A method and application for the determination of multiple components based on HPLC dual-wavelength gradient elution

Abstract

This invention discloses a method and application for the determination of multiple components based on HPLC dual-wavelength gradient elution. The method includes: setting chromatographic conditions; using octadecylsilane-bonded silica gel as the packing material and acetonitrile-0.5% triethylamine solution as the mobile phase; preparing a mixed reference solution, a test solution, and a negative sample solution; accurately injecting the solutions into the HPLC instrument; determining and recording the chromatograms according to the chromatographic conditions; calculating the content based on peak area using the external standard method; and performing linear regression on the peak area y with the solution's mass concentration x. The HPLC dual-wavelength gradient elution method established in this invention can simultaneously determine the content of four components—chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate—in imipramine cream. It exhibits strong specificity, good linearity, good precision and repeatability, and high recovery rate, improving quality control standards and comprehensively evaluating the overall quality of the product.

Description

Technical Field

[0001] This invention belongs to the field of chemical detection, specifically relating to a method and application for determining the content of multiple components based on HPLC dual-wavelength gradient elution. Background Technology

[0002] Imibetasol propionate cream is a dermatological compound hormone preparation with anti-inflammatory, antibacterial, and antipruritic effects. It is mainly used for skin diseases such as eczema and has proven effective through long-term clinical use in dermatology. Imibetasol propionate cream is composed of more than ten raw and auxiliary materials. The original content determination method (Shanghai Medical Institution Quality Standard SYZ-HF-003-2008) was single-wavelength isocratic elution, which only quantitatively controlled two components, clobetasol propionate and miconazole nitrate, and could not fully reflect its intrinsic quality.

[0003] In existing technologies, such as:

[0004] An Zhenzhen, Yang Yun, Ren Lei, et al. Simultaneous determination of three components in compound diphenhydramine nasal drops by RP-HPLC [J]. China Pharmacy, 2021, 28(18):2573-2575.

[0005] Chen Fangjian, Wang Xiaojun, Lu Songwei, et al. Determination of the contents of ketoconazole, clobetasol propionate and neomycin sulfate in compound ketoconazole cream [J]. Pharmaceutical Service and Research, 2018, 18(3):203-206.

[0006] Sun Ying, Guo Zhaohui, Zhao Jianbang. Determination of chloramphenicol content in chloramphenicol sulfoxide liniment by HPLC [J]. Chinese Pharmacy, 2014, 28(10):1140-1142.

[0007] Xu Yan, Zhong Mingkang, Li Guiqiong, et al. Simultaneous determination of urea and miconazole in Kanglizhi ointment by HPLC [J]. Chinese Journal of Clinical Pharmacy, 2019, 28(2):126-129.

[0008] Liu Yiqing. Determination of the content of neomycin sulfate and miconazole nitrate in imipramine cream by HPLC-ELSD method [J]. Pharmaceutical and Clinical Research, 2017, 25(4):308-310, etc., and there is no comprehensive evaluation of the four main components in imipramine cream. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the main objective of this invention is to provide a method for determining the content of multiple components based on HPLC dual-wavelength gradient elution, thereby establishing an HPLC dual-wavelength gradient elution method that can simultaneously determine the content of multiple components in imidocarbamate cream.

[0010] Another objective of this invention is to provide the application of the above-mentioned multi-component content determination method based on HPLC dual-wavelength gradient elution to simultaneously determine the content of four components—chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate—in imipramine cream, thereby improving quality control standards, optimizing quality criteria, and comprehensively evaluating its overall quality.

[0011] To achieve the above objectives, the present invention adopts the following technical solution:

[0012] This invention provides a method for determining the content of multiple components based on HPLC dual-wavelength gradient elution, comprising the following steps:

[0013] (1) Setting the chromatographic conditions for the liquid chromatography column, including: using octadecylsilane-bonded silica gel as the packing material and acetonitrile-0.5% triethylamine solution as the mobile phase, the gradient elution program of the mobile phase is shown in the table below:

[0014]

[0015] The parameters are set as follows: Flow rate: 1.0 mL·min -1 Column temperature: 25℃; Detection wavelength: 240, 277nm; Injection volume: 20μL;

[0016] (2) Preparation of mixed reference solution: Weigh chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate reference standards accurately, add methanol to prepare a stock solution containing 0.5 mg chloramphenicol, 2 mg diphenhydramine hydrochloride, 0.05 mg clobetasol propionate and 1 mg miconazole nitrate per 1 mL; accurately transfer 1 mL of the above stock solution into a 10 mL volumetric flask, add the mobile phase described in step (1) to dilute to the mark, shake well, and prepare a mixed reference solution containing 50 μg chloramphenicol, 200 μg diphenhydramine hydrochloride, 5 μg clobetasol propionate and 100 μg miconazole nitrate per 1 mL;

[0017] (3) Preparation of test solution: Accurately weigh 0.5 g of imidimidine cream sample, place it in a 50 mL volumetric flask, add methanol, heat and shake in an 80 °C water bath for 10 min to dissolve completely, cool in an ice bath, place to room temperature, dilute with methanol to the mark, shake well, and filter.

