Method for detecting content of minoxidil liniment

Abstract

The invention discloses a method for detecting content of minoxidil liniment.The method belongs to the field of medicine analysis and detection.The detection method includes the steps that an octadecyl silane bonded silica gel chromatographic column is adopted, a methanol-phosphate buffer mixed solution serves as a mobile phase, the flow velocity is 0.9 mL / min to 1.1 mL / min, the column temperature is 35 DEG C to 40 DEG C, and the detection wavelength of a sample is 278 nm to 282nm. According to the method, the mobile phase without acid is used as the diluent, so that the solution system is more stable, the detection repeatability is good, the sensitivity is high, and the detection result is more accurate. According to the method disclosed by the invention, methodological verification tests are respectively a linearity and range test, a recovery rate test, a repeatability test, a precision test and a stability test for verifying 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 method for detecting the content of minoxidil ointment, which adopts a high performance liquid chromatograph for direct determination. Background Art

[0002] Minoxidil is a peripheral vasodilator that can be used to treat hypertension, but in clinical applications, it has been found that this drug also has the effect of stimulating hair growth. When used topically for a long time, it can be used to treat seborrheic alopecia and alopecia areata. The 2020 edition of the "Chinese Pharmacopoeia" stipulates that the minoxidil content is determined by potentiometric titration, but minoxidil lotion is a liquid preparation prepared from minoxidil, polyol solvents and purified water. In order to further improve the quality control of minoxidil lotion and ensure its drug quality, it is very necessary to establish a method for detecting the content in minoxidil lotion. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a method for detecting the content of the main component in minoxidil ointment with low detection cost, stable detection solution system and accurate detection result, in view of the problems that the current detection method for the content of the main component in minoxidil ointment has high detection cost, unstable detection solution system and inaccurate detection result. The method comprises the following steps:

[0004] Step 1: Set the chromatographic conditions:

[0005] 1. Chromatographic column: UltimateLP-C18 (4.6×250mm, 5μm); UltimateAQ-C18 (4.6×250mm, 5μm) 2. Column temperature: 30℃~40℃;

[0006] 3. Flow rate: 0.9-1.1 ml per minute;

[0007] 4. Detection wavelength: 278nm~282nm;

[0008] 5. Mobile phase: methanol: phosphate buffer = 600:400;

[0009] 6. Diluent: methanol: water: glacial acetic acid = 500:500:2;

[0010] Step 2: Prepare reference solution:

[0011] a. Weigh about 25 mg of minoxidil reference substance, accurately weigh, place in a 50 ml volumetric flask, dissolve with diluent and dilute to scale, shake well, as a stock solution;

[0012] b. Accurately measure 5.0 ml of the above stock solution and place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready;

[0013] Step 3: Prepare the test solution:

[0014] a. Accurately measure 2.0 ml of sample solution (equivalent to 100 mg of minoxidil), place it in a 200 ml volumetric bottle, add diluent to the scale, and shake well;

[0015] b. Accurately measure 5.0 ml of the above solution and place it in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain;

[0016] Step 4: Determination and calculation:

[0017] Accurately pipette 4 μl of the reference solution prepared in step 2 and the test solution prepared in step 3 into the liquid chromatograph for determination. The minoxidil peak area RSD in the reference solution is ≤ 2.0%. Record the result and calculate the minoxidil content in the ointment using the peak area calculated by the external standard method.

[0018] As an optimization: the column temperature is 35°C.

[0019] As an optimization: the flow rate is 1.0 mL per minute.

[0020] As an optimization: the detection wavelength is 280 nm.

[0021] As an optimization, in the mobile phase, methanol:phosphate buffer=600:400.

[0022] As an optimization: the phosphate buffer in the mobile phase is prepared as follows: 6.85 g of dipotassium hydrogen phosphate is weighed and placed in 1000 ml of purified water, stirred to completely dissolve it, adjusted the pH value to 3.3 with phosphoric acid, and filtered through a 0.45 μm water membrane.

[0023] Beneficial effect: The method of the present invention uses a mobile phase without acid as a diluent, which makes the solution system more stable, has good detection repeatability, high sensitivity, and more accurate detection results. The method of the present invention has been verified by methodological validation tests, including linearity and range test, recovery test, repeatability test, precision test and stability test, and the results show that the method is accurate and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a chromatogram of a minoxidil reference solution measured by the minoxidil ointment content detection method provided by the present invention;

[0025] Figure 2 is a chromatogram of a test solution measured by the minoxidil liniment content detection method provided by the present invention;

[0026] Figure 3 is a standard curve diagram of the minoxidil liniment content detection method provided by the present invention; DETAILED DESCRIPTION

[0027] 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, not all of them.

