholino-1,2,5-thiadiazol-3(2H)-one 1-oxide;

[0024] Impurity H: 2-(3-(tert-butylamino)-2-hydroxypropyl)-4-morpholino-1,2,5-thiadiazol-3(2H)-one;

[0025] Impurity I: 1-(ethylamino)-3-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-2-propanol;

[0026] Impurity J: 3,3'-((1,2,5-thiadiazole-3,4-diyl)bis(oxy))bis(1-(tert-butylamino)propanol).

[0027] Preferably, the gradient elution is set as follows:

[0028] 0 - 20 min: 45 - 50% A → 27 - 33% A;

[0029] 20 - 30 min: 27 - 33% A → 27 - 33% A;

[0030] 30 - 31 min: 27 - 33% A → 45 - 50% A;

[0031] 31 - 40 min: 45 - 50% A → 45 - 50% A.

[0032] Preferably, the gradient elution is set as follows:

[0033] 0 - 20 min: 48% A → 30% A;

[0034] 20 - 30 min: 30% A → 30% A;

[0035] 30 - 31 min: 30% A → 48% A;

[0036] 31 - 40 min: 48% A → 48% A.

[0037] Preferably, the flow rate of the gradient elution is 0.9 - 1.1 ml / min, more preferably 1.0 ml / min.

[0038] Preferably, in the test solution, the timolol content is 1 mg / ml.

[0039] Preferably, in the control solution, the timolol content is 2 μg / ml.

[0040] Preferably, the pH value of the sodium octanesulfonate buffer solution is 2.8 - 3.2; more preferably 3.0.

[0041] Preferably, the concentration of sodium octanesulfonate in the sodium octanesulfonate buffer solution is 4.3 - 4.34 g / L; more preferably 4.32 g / L.

[0042] Preferably, the sodium octanesulfonate buffer solution is adjusted by adding glacial acetic acid to an aqueous solution of sodium octanesulfonate.

[0043] Preferably, the particle size of the packing particles of octadecylsilyl-bonded silica gel is 2-5 μm; the column temperature of the chromatographic column used in the HPLC is 33-37 °C.

[0044] Preferably, the detection wavelength of the detector used in the HPLC is 295 ± 2 nm.

[0045] In summary, the present application has the following beneficial effects:

[0046] The method of the present invention can effectively separate the related impurities of besifloxacin and timolol maleate eye drops and determine their contents, and the method has the advantages of strong specificity, high sensitivity, high efficiency and accuracy, good repeatability and durability, simple operation, stable and reliable results, etc. It is of great significance for realizing the quality control of besifloxacin and timolol maleate eye drops. Description of the Drawings

[0047] Figure 1 It is the chromatogram of the system suitability solution under the conditions of Example 1 of the present application.

[0048] Figure 2 The chromatogram of the system suitability solution under the conditions of Control Example 1.

[0049] Figure 3 The chromatogram of the system suitability solution under the conditions of Control Example 2.

[0050] Figure 4 The chromatogram of the system suitability solution under the conditions of Control Example 3. Detailed Description of the Invention

[0051] Hereinafter, the preferred embodiments of the present invention will be described in detail with reference to the drawings. For the experimental methods without specific conditions indicated in the preferred embodiments, they are usually carried out under conventional conditions. The examples given are for better explaining the content of the present invention, but the content of the present invention is not limited to the given examples. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation schemes according to the above-mentioned invention content still fall within the protection scope of the present invention.

[0052] Examples

[0053] Example 1, a method for separating and determining related impurities of besifloxacin and timolol maleate eye drops:

[0054] 1. Chromatographic conditions

[0055] Chromatographic column: Kromasil 100-5 C18, 250 mm × 4.6 mm, 5 μm, mobile phase A: 4.32 g / L sodium octanesulfonate solution (take 4.32 g of sodium octanesulfonate, add 1000 ml of water to dissolve it, and adjust the pH value to 3.0 with glacial acetic acid), mobile phase B: methanol, gradient elution is carried out, and the gradient elution settings are as follows:

[0056]

[0057] Flow rate: 1 ml / min, column temperature: 35 °C, detection wavelength: 295 nm, injection volume: 20 μl.

