Quantitative analysis method for dissolution rate of isavuconazole sulfate capsule based on high performance liquid chromatography

Abstract

The invention discloses a quantitative analysis method for dissolution rate of isavuconazole sulfate capsules based on high performance liquid chromatography. The quantitative analysis method comprises the following steps: step 1, setting chromatographic conditions; 2, preparing a reference substance solution; step 3, preparing a test solution; and 4, determining the content of isavuconazole in the test solution. The high performance liquid chromatography is used for detecting the dissolution amount of the sample, so that the solution detection repeatability is good, the sensitivity is high, and the detection result is more accurate. A linearity and range test, a recovery rate test, a repeatability test and a precision test are respectively carried out to verify the method, and the result shows that the method is accurate and reliable.

Description

Technical Field

[0001] The invention belongs to the field of drug analysis and detection, and specifically relates to a quantitative analysis method for the dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography, which is determined by a high performance liquid chromatograph. Background Art

[0002] Isavuconazole sulfate is a drug used to treat fungal infections, and its dissolution test is an important indicator for evaluating the quality of its preparations. The traditional dissolution test method uses ultraviolet spectrophotometry, but this method has limitations in detection specificity, sensitivity, and quantitative accuracy. Therefore, an improved detection method based on high performance liquid chromatography (HPLC) is needed to improve the accuracy and efficiency of quantitative detection of isavuconazole sulfate capsules and ensure their drug quality. Summary of the invention

[0003] Purpose of the invention: The technical problem to be solved by the present invention is to provide a quantitative detection method for the dissolution of isavuconazole sulfate capsules with high detection sensitivity, strong specificity, stable analysis results and good result repeatability, in view of the problems of insufficient detection specificity and poor detection result repeatability in the current quantitative detection method for the dissolution of isavuconazole sulfate capsules.

[0004] Technical solution: A quantitative analysis method for the dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography, comprising the following steps:

[0005] Step 1: Set the chromatographic conditions as follows:

[0006] Chromatographic column: Octadecylsilane bonded silica gel is used as filler;

[0007] Column temperature: 35℃~45℃;

[0008] Flow rate: 0.8-1.0 mL per minute;

[0009] Detection wavelength: 250nm;

[0010] The mobile phases were as follows: mobile phase A: water: acetonitrile: trifluoroacetic acid = 99:1:0.15, 99:1:0.2 or 99:1:0.22; mobile phase B: acetonitrile: trifluoroacetic acid = 100:0.1; the initial ratio of mobile phase A:B was 73:27;

[0011] The gradient elution conditions were as follows:

[0012]

[0013] The diluents are as follows: Diluent 1: Dissolution medium mixed with 0.1 mol / L hydrochloric acid at a ratio of 1:1 (v / v), used for reference sample dissolution and quantitative dilution of reference sample solution; Diluent 2: 0.1 mol / L hydrochloric acid, used for quantitative dilution of the dissolution solution after filtration in the preparation of the test sample solution;

[0014] Step 2, prepare the reference solution: weigh 20 mg of isavuconazole sulfate reference substance, accurately weigh it, put it in a 200 mL volumetric flask, dissolve it with diluent 1 and quantitatively dilute it to the scale, shake it well, and obtain the reference solution;

[0015] Step 3: Prepare the test solution: place 6 test samples in a sinker basket, put them in a dissolution cup, add 900 mL of dissolution medium, and rotate and dissolve at a speed of 75 revolutions per minute for 60 minutes; take 10 mL of the dissolution solution, filter it with a filter membrane, accurately measure 2 mL of the filtrate, add 2 mL of diluent 2 and dilute it quantitatively to obtain the test solution;

[0016] Step 4. Determine the content of isavuconazole in the test solution: Accurately pipette 5 μl of the reference solution prepared in step 2 and the test solution prepared in step 3, inject them into the liquid chromatograph, and calculate the peak area by the external standard method to obtain the content of isavuconazole in the test solution.

[0017] As an optimization: in the chromatographic conditions described, the column temperature is 40°C.

[0018] As an optimization: in the chromatographic conditions, the flow rate is 0.9 mL per minute.

[0019] As an optimization: in the chromatographic conditions, the mobile phase A: water: acetonitrile: trifluoroacetic acid is 99:1.0:0.2.

[0020] As an optimization: in the chromatographic conditions, the initial ratio of the mobile phase A:B is: 71:29 or 75:25.