[0018] (4) Preparation of negative sample solution: Prepare a sample lacking chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate according to the prescription of imipramine cream, and prepare a negative sample solution according to the preparation method of the test sample solution.

[0019] (5) Accurately measure the mixed reference solution, test solution and negative sample solution, inject them into the liquid chromatograph, determine and record the chromatogram according to the chromatographic conditions, calculate the content by peak area according to the external standard method, and take the average value of two parallel tests.

[0020] (6) Perform linear regression on the peak area y of the solution with the mass concentration x of the solution described in step (5).

[0021] Preferably, the test sample chromatogram has a chromatographic peak at the same retention time as the mixed reference standard chromatogram, the negative sample has no corresponding peak, the theoretical plate number is greater than 5000, and the resolution with adjacent chromatographic peaks is greater than 1.5.

[0022] Preferably, the acetonitrile-0.5% triethylamine solution is prepared by adjusting the pH to 6.40 with phosphoric acid, shaking well, and filtering through a 0.45 μm filter membrane.

[0023] Preferably, 240nm is the detection wavelength for clobetasol propionate, diphenhydramine hydrochloride, and miconazole nitrate, and 277nm is the detection wavelength for chloramphenicol.

[0024] As a preferred embodiment, the regression equation and linear range in step (6) are shown in the table below.

[0025] Element Regression equation r value <![CDATA[Linear range / μg·mL -1 > Chloramphenicol y = 30.69x + 37.99 1 10.06～5003.0 diphenhydramine hydrochloride y = 2.04x + 33.06 0.9999 40.16～2008.0 Clobetasol propionate y = 38.25x + 1.65 1 1.01～50.3 Miconazole Nitrate y = 3.69x + 42.12 0.9997 20.08～1004.0

[0026] This invention also provides the application of the above-mentioned multi-component content determination method based on HPLC dual-wavelength gradient elution to simultaneously determine the content of four components in imipramine cream: chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. This invention establishes a multi-component content determination method using HPLC dual-wavelength gradient elution, which has strong specificity, good linearity, good precision and repeatability, and high recovery rate.

[0029] 2. The HPLC dual-wavelength gradient elution method for determining the content of multiple components established in this invention can simultaneously determine the content of multiple components in imipramine cream, such as simultaneously determining the content of two or more components in chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate in imipramine cream, thereby improving the quality control level of imipramine cream, optimizing quality standards, and comprehensively evaluating its overall quality. Attached Figure Description

[0030] Figure 1 The following are HPLC chromatograms of the reference standard, imidocarbamate cream test sample, and negative sample in the examples; wherein: 1. chloramphenicol; 2. diphenhydramine hydrochloride; 3. clobetasol propionate; 4. miconazole nitrate. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to embodiments, but the present invention is not limited to these embodiments.

[0032] Example 1

[0033] This embodiment provides a method for determining the content of multiple components based on HPLC dual-wavelength gradient elution, the steps of which are as follows:

[0034] (1) Chromatographic conditions: The column was packed with octadecylsilane-bonded silica gel, and the mobile phase was acetonitrile-0.5% triethylamine solution (adjusted to pH 6.40 with phosphoric acid, shaken well, and filtered through a 0.45 μm filter membrane). The gradient elution program is shown in Table 1. Flow rate: 1.0 mL·min -1 Column temperature: 25℃, detection wavelength: 240, 277nm, injection volume: 20μL.

[0035] Table 1: Mobile Phase Gradient Elution Program

[0036]

[0037]

[0038] (2) Solution preparation

[0039] 2.1 Mixed Reference Solution: Weigh appropriate amounts of chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate reference standards, accurately, and dissolve in methanol to prepare a stock solution containing approximately 0.5 mg chloramphenicol, 2 mg diphenhydramine hydrochloride, 0.05 mg clobetasol propionate, and 1 mg miconazole nitrate per 1 mL. Accurately transfer 1 mL of the above stock solution to a 10 mL volumetric flask, dilute to the mark with mobile phase, and mix well to prepare a mixed reference solution containing approximately 50 μg chloramphenicol, 200 μg diphenhydramine hydrochloride, 5 μg clobetasol propionate, and 100 μg miconazole nitrate per 1 mL.