[0058] 2. Methods and Results

[0059] 2.1 Preparation of Solutions

[0060] Test solution: Dilute the test sample with water to prepare a solution containing 1 mg of timolol in 1 ml of water;

[0061] Reference solution: Weigh an appropriate amount of timolol maleate reference substance accurately, dissolve it in water and quantitatively dilute to prepare a solution containing approximately 2 μg of timolol in 1 ml;

[0062] System suitability solution (spiked test solution): Take besifloxacin ophthalmic solution and impurities B, C, D, E, F, G, H, I, J, dissolve and dilute with water to prepare a solution containing 1 mg of timolol and 2 μg of each impurity in 1 ml of water;

[0063] Blank excipient solution: Weigh appropriate amounts of sodium chloride, disodium hydrogen phosphate heptahydrate, citric acid, benzalkonium chloride and bimatoprost accurately, dissolve them in water and dilute to prepare a solution containing approximately 6.8 mg of sodium chloride, 2.68 mg of disodium hydrogen phosphate heptahydrate, 0.14 mg of citric acid, 0.05 mg of benzalkonium chloride and 0.3 mg of bimatoprost in 1 ml. Accurately pipette 1 ml of the above solution into a 5-ml volumetric flask and dilute to the mark with water.

[0064] 2.2 Specificity

[0065] Precisely measure and inject the above blank excipient solution and system suitability solution, and record the chromatogram.

[0066] The blank excipient solution showed no interference with each impurity; if Figure 1As shown, in the system suitability solution, the elution order is as follows: the retention time of maleic acid (Rt) is 1.9 min, impurity G (retention time Rt 2.841 min), impurity D (retention time Rt 5.615 min), impurity E (retention time Rt 8.120 min), impurity H (retention time Rt 10.102 min), impurity B (retention time Rt 11.321 min), impurity I (retention time Rt 12.774 min), impurity F (retention time Rt 13.806 min), the retention time of timolol (Rt) is 14.439 min, impurity J (retention time Rt 19.576 min), impurity C (retention time Rt 24.170 min); the resolution of each impurity and timolol is greater than 1.5.

[0067] 2.3 Repeatability

[0068] Precisely measure the above-mentioned control solution and the spiked test solution, inject the samples, and record the chromatograms. Calculate the RSD of the contents of each related substance in 6 spiked test solutions by the external standard method of the main component with correction factor. The RSD of the contents of each detected related impurity in 6 spiked test solutions is less than 10.0%, meeting the requirements for the detection of related substances by high performance liquid chromatography.

[0069] 2.4 Intermediate precision The intermediate precision test was carried out by different analysts, on different dates, using different instruments, but with the same method. The RSD of the contents of each detected related impurity in 12 spiked test solutions is less than 10.0%, meeting the requirements for the detection of related substances by high performance liquid chromatography.

[0070] 2.4 Linearity

[0071] Take besifloxacin and timolol eye drops and its impurities B, C, D, E, F, G, H, I, J, dissolve them in water and dilute to prepare a mixed solution containing 10 μg of timolol, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I and impurity J per 1 ml as the linear stock solution. Precisely transfer 2.5 ml of the linear stock solution into 50 ml volumetric flasks, 2.5 ml into 25 ml volumetric flasks, 7.5 ml into 50 ml volumetric flasks, 2 ml into 10 ml volumetric flasks, 4 ml into 10 ml volumetric flasks, 6 ml into 10 ml volumetric flasks respectively, and dilute to the mark with water as the linear series solutions. Take the quantitation limit solution as the lowest point of the linearity. Perform linear regression with the peak area against the concentration to obtain the linear equation (see Table 1). Timolol and related impurities have good linear relationships within the linear range.

[0072] Table 1. Results of linearity determination

[0073]

[0074]

[0075] 2.5 Detection Limit and Quantitation Limit

[0076] Appropriately weigh timolol and each impurity reference substance, and prepare a series of solutions (see Table 2). When S / N ≥ 10, it is used as the quantitation limit solution; when S / N ≥ 3, it is used as the detection limit solution. The results are shown in Table 2.