[0028] If no specific technology or conditions are specified in the examples, the technology or conditions described in the literature in this field or the product instructions shall be followed. The instruments used, etc., for which the manufacturer is not specified, are all conventional products that can be purchased through regular channels. The methods described are conventional methods unless otherwise specified, and the raw materials described 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 in this field or the product instructions shall be followed. Example 1: A method for detecting the content of minoxidil ointment, as follows:

[0029] 1. Instruments: Agilent 1260 high performance liquid chromatograph; Sartorius SQP electronic balance; Yuexu chromatographic columns Ultimate LP-C18 (4.6×250mm, 5μm); Ultimate AQ-C18 (4.6×250mm, 5μm)

[0030] 2. Chromatographic conditions: The chromatographic columns were Ultimate LP-C18 (4.6×250mm, 5μm); Ultimate AQ-C18 (4.6×250mm, 5μm), with methanol-phosphate buffer (6.85g of potassium dihydrogen phosphate was weighed and placed in 1000ml of purified water, stirred to completely dissolve it, the pH value was adjusted to 3.3 with phosphoric acid, and filtered through a 0.45μm aqueous membrane) (600:400) as the mobile phase for isocratic elution; methanol was used as the needle washing solution; the flow rate was 1.0ml per minute; the detection wavelength was 280nm; and the column temperature was 35℃.

[0031] 3. Diluent: methanol-water-glacial acetic acid (500:500:2).

[0032] 4. Preparation of reference solution: Weigh about 25 mg of minoxidil reference substance, accurately weigh, place in a 50 ml volumetric flask, dissolve with diluent and dilute to scale, shake well, as stock solution. Accurately measure 5.0 ml of the above stock solution and place in a 50 ml volumetric flask, dilute to scale with diluent, shake well, and the solution is ready.

[0033] 5. Preparation of test solution: Accurately measure 2.0 ml of sample solution (equivalent to 100 mg of minoxidil), place it in a 200 ml volumetric flask, add diluent to the mark, and shake well. Accurately measure 5.0 ml of the above solution in a 50 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0034] 6. Determination steps: Accurately pipette 4 μl of reference solution and test solution respectively and inject them into liquid chromatograph for determination. Calculate the peak area according to the external standard method to obtain the content in minoxidil ointment.

[0035] 7. Comparison between the existing method and the method of the present invention: The present method uses a mobile phase without acid as a diluent, which makes the solution system more stable, the detection repeatability is good, the sensitivity is high, and the detection result is more accurate.

[0036] The relevant performance test experiments and experimental results in the present invention are as follows:

[0037] 1. Linearity and range test: Accurately weigh 20 mg (content 99.6%) of minoxidil reference substance, place it in a 200 ml volumetric flask, dissolve it with diluent and make up to the scale, shake well, and use it as the linear stock solution. Accurately pipette an appropriate amount of the linear stock solution, dilute it with diluent to prepare reference substance solutions with concentrations of approximately 20.0 μg / ml, 30.0 μg / ml, 40.0 μg / ml, 50.0 μg / ml, 60.0 μg / ml, and 80.0 μg / ml, respectively, inject them into the liquid chromatograph, and measure them under the above chromatographic conditions. The results are shown in Table 1. Draw a standard curve with minoxidil concentration (μg / ml) as the horizontal axis and the corresponding peak area as the vertical axis (see Figure 3 ). The results showed that minoxidil showed a good linear relationship within the range, and the regression equation was y=19470x+17.12, r=0.999.

[0038] Table 1 Linearity test results

[0039]

[0040]

[0041] 1. Precision test:

[0042] The minoxidil reference solution was accurately pipetted and injected 6 times in succession with an injection volume of 4 μl. The chromatogram was recorded, the minoxidil peak area was determined, and the relative standard deviation (RSD) was calculated. The results are shown in Table 2. The RSD (n=6) was 0.17%, indicating that the method had good precision.

[0043] Table 2 Precision test results

[0044]

[0045] 3. Repeatability test:

[0046] Take 6 samples of the same batch number (240701) and measure them according to the preparation of the test solution and the chromatographic conditions. The content of the 6 test samples is measured. The results are shown in Table 3. The average content is 101.7% and the RSD is 0.41%, indicating that this method has good repeatability.