[0021] Beneficial effect: The present invention uses high performance liquid chromatography to detect the sample dissolution amount, so that the solution detection has good repeatability, high sensitivity, and more accurate detection results. The present invention conducts linearity and range tests, recovery tests, repeatability tests, and precision tests to verify the method, and the results show that the method is accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention discloses a chromatogram of a reference solution measured by the quantitative detection method for dissolution of isavuconazole sulfate capsules provided by the present invention.

[0023] Figure 2 The present invention provides a chromatogram of a test solution measured by the quantitative detection method for the dissolution of isavuconazole sulfate capsules.

[0024] Figure 3 The linearity and range curves of the quantitative detection method for dissolution of isavuconazole sulfate capsules provided by the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention but not all of them.

[0026] If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature of the field or the product instructions are used. If the manufacturer of the instruments used is not specified, they are all conventional products that can be purchased through regular channels. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified. If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature of the field or the product instructions are used. Specific embodiment 1

[0028] Instruments: Thermo U3000 high performance liquid chromatograph; Sartorius MSE125P electronic balance.

[0029] Chromatographic conditions: Octadecylsilane bonded silica gel as filler, water: acetonitrile: trifluoroacetic acid (99:1:0.2) as mobile phase A, acetonitrile: trifluoroacetic acid (100:0.1) as mobile phase B, gradient elution according to the following table; flow rate 0.9 ml per minute; column temperature 40°C; detection wavelength 250 nm. Mobile phase gradient:

[0030]

[0031] Thinner:

[0032] Diluent 1: Dissolution medium mixed with 0.1 mol / L hydrochloric acid at a ratio of 1:1 (v / v) for reference substance dissolution and quantitative dilution of reference substance solution;

[0033] Diluent 2: 0.1 mol / L hydrochloric acid, used for quantitative dilution of the dissolved solution after filtration in the preparation of the test solution.

[0034] Preparation of reference solution: Weigh approximately 20 mg of isavuconazole sulfate reference substance, accurately weigh, place in a 200 mL volumetric flask, dissolve with diluent 1 and quantitatively dilute to scale, shake well, and obtain a reference solution.

[0035] Preparation of test sample solution: Place 6 test sample pellets in a sinker basket, put into a dissolution cup, add 900 mL of dissolution medium, and rotate and dissolve at 75 rpm for 60 min; take 10 mL of the dissolution solution, filter it with a filter membrane, accurately measure 2 mL of the filtrate, add 2 mL of diluent 2 for quantitative dilution, and obtain the test sample solution.

[0036] Determination steps: Accurately pipette 5 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. Calculate the peak area according to the external standard method to obtain the content of isavuconazole in the test solution. Specific embodiment 2

[0038] Instruments: Thermo U3000 high performance liquid chromatograph; Sartorius MSE125P electronic balance.

[0039] Chromatographic conditions: Octadecylsilane bonded silica gel as filler, water: acetonitrile: trifluoroacetic acid (99:1:0.22) as mobile phase A, acetonitrile: trifluoroacetic acid (100:0.1) as mobile phase B, gradient elution according to the following table; flow rate 1.0 ml per minute; column temperature 35°C; detection wavelength 250 nm. Mobile phase gradient:

[0040]

[0041] Thinner:

[0042] Diluent 1: Dissolution medium mixed with 0.1 mol / L hydrochloric acid at a ratio of 1:1 (v / v) for reference substance dissolution and quantitative dilution of reference substance solution;

[0043] Diluent 2: 0.1 mol / L hydrochloric acid, used for quantitative dilution of the dissolved solution after filtration in the preparation of the test solution.

[0044] Preparation of reference solution: Weigh approximately 20 mg of isavuconazole sulfate reference substance, accurately weigh, place in a 200 mL volumetric flask, dissolve with diluent 1 and quantitatively dilute to scale, shake well, and obtain a reference solution.

[0045] Preparation of test sample solution: Place 6 test sample pellets in a sinker basket, put into a dissolution cup, add 900 mL of dissolution medium, and rotate and dissolve at 75 rpm for 60 min; take 10 mL of the dissolution solution, filter it with a filter membrane, accurately measure 2 mL of the filtrate, add 2 mL of diluent 2 for quantitative dilution, and obtain the test sample solution.

[0046] Determination steps: Accurately pipette 5 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. Calculate the peak area according to the external standard method to obtain the content of isavuconazole in the test solution. Specific embodiment 3

[0048] Instruments: Thermo U3000 high performance liquid chromatograph; Sartorius MSE125P electronic balance.

