Method for measuring content of ethylparaben in urea vitamin E cream

Abstract

The invention provides a method for determining the content of ethylparaben in urea vitamin E cream. According to the method, HPLC (High Performance Liquid Chromatography) is adopted to detect a test solution, a chromatographic column takes octadecylsilane chemically bonded silica as a filling agent, a solution of acetonitrile and 0.5% acetic acid in a volume ratio of (18: 82)-(30: 70) is taken as a mobile phase, and good separation of an ethylparaben peak, an auxiliary material peak and vitamin E can be realized. The determination method disclosed by the invention is high in accuracy, good in precision, strong in specificity and good in stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of preservative content determination in pharmaceutical preparations, and particularly relates to a method for determining the content of hydroxyphenyl ethyl ester in urea and vitamin E cream. BACKGROUND

[0002] Urea and vitamin E cream is a non-prescription drug for external use, and is clinically used for treating fissures caused by hand and foot fissure and keratotic tinea manuum and tinea pedis. The main components of urea and vitamin E cream (specification: 25 grams: urea 3.75 grams and vitamin E 0.25 gram, approval number: State Drug Standard Code H20073634) are urea and vitamin E, and the auxiliary materials are cetostearyl alcohol, white vaseline, light liquid paraffin, monodiglyceride, white peak wax, polysorbate 80, sodium dodecyl sulfate, hydroxyphenyl ethyl ester, glycerol, three-flower fragrance, and purified water.

[0003] At present, there is no quantitative determination method for the bacteriostatic agent hydroxyphenyl ethyl ester in urea and vitamin E cream. With the increasing requirements of drug quality control in China, it is necessary to establish a quantitative detection method for the bacteriostatic agent hydroxyphenyl ethyl ester in urea and vitamin E cream to perfect the comprehensive evaluation of its quality indicators.

[0004] There are methods for detecting hydroxyphenyl ethyl ester in other drug cream varieties in the prior art, including Biaxin gel, triamcinolone acetonide and econazole cream, danpi phenol ointment, compound dexamethasone acetate cream, compound dexamethasone acetate gel, ofloxacin gel, skin cleansing cream, miconazole nitrate cream, betamethasone urea cream, diclofenac cream, hydroxymethylazoline hydrochloride eye gel, fluconazole moxifloxacin eye gel, Wei Yi cream, diclofenac sodium gel, and the like. However, due to the differences in the main components and auxiliary materials of different creams and gels, the known cream varieties cannot be used for the determination of the content of hydroxyphenyl ethyl ester in urea and vitamin E cream. SUMMARY

[0005] The present application provides a method for determining the content of hydroxyphenyl ethyl ester in urea and vitamin E cream, which adopts HPLC.

[0006] The determination method comprises the following steps: preparing a test sample solution, and detecting the test sample solution by HPLC. The HPLC detection conditions include that the chromatographic column is octadecylsilane bonded silica gel as the filler, and acetonitrile-0.5% acetic acid solution is used as the mobile phase.

[0007] In some embodiments of the present invention, the chromatographic column is either an Agilent Eclipse XDB-C18 or a Waters XBridge Shield RP18. In one embodiment, the chromatographic column used is an Agilent Eclipse XDB-C18, 4.6 mm × 150 mm, 5 μm. In another embodiment, the chromatographic column used is a Waters XBridge Shield RP18, 4.6 mm × 150 mm, 5 μm. The inventors of the present invention have experimentally confirmed that both the Agilent Eclipse XDB-C18 (4.6 mm × 150 mm, 5 μm) and the Waters XBridge Shield RP18 (4.6 mm × 150 mm, 5 μm) can achieve good separation of the ethylparaben peak from other chromatographic peaks (resolution > 1.5), see [link to relevant documentation]. Figure 1 Therefore, columns comparable in performance to the Agilent Eclipse XDB-C18 or Waters XBridge Shield RP18 can be selected.

[0008] According to the present invention, the mobile phase is a solution of acetonitrile and 0.5% acetic acid in a volume ratio of (18:82) to (30:70), for example: a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 30:70, a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 22:78, a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 20:80, and a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 18:82.

[0009] According to the present invention, the chromatographic conditions further include: column temperature: 28–32°C. In one embodiment of the present invention, the column temperature is 30°C.

[0010] According to the present invention, the chromatographic conditions further include a flow rate of 0.9–1.1 mL / min. In one embodiment of the present invention, the flow rate is 1.0 mL / min.

[0011] According to the present invention, the chromatographic conditions further include a detection wavelength of 252 nm to 256 nm. In one embodiment of the present invention, the detection wavelength is 254 nm.

[0012] According to the present invention, the preparation method of the test solution is as follows: take the test sample (urea vitamin E cream), add diluent at a final concentration of 15-25 mg / mL, heat and shake in a water bath at 60-70°C for 9-16 minutes, cool to room temperature, filter and take the filtrate, or centrifuge and take the supernatant as the test solution. The diluent is methanol or ethanol.