[0077] Table 2. Results of Quantitation Limit and Detection Limit

[0078]

[0079] 2.6 Accuracy

[0080] Take besifloxacin and timolol eye drops, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J, dissolve and dilute with water to prepare a solution containing 1 mg of timolol and 1 μg of each impurity per 1 ml as the 50% recovery solution; take besifloxacin and timolol eye drops and its impurities B, C, D, E, F, G, H, I, and J, dissolve and dilute with water to prepare a solution containing 1 mg of timolol and 2 μg of each impurity per 1 ml as the 100% recovery solution; take besifloxacin and timolol eye drops and its impurities B, C, D, E, F, G, H, I, and J, dissolve and dilute with water to prepare a solution containing 1 mg of timolol and 3 μg of each impurity per 1 ml as the 150% recovery solution; each concentration is in parallel for 3 portions. Take the recovery solution and the reference solution, inject for analysis, and record the chromatogram. Calculate the recovery rate and RSD of each impurity by the external standard method of the main component with a correction factor. The results show that the recovery rates of each impurity at each concentration are between 80.0% and 120.0%, and the RSDs are all less than 10.0%, meeting the requirements for the detection of related substances by high performance liquid chromatography. The results are shown in Table 3.

[0081] Table 3. Results of Accuracy

[0082]

[0083] 3. Conclusion

[0084] Under these chromatographic conditions, besifloxacin and timolol eye drops and their related impurities can be completely separated. This method has strong specificity, high sensitivity, and the accuracy, repeatability, and intermediate precision all meet the technical requirements of drug quality research standards, and the obtained results are stable and reliable.

[0085] Control Example

[0086] Control Example 1

[0087] 1. Chromatographic Conditions

[0088] Chromatographic column: Waters Symmetry C18 (3.9 mm × 150 mm, 5 μm), mobile phase A: 4.32 g / L sodium octylsulfonate buffer (pH 3.0) - methanol (50:50), mobile phase B: methanol, gradient elution was performed, and the gradient elution settings were as follows:

[0089]

[0090]

[0091] Flow rate: 1.2 ml / min, column temperature: 35 °C, detection wavelength: 295 nm, injection volume: 20 μl.

[0092] 2. Method

[0093] Take timolol maleate and betaxolol ophthalmic solution, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity I, and impurity J, dissolve and dilute with water to prepare a solution containing 1 mg of timolol and 2 μg of each related impurity per 1 ml as the system suitability solution. Precisely measure 20 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 2 .

[0094] 3. Conclusion: Under these chromatographic conditions, impurity F and impurity I overlap (Rt 5.8 min), impurity G (Rt 1.0 min) elutes earlier, and it does not reach baseline separation from the maleic acid peak (Rt 0.7 min).

[0095] Control Example 2

[0096] 1. Chromatographic conditions:

[0097] Chromatographic column: ACE Excel 1.7 C18 - PFP (2.1 mm × 100 mm, 1.7 μm), mobile phase A: trifluoroacetic acid - water (0.5:1000), mobile phase B: trifluoroacetic acid - acetonitrile (0.5:1000), gradient elution was performed, and the gradient elution settings were as follows:

[0098] Time (min) A(％) B(％) 0 84 16 2.4 84 16 8 20 80 8.1 84 16 11 84 16

[0099] Flow rate: 0.1 ml / min, column temperature: 30 °C, detection wavelength: 295 nm, injection volume: 2.5 μl.

[0100] 2. Method

[0101] Take Besifloxacin and Timolol Eye Drops and impurities B, C, D, E, F, G, H, I, and J, dissolve and dilute them with water to prepare a solution containing 1 mg of timolol and 2 μg of each related impurity per 1 ml as the system suitability solution. Precisely measure 20 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 3 。

[0102] 3. Conclusion: Under these chromatographic conditions, the impurities were not completely separated.

[0103] Control Example 3

[0104] 1. Chromatographic conditions:

[0105] Chromatographic column: Waters Xbridge-C18 (4.6 mm × 150 mm, 3.5 μm), mobile phase A: 0.01% phosphoric acid buffer solution (pH 3.0) - methanol (80:20), mobile phase B: acetonitrile - methanol (70:30), perform gradient elution, and the gradient elution settings are as follows:

[0106] Time (min) A(％) B(％) 0 82 18 5 82 18 5.1 60 40 16 60 40 17 82 18 25 82 18

[0107] Flow rate: 1.0 ml / min, column temperature: 30 °C, detection wavelength: 295 nm, injection volume: 5 μl.

[0108] 2. Method:

[0109] Take Besifloxacin and Timolol Eye Drops and impurities B, C, D, E, F, G, H, I, and J, dissolve and dilute them with water to prepare a solution containing 1 mg of timolol and 2 μg of each impurity per 1 ml as the system suitability solution. Precisely measure 5 μl and inject it into the liquid chromatograph, record the chromatogram, and the chromatogram of the system suitability solution is shown in the appendix Figure 4 。

[0110] 3. Conclusion: Under these chromatographic conditions, the peak shape of the main peak is poor.