[0049] Chromatographic conditions: Octadecylsilane bonded silica gel as filler, water: acetonitrile: trifluoroacetic acid (99:1:0.15) as mobile phase A, acetonitrile: trifluoroacetic acid (100:0.1) as mobile phase B, gradient elution according to the following table; flow rate 0.8 ml per minute; column temperature 45°C; detection wavelength 250 nm. Mobile phase gradient:

[0050]

[0051] Thinner:

[0052] Diluent 1: Dissolution medium mixed with 0.1 mol / L hydrochloric acid at a ratio of 1:1 (v / v) for reference substance dissolution and quantitative dilution of reference substance solution;

[0053] Diluent 2: 0.1 mol / L hydrochloric acid, used for quantitative dilution of the dissolved solution after filtration in the preparation of the test solution.

[0054] Preparation of reference solution: Weigh approximately 20 mg of isavuconazole sulfate reference substance, accurately weigh, place in a 200 mL volumetric flask, dissolve with diluent 1 and quantitatively dilute to scale, shake well, and obtain a reference solution.

[0055] Preparation of test sample solution: Place 6 test sample pellets in a sinker basket, put into a dissolution cup, add 900 mL of dissolution medium, and rotate and dissolve at 75 rpm for 60 min; take 10 mL of the dissolution solution, filter it with a filter membrane, accurately measure 2 mL of the filtrate, add 2 mL of diluent 2 for quantitative dilution, and obtain the test sample solution.

[0056] Determination steps: Accurately pipette 5 μl of the reference solution and the test solution respectively, inject into the liquid chromatograph, and determine. Calculate the peak area according to the external standard method to obtain the content of isavuconazole in the test solution.

[0057] The present invention has carried out the following methodological verification test:

[0058] 1. Linearity and range test

[0059] Accurately weigh about 50 mg (content 94.0%) of isavuconazole sulfate reference substance, place it in a 50 mL volumetric flask, add solvent (acetonitrile: water: trifluoroacetic acid = 50:50:0.05) to dissolve and dilute to the scale, shake well, and use as the linear reserve solution. Accurately draw an appropriate amount of linear reserve solution, dilute with solvent to prepare reference substance solutions with concentrations of approximately 40 μg / mL, 100 μg / mL, 160 μg / mL, 200 μg / mL, 240 μg / mL, and 500 μg / mL, respectively, inject them into the liquid chromatograph, and measure according to the above chromatographic conditions. The results are shown in Table 1. Use the isavuconazole sulfate concentration (μg / mL) as the horizontal axis and the corresponding peak area as the vertical axis to draw a standard curve (see Figure 3 ). The results showed that isavuconazole sulfate showed a good linear relationship within the concentration range, and the regression equation was y=0.0664x+0.1030, r=1.0000.

[0060] Table 1 Linearity test results

[0061]

[0062] 2. Precision test

[0063] The isavuconazole sulfate reference solution was accurately drawn and injected 6 times in succession with an injection volume of 5 μl. The chromatogram was recorded, the isavuconazole sulfate peak area was determined, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2. The RSD of the main peak retention time was 0.1%, and the RSD of the peak area was 0.1%, indicating that the precision of this method was good.

[0064] Table 2 Precision test results

[0065]

[0066] 3. Repeatability test

[0067] Two reference solutions were prepared according to the preparation of the reference solution, and 6 samples of the same batch were taken. The isavuconazole content of the 6 test samples was determined according to the preparation of the test solution and the chromatographic conditions. The results are shown in Table 3. The average content was 100.7% and the RSD was 0.1%, indicating that this method has good repeatability.

[0068] Table 3 Repeatability test results

[0069]

[0070] 4. Accuracy test

[0071] Weigh 4.02g of blank auxiliary material accurately, transfer it to a 200mL volumetric flask, add about 100mL of solvent (acetonitrile: water: trifluoroacetic acid = 50:50:0.05), ultrasonicate and shake for about 10 minutes, dilute to scale with solvent, mix well, take about 10mL of solution, centrifuge at 10,000 rpm for 10 minutes, take the supernatant as blank auxiliary material stock solution. Accurately measure 1mL of blank auxiliary material stock solution, place it in a 50ml volumetric flask, dilute to scale with solvent, shake well, and obtain blank auxiliary material solution.