[0047] Table 3 Repeatability test results

[0048]

[0049]

[0050] 4. Stability test

[0051] The reference solution and the test solution were accurately aspirated, and the minoxidil peak area was measured at room temperature at 0, 2, 6, 12, and 28 hours after preparation, respectively. 4 μl was injected each time, and the peak area RSD of the minoxidil reference solution within 28 hours was 0.55%; the peak area RSD of the test solution was 2.86%. The peak area RSD of the test solution at 11 hours was 1.44%. The test results are shown in Table 4, indicating that the minoxidil reference solution is stable at room temperature for 28 hours and the test solution is stable at room temperature for 12 hours.

[0052] Table 4 Stability test results

[0053]

[0054] 5. Recovery test

[0055] Measure 18ml of anhydrous ethanol and 30ml of propylene glycol into a 60ml volumetric flask, dilute with purified water and make up to the mark, shake well to obtain a blank excipient solution. Accurately weigh about 40mg of minoxidil reference substance and 1ml of blank excipient solution, place in a 100ml volumetric flask, and add diluent to make up to the mark. Take 5ml, place in a 50ml volumetric flask, add diluent to dissolve and make up to the mark, and prepare a test solution with an accuracy of approximately 40μg / ml. Accurately weigh about 50mg of minoxidil reference substance and 1ml of blank excipient solution, place in a 100ml volumetric flask, add diluent to make up to the mark. Take 5ml, place in a 50ml volumetric flask, add diluent to dissolve and make up to the mark, and prepare a test solution with an accuracy of approximately 50μg / ml. Accurately weigh about 60 mg of minoxidil reference substance and 1 ml of blank auxiliary solution, place in a 100 ml volumetric flask, add diluent to the scale. Take 5 ml, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, and prepare a test solution with an accuracy of about 60 μg / ml. Prepare 3 test solutions for each concentration, and perform recovery determination according to the content determination method. The recovery rate is measured, and the RSD (n=9) is 0.60%. The results are shown in Table 5, indicating that this method has a good recovery rate.

[0056] Table 5 Recovery test results

[0057]

[0058] In summary, the method of the present invention has been subjected to methodological validation tests, specifically: linearity and range test, recovery test, repeatability test, precision test and stability test to validate the method, and the results show that the method is accurate and reliable.

[0059] Although this patent has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of this patent should be included in the scope of protection of this patent.

Claims

1. A method for detecting the content of minoxidil liniment, characterized in that: The process includes the following steps: Step 1: Setting the chromatographic conditions: a). Chromatographic column: Ultimate LP-C18 (4.6×250mm, 5μm); Ultimate AQ-C18 (4.6×250mm, 5μm) b). Column temperature: 30℃~40℃; c). Flow rate: 0.9-1.1 ml per minute; d). Detection wavelength: 278nm~282nm; e). Mobile phase: methanol: phosphate buffer = 600:400; f). Diluent: methanol: water: glacial acetic acid = 500: 500: 2; Step 2: Prepare reference solution: a. Weigh about 25 mg of minoxidil reference substance, accurately weigh, place in a 50 ml volumetric flask, dissolve with diluent and dilute to scale, shake well, as a stock solution; b. Accurately measure 5 ml of the above stock solution and place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready; Step 3: Prepare the test solution: (a) Accurately measure 2 ml of sample solution (equivalent to 100 mg of minoxidil), place it in a 200 ml volumetric flask, add diluent to the mark, and shake well; (b) Accurately measure 5 ml of the above solution and place it in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready; Step 4: Determination and calculation: Accurately pipette 4 μl of the reference solution prepared in step 2 and the test solution prepared in step 3 into the liquid chromatograph for determination. Record the minoxidil peak area RSD of the reference solution if it is ≤ 2.0%. Calculate the minoxidil content in the ointment using the peak area using the external standard method.

2. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: The column temperature phosphate buffer solution is prepared as follows: 6.85 g of dipotassium hydrogen phosphate is weighed and placed in 1000 ml of purified water, stirred to completely dissolve, the pH value is adjusted to 3.3 with phosphoric acid, and filtered through a 0.45 μm water membrane.

3. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: The column temperature is 35°C.

4. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: The flow rate is 1.0 mL per minute.

5. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: The detection wavelength is 230nm.

6. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: In the mobile phase, methanol:phosphate buffer=600:

400.

7. The method for detecting the content of minoxidil liniment according to claim 1, characterized in that: The method for detecting the content of minoxidil in ointment described in 1 is characterized in that: in the phosphate buffer, the pH value of the phosphate buffer is 3.2-3.4.

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