[0111] This specific embodiment is only an interpretation of the present application and does not limit the present application. Those skilled in the art can make modifications without creative contributions to this embodiment after reading this specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for the separation and determination of related impurities in besifloxacin and timolol eye drops, characterized in that, The HPLC method is adopted and includes the following steps: Prepare the test solution: Dissolve and dilute the test sample with water to obtain it; Prepare the control solution: Dissolve and dilute timolol maleate with water to obtain it; Prepare the system suitability solution: Take besifloxacin ophthalmic solution and its related impurities, mix them and dilute with water to obtain it; Prepare the blank excipient solution: Take sodium chloride, disodium hydrogen phosphate heptahydrate, citric acid, benzalkonium chloride and bimatoprost, dissolve and dilute with water to obtain it; Determine the retention time: Inject the blank excipient solution and the system suitability solution for analysis to determine the retention time of the related impurities in besifloxacin ophthalmic solution; Separate and detect the target substance: Inject the test solution and the control solution for detection, and calculate the content of the related impurities in the test solution by the external standard method of the main component; The HPLC uses octadecylsilane chemically bonded silica as the stationary phase, sodium octanesulfonate buffer solution as mobile phase A, and methanol as mobile phase B for gradient elution; the pH value of the sodium octanesulfonate buffer solution is 2.8 - 3.2; the concentration of sodium octanesulfonate in the sodium octanesulfonate buffer solution is 4.3 - 4.34 g / L; the gradient elution is set as follows: 0 - 20 min: 45 - 50% A → 27 - 33% A; 20 - 30 min: 27 - 33% A → 27 - 33% A; 30 - 31 min: 27 - 33% A → 45 - 50% A; 31 - 40 min: 45 - 50% A → 45 - 50% A; The flow rate of the gradient elution is 0.9 - 1.1 ml / min; Among them, the related impurities include: Impurity B: 3-(tert-butylamino)-2-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-1-propanol maleate; Impurity C: N-(tert-butyl)-2,3-bis((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-1-propanamine maleate; Impurity D: 4-morpholino-1,2,5-thiadiazol-3-ol; Impurity E: (S,Z)-4-((1-(tert-butylamino)-3-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)propan-2-yl)oxy)-4-oxobut-2-enoic acid; Impurity F: 4-(4-chloro-1,2,5-thiadiazol-3-yl)morpholine; Impurity G: 4-morpholino-1,2,5-thiadiazol-3(2H)-one 1-oxide; Impurity H: 2-(3-(tert-butylamino)-2-hydroxypropyl)-4-morpholino-1,2,5-thiadiazol-3(2H)-one; Impurity I: 1-(ethylamino)-3-((4-morpholino-1,2,5-thiadiazol-3-yl)oxy)-2-propanol; Impurity J: 3,3'-((1,2,5-thiadiazole-3,4-diyl)bis(oxy))bis(1-(tert-butylamino)propanol).

2. The method for separating and determining related impurities of besifloxacin and timolol maleate eye drops according to claim 1, characterized in that, The gradient elution is set as follows: 0 - 20 min: 48% A → 30% A; 20 - 30 min: 30% A → 30% A; 30 - 31 min: 30% A → 48% A; 31 - 40 min: 48% A → 48% A.

3. The method for separating and determining related impurities of besifloxacin and timolol maleate eye drops according to claim 1, characterized in that, In the test solution, the content of timolol is 1 mg / ml; in the control solution, the content of timolol is 2 μg / ml.

4. The separation and determination method of related impurities of bemesulthiamolol eye drops according to claim 1, wherein, The pH value of the sodium octanesulfonate buffer solution is 3.

0.

5. The separation and determination method of related impurities of besifloxacin and timolol maleate eye drops according to claim 1, characterized in that The particle size of the packing particles of the octadecylsilyl silica gel is 2 - 5 μm; the column temperature of the chromatographic column used in the HPLC is 33 - 37 °C.

6. The separation and determination method of related impurities of besifloxacin and timolol maleate eye drops according to claim 1, characterized in that, The detection wavelength of the detector used in the HPLC is 295 ± 2 nm.

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