[0072] Accurately measure 50 mg of isavuconazole sulfate reference substance, place it in a 50 mL volumetric flask, add solvent to dissolve and dilute to the mark, shake well, and use it as the isavuconazole sulfate reference substance stock solution. Accurately pipette 2 mL of the reference substance stock solution, place it in a 10 mL volumetric flask, add solvent to dilute to the mark, shake well, and use it as the isavuconazole sulfate reference substance solution.

[0073] Accurately weigh an appropriate amount of isavuconazole sulfate reference substance, place it in a 50mL volumetric flask, add about 1g of blank excipients, about 25mL of solvent, ultrasonically shake for about 10 minutes, dilute to scale with solvent, and mix well. Take about 10mL of solution, centrifuge at 10,000 rpm for 10 minutes, take the supernatant as the test sample stock solution. Accurately measure 2mL of the test sample stock solution, place it in a 10mL volumetric flask, dilute to scale with solvent, shake well, and obtain the accuracy solution. The specific amount of reference substance added is shown in the table below.

[0074]

[0075] Three test solutions were prepared for each concentration and calculated by peak area using the external standard method. At the limit level of 20% to 250% of the labeled concentration, the average recovery of isavuconazole sulfate was between 100.9% and 100.3%, the total average recovery was 101.1%, and the RSD was 0.5%. The results are shown in Table 4, indicating that this method has a good recovery rate.

[0076] Table 4 Recovery test results

[0077]

[0078] The above-mentioned linearity and range test, recovery test, repeatability test and precision test conducted by the present invention are used to verify the method, and the results show that the method is accurate and reliable. Compared with the existing method, the method of the present invention uses high performance liquid chromatography to detect the sample dissolution amount, so that the solution detection has good repeatability, high sensitivity and more accurate detection results.

Claims

1. A quantitative analysis method for the dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography, characterized in that: The steps include: Step 1: Set the chromatographic conditions as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as filler; Column temperature: 35℃~45℃; Flow rate: 0.8-1.0 mL per minute; Detection wavelength: 250nm; The mobile phases were as follows: mobile phase A: water: acetonitrile: trifluoroacetic acid = 99:1:0.15, 99:1:0.2 or 99:1:0.22; mobile phase B: acetonitrile: trifluoroacetic acid = 100:0.1; the initial ratio of mobile phase A:B was 73:27; The gradient elution conditions were as follows: The diluents are as follows: Diluent 1: Dissolution medium mixed with 0.1 mol / L hydrochloric acid at a ratio of 1:1 (v / v), used for reference sample dissolution and quantitative dilution of reference sample solution; Diluent 2: 0.1 mol / L hydrochloric acid, used for quantitative dilution of the dissolution solution after filtration in the preparation of the test sample solution; Step 2, prepare the reference solution: weigh 20 mg of isavuconazole sulfate reference substance, accurately weigh it, put it in a 200 mL volumetric flask, dissolve it with diluent 1 and quantitatively dilute it to the scale, shake it well, and obtain the reference solution; Step 3: Prepare the test solution: place 6 test samples in a sinker basket, put them in a dissolution cup, add 900 mL of dissolution medium, and rotate and dissolve at a speed of 75 revolutions per minute for 60 minutes; take 10 mL of the dissolution solution, filter it with a filter membrane, accurately measure 2 mL of the filtrate, add 2 mL of diluent 2 and dilute it quantitatively to obtain the test solution; Step 4. Determine the content of isavuconazole in the test solution: Accurately pipette 5 μl of the reference solution prepared in step 2 and the test solution prepared in step 3, inject them into the liquid chromatograph, and calculate the peak area by the external standard method to obtain the content of isavuconazole in the test solution.

2. The method for quantitative analysis of dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography according to claim 1, characterized in that: In the chromatographic conditions, the column temperature is 40°C.

3. The quantitative analysis method for dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography according to claim 1, characterized in that: In the chromatographic conditions, the flow rate was 0.9 mL per minute.

4. The quantitative analysis method for dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography according to claim 1, characterized in that: In the chromatographic conditions, mobile phase A: water: Acetonitrile:trifluoroacetic acid is 99:1.0:0.

2.

5. The method for quantitative analysis of dissolution of isavuconazole sulfate capsules based on high performance liquid chromatography according to claim 1, characterized in that: In the chromatographic conditions, the initial ratio of mobile phase A:B is 71:29 or 75:25.

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