[0013] In some embodiments of the present invention, the solution cooled to room temperature is further cooled in an ice-water bath for 30 to 60 minutes before filtration or centrifugation.

[0014] In a preferred embodiment of the invention, the final test solution is obtained by filtration. A nylon filter membrane is preferably used.

[0015] In a preferred embodiment of the present invention, the first 3 to 20% of the volume of the filtered filtrate is discarded, and the remaining 80% to 97% of the volume of the subsequent filtrate is used as the final test solution for determination.

[0016] According to the present invention, the detection method further includes preparing a reference solution, wherein the preparation method is as follows: ethylparaben reference standard is weighed and diluted with a diluent to prepare a solution with a final concentration of 0.01 mg / mL to 0.06 mg / mL. In one embodiment of the present invention, the final concentration of the reference solution is 0.04 mg / mL. The diluent is methanol or ethanol.

[0017] According to the present invention, the detection method further includes calculating the content of ethylparaben in urea vitamin E cream by peak area using the external standard method, that is, the number of milligrams of ethylparaben contained in each gram of urea vitamin E cream, calculated by the following formula:

[0018]

[0019]

[0020] In the formula:

[0021] -C X The concentration of ethylparaben in the test solution is in mg / mL;

[0022] -C R The concentration of ethylparaben in the reference solution is in mg / mL.

[0023] -A X The peak area of ​​ethylparaben in the chromatogram of the test solution;

[0024] -A R The peak area of ​​ethylparaben in the chromatogram of the reference solution;

[0025] -V represents the volume of the test solution, in mL;

[0026] -W represents the sample weight, in grams.

[0027] Those skilled in the art will understand that, regarding the volume of the test sample solution "V" mentioned above, when the test sample is dissolved once by adding solvent during preparation to obtain the final test sample solution, then V (volume of the test sample solution) is the final volume after this addition. For example: if 1g of the test sample is weighed, dissolved in methanol, and then diluted to a volume of 50mL, and this solution is used as the final test sample solution, then V is 50mL. If the test sample is diluted multiple times by adding solvent during preparation to obtain the final test sample solution, then V (volume of the test sample solution) is the total volume of the test sample solution obtained after calculating the dilution factor. For example: if 2g of the test sample is weighed, dissolved in methanol, and then diluted to a volume of 50mL, and 5mL of this solution is diluted with methanol to 10mL, and this diluted solution is used as the final test sample solution, then V is 100mL (i.e., 50mL × (10mL ÷ 5mL)). The same understanding applies to the "volume of the reference solution" mentioned in this application.

[0028] In one embodiment of the present invention, the method includes:

[0029] (1) Chromatographic conditions: The column was packed with octadecylsilane-bonded silica gel; the mobile phase was acetonitrile-0.5% acetic acid solution (volume ratio 20:80); the column temperature was 30℃; the flow rate was 1mL / min; the wavelength was 254nm; and the injection volume was 10μL.

[0030] (2) Sample preparation

[0031] Preparation of reference solution: Accurately weigh 10 mg of ethylparaben reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of diluent, sonicate to dissolve, let it cool to room temperature, dilute to the mark with diluent, and shake well; accurately measure 5 mL into a 25 mL volumetric flask, and dilute to the mark with diluent.

[0032] Preparation of test solution: Take 1g of test sample, place it in a 50mL volumetric flask, add an appropriate amount of diluent, heat and shake in a 65℃ water bath for 10 minutes, cool to room temperature, dilute to the mark with diluent, shake well, place in an ice water bath to cool for more than 30 minutes, take it out, shake well, and filter quickly. Discard 2mL of the initial filtrate and take the subsequent filtrate.

[0033] (3) Determination: Accurately measure 10 μL each of the above reference solution and test solution, inject them into the liquid chromatograph, record the chromatogram, and calculate the ethylparaben content according to the external standard method. The calculation formula is:

[0034]

[0035] In the formula:

[0036] -C XThe concentration of ethylparaben in the test solution is in mg / mL;

[0037] -0.04 represents the concentration of ethylparaben in the reference solution, which is 0.04 mg / mL;

[0038] -A X The peak area of ​​ethylparaben in the chromatogram of the test solution;

[0039] -A R The peak area of ​​ethylparaben in the chromatogram of the reference solution;

[0040] -50 represents the volume of the test solution, which is 50 mL.

[0041] -1 represents the sample weight of 1g.

[0042] In this invention, "and / or" will be considered as a specific disclosure of each of the two specified features or components having or not having the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0043] "Comprising" and "including" have the same meaning and are intended to be open and allow, but do not require, the inclusion of additional elements or steps. When the terms "comprising" or "including" are used herein, the terms "consisting of" and / or "substantially consisting of" are also included and disclosed.

[0044] In this invention, “about” represents a range of ±10% of the corresponding value.