[0040] 2.2 Test solution: Accurately weigh 0.5 g of imidimidine cream sample, place it in a 50 mL volumetric flask, add an appropriate amount of methanol, heat and shake in an 80 °C water bath for 10 min to dissolve completely, cool in an ice bath, let it stand to room temperature, dilute with methanol to the mark, shake well, filter, and the solution is obtained.

[0041] 2.3 Negative Sample Solution: Prepare a sample lacking chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate according to the formulation of imipramine cream, and prepare a negative sample solution according to the preparation method of the test solution.

[0042] (3) Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatograph, record the chromatogram, calculate the content by peak area according to the external standard method, and take the average value of two parallel tests.

[0043] (4) System suitability requirements: The test sample chromatogram has a chromatographic peak at the same retention time as the reference sample chromatogram, while the negative sample does not have a corresponding peak; the theoretical plate number is greater than 5000, and the resolution with adjacent chromatographic peaks is greater than 1.5.

[0044] (5) Selection of detection wavelength: Full-wavelength scanning revealed that the maximum absorption wavelengths of the four reference standards were 277 nm for chloramphenicol, 240 nm for clobetasol propionate, 258 nm for diphenhydramine hydrochloride, and 230 nm for miconazole nitrate, consistent with the description in the Chinese Pharmacopoeia. Using the above wavelengths as detection wavelengths, the results showed that the peak shape of diphenhydramine hydrochloride was better at 240 nm than at 258 nm, and the absorption of miconazole nitrate was close and the peak shape was good at 240 nm and 230 nm. Therefore, 240 nm was selected as the detection wavelength for clobetasol propionate, diphenhydramine hydrochloride, and miconazole nitrate. Chloramphenicol showed interference at 240 nm, but less interference and a higher response at 277 nm. Therefore, 277 nm was selected as the detection wavelength for chloramphenicol.

[0045] (6) Selection of mobile phase: RP-HPLC with octadecylsilane-bonded silica gel as the stationary phase is suitable for the retention of the four components in chloramphenicol cream. Based on previous work, phosphoric acid solution was selected as the mobile phase. At the same time, the addition of a small amount of triethylamine to inhibit the adsorption of nitrogen-containing compounds and silanol groups can significantly improve the tailing of chromatographic peaks and stabilize chromatographic retention behavior. The retention of the four components varies greatly, and the cream matrix components can easily interfere with the determination. Different proportions and compositions of acetonitrile-water mobile phase systems and methanol-water mobile phase systems were tested. It was found that the equal proportion mobile phase system is difficult to balance retention time and specificity. Therefore, this embodiment adopts a gradient elution method, which can effectively separate chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate, while shortening the elution time of excipients in chloramphenicol cream and saving analysis time. The final mobile phase was determined to be acetonitrile-0.5% triethylamine solution (adjusted to pH 6.40 with phosphoric acid), and a gradient elution method was adopted. Under these conditions, the peak shapes of each component were good, the negative control solution did not interfere with the target component, the resolution met the requirements of quantitative analysis, and it can be well used for the content determination of imidimidine cream.

[0046] (7) Selection of extraction method: The sample preparation method directly affects the results of high performance liquid chromatography (HPLC). The effects of different extraction solvents, extraction methods, and extraction times on the content determination of four active ingredients in imipramine cream were tested. The results showed that using methanol as the extraction solvent and heating and shaking in an 80℃ water bath for 10 min resulted in high recovery rate, no interference from negative sample solutions, and was simple and rapid, making it suitable for the content determination of imipramine cream.

[0047] Example of effect 1

[0048] The method described in Example 1, which uses HPLC dual-wavelength gradient elution to simultaneously determine the contents of four components—chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate—in imipramine cream has the following advantages:

[0049] (1) High specificity: Mixed reference solution, test solution, and negative sample solution were tested under the above chromatographic conditions. The results showed that the sample chromatogram had peaks at the same retention times as the reference chromatogram, while the negative sample had no corresponding peaks. This indicates that other components in the sample did not interfere with the determination of chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate. The theoretical plate numbers were all greater than 5000. The peak shapes of the analytes were good, and the resolution with adjacent peaks was greater than 1.5, indicating that the method has good specificity. Figure 1 As shown.

[0050] (2) Good linearity: Accurately pipette each reference solution and determine it according to the chromatographic conditions. Perform linear regression on the peak area y with the reference concentration x to obtain the regression equation, as shown in Table 2.

[0051] Table 2: Regression Equation and Linear Range

[0052] Element Regression equation r value <![CDATA[Linear range / μg·mL -1 > Chloramphenicol y = 30.69x + 37.99 1 10.06～5003.0 diphenhydramine hydrochloride y = 2.04x + 33.06 0.9999 40.16～2008.0 Clobetasol propionate y = 38.25x + 1.65 1 1.01～50.3 Miconazole Nitrate y = 3.69x + 42.12 0.9997 20.08～1004.0

[0053] (3) Good precision: The mixed reference solution was injected 6 times consecutively, and the peak area was measured. The RSD (n=6) of the peak areas of chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate were 1.7%, 0.5%, 0.7% and 0.6%, respectively. This indicates good precision.

[0054] (4) Good repeatability: Six test solutions were prepared in parallel according to the test sample, and the results were determined and calculated according to the chromatographic conditions. The RSDs of chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate were 0.3%, 0.3%, 0.6% and 0.7%, respectively, indicating that the method has good repeatability.

[0055] (5) High recovery rate: Take 6 negative samples, each about 0.5g, and accurately add 5mL of mixed reference solution to prepare a sample solution with a content of 100% of the labeled amount. Determine the content and calculate the recovery rate according to the above chromatographic conditions. The results are shown in Table 3.

[0056] Table 3: Results of the recovery test

[0057] Element Amount added / μg Actual measurement / μg Recovery rate / % Chloramphenicol 251.5 243.9±0.8 97.0±0.3 diphenhydramine hydrochloride 1004.0 986.4±5.5 98.2±0.5 Clobetasol propionate 25.2 24.9±0.2 98.8±1.0 Miconazole Nitrate 502.0 491.4±4.0 97.9±0.8

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining the content of multiple components based on HPLC dual-wavelength gradient elution, characterized in that, Includes the following steps: (1) Setting the chromatographic conditions for the liquid chromatography column, including: using octadecylsilane-bonded silica gel as the packing material and acetonitrile-0.5% triethylamine solution as the mobile phase, the gradient elution program of the mobile phase is shown in the table below: Parameter settings include: Flow rate: 1.0 mL·min -1 Column temperature: 25℃; Detection wavelength: 240, 277nm; Injection volume: 20μL; (2) Preparation of mixed reference solution: Weigh chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate reference standards accurately, add methanol to prepare a stock solution containing 0.5 mg chloramphenicol, 2 mg diphenhydramine hydrochloride, 0.05 mg clobetasol propionate and 1 mg miconazole nitrate per 1 mL; accurately transfer 1 mL of the above stock solution into a 10 mL volumetric flask, add the mobile phase described in step (1) to dilute to the mark, shake well, and prepare a mixed reference solution containing 50 μg chloramphenicol, 200 μg diphenhydramine hydrochloride, 5 μg clobetasol propionate and 100 μg miconazole nitrate per 1 mL; (3) Preparation of test solution: Accurately weigh 0.5 g of imidimidine cream sample, place it in a 50 mL volumetric flask, add methanol, heat and shake in an 80 °C water bath for 10 min to dissolve completely, cool in an ice bath, place to room temperature, dilute with methanol to the mark, shake well, and filter. (4) Preparation of negative sample solution: Prepare a sample lacking chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate and miconazole nitrate according to the prescription of imipramine cream, and prepare a negative sample solution according to the preparation method of the test sample solution described above. (5) Accurately measure the mixed reference solution, test solution and negative sample solution, inject them into the liquid chromatograph, determine and record the chromatogram according to the chromatographic conditions, calculate the content by peak area according to the external standard method, and take the average value of two parallel tests. (6) Perform linear regression on the peak area y of the solution with the mass concentration x of the solution described in step (5).

2. The method for determining the content of multiple components based on HPLC dual-wavelength gradient elution according to claim 1, characterized in that, The test sample chromatogram showed a chromatographic peak at the same retention time as the mixed reference standard chromatogram, while the negative sample chromatogram showed no corresponding peak. The theoretical plate number of all samples was greater than 5000, and the resolution with adjacent chromatographic peaks was greater than 1.

5.

3. The method for determining the content of multiple components based on HPLC dual-wavelength gradient elution according to claim 1, characterized in that, The acetonitrile-0.5% triethylamine solution was adjusted to pH 6.40 with phosphoric acid, shaken well, and filtered through a 0.45 μm filter membrane.

4. The method for determining the content of multiple components based on HPLC dual-wavelength gradient elution according to claim 1, characterized in that, 240nm is the detection wavelength for clobetasol propionate, diphenhydramine hydrochloride, and miconazole nitrate, while 277nm is the detection wavelength for chloramphenicol.

5. The method for determining the content of multiple components based on HPLC dual-wavelength gradient elution according to claim 1, characterized in that, The regression equation and linear range in step (6) are shown in the table below: 。 6. The application of the multi-component content determination method based on HPLC dual-wavelength gradient elution as described in any one of claims 1 to 5 in the simultaneous determination of the contents of four components—chloramphenicol, diphenhydramine hydrochloride, clobetasol propionate, and miconazole nitrate—in imipramine cream.

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