[0045] Those skilled in the art will understand that the volume ratio of acetonitrile to 0.5% acetic acid in the mobile phase is defined as the final total volume of the mobile phase being 100%. Therefore, a mobile phase consisting of a solution with a volume ratio of acetonitrile to 0.5% acetic acid of (18:82) to (30:70) includes various volume ratios within this range, where the sum of the final volume ratios of the two is 100%. Furthermore, those skilled in the art will understand that a volume ratio of acetonitrile to 0.5% acetic acid of (18:82) to (30:70) is equivalent to a volume ratio of acetonitrile to 0.5% acetic acid of (1.8:8.2) to (3:7). This understanding also applies to mobile phases composed of other different solvents in different volume ratios. This is common knowledge in the art. Attached Figure Description

[0046] Figure 1 HPLC chromatograms of Agilent Eclipse XDB-C18 columns (4.6 mm × 150 mm, 5 μm) and Waters XBridgeShield RP18 columns (4.6 mm × 150 mm, 5 μm).

[0047] Figure 2 HPLC chromatogram using methanol as the mobile phase

[0048] Figure 3 HPLC chromatogram using a methanol:water solution with a volume ratio of 9:1 as the mobile phase.

[0049] Figure 4 HPLC chromatogram using a mobile phase composed of 1% glacial acetic acid and methanol in a volume ratio of 3:7.

[0050] Figure 5 HPLC chromatogram using a solution of 1% glacial acetic acid and methanol in a volume ratio of 7:3 as the mobile phase.

[0051] Figure 6 HPLC chromatogram using a mobile phase composed of 1% glacial acetic acid and methanol in a 5:5 volume ratio.

[0052] Figure 7 HPLC chromatogram of a mobile phase consisting of acetonitrile and 0.5% acetic acid in a volume ratio of 3:7.

[0053] Figure 8 HPLC chromatogram of a mobile phase consisting of acetonitrile and 0.5% acetic acid in a volume ratio of 2:8.

[0054] Figure 9 HPLC chromatograms of blank solvent, blank excipient, and test sample

[0055] Figure 10 Linear graph of test concentration versus peak area Detailed Implementation

[0056] The present invention will be further described below with reference to embodiments. It should be noted that the embodiments are not intended to limit the scope of protection of the present invention. Those skilled in the art will understand that any improvements and variations made based on the present invention are within the scope of protection of the present invention. Unless otherwise specified, the compounds, devices, and materials used in these embodiments are commercially available.

[0057] The urea vitamin E cream sample used in the following examples has a batch number of 20220501, which is a near-expiration sample. Ethylparaben will degrade during the storage of urea vitamin E cream. Therefore, the content of ethylparaben in this batch of products has degraded to about 1.4 mg / g (equivalent to 70% of the initial ethylparaben content of 2 mg / g in the product) at the time of testing in this application.

[0058] Example 1:

[0059] 1. Mobile phase

[0060] Various organic solvents and solvent combinations were tested, and it was found that methanol as the mobile phase could not separate the ethylparaben peak from the excipient peak. Figure 2 Using a solution of methanol and water in a volume ratio of 9:1 as the mobile phase, the peak separation between ethylparaben and excipients was <1.5. Figure 3 Using a solution of 1% glacial acetic acid and methanol in a volume ratio of 3:7 as the mobile phase, the separation of ethylparaben and excipient peaks was not ideal, and the peak elution time was too fast. Figure 4 Using a solution of 1% glacial acetic acid and methanol in a volume ratio of 7:3 as the mobile phase, ethylparaben elutes too slowly. Figure 5 Using a solution of 1% glacial acetic acid and methanol in a 5:5 volume ratio as the mobile phase, the resolution between ethylparaben (RT = 15.0961 min) and the excipient peak (RT = 13.7094 min) was 1.52. Figure 6 Using a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 3:7 as the mobile phase, the separation of the ethylparaben peak and the excipient peak was better than that of the system mainly composed of methanol. Figure 7 Using a solution of acetonitrile and 0.5% acetic acid at a volume ratio of 20:80 as the mobile phase, the ethylparaben peak showed good separation from the excipient peak and vitamin E peak. Figure 8 ).

[0061] 2. Preparation method and conditions of the test sample

[0062] To effectively extract the analyte from the sample, various solvents were tested. Recovery tests showed that methanol or ethanol, as diluents, could completely extract the analyte, and the sample solution was clear after filtration. However, using other mobile phases mentioned in point 1 above as diluents failed to effectively extract the analyte, and the sample solution remained turbid after centrifugation or filtration, making it unsuitable for HPLC analysis.

[0063] The preparation method and conditions for the test samples were explored using methanol as a diluent. The screening conditions and experimental results are shown in Table 1.

[0064] Table 1. Preparation methods and conditions of the test samples and their results

[0065]

[0066] Example 2: Method Establishment and Validation

[0067] Information sheet for test sample and reference sample:

[0068]

[0069] 1) Detection method:

[0070] Chromatographic columns: Agilent Eclipse XDB-C18 (4.6mm×150mm, 5μm), Waters XBridgeShield RP18 (4.6mm×150mm, 5μm)

[0071] Column temperature: 30℃;

[0072] Flow rate: 1.0 mL / min;

[0073] Detection wavelength: 254nm;

[0074] Injection volume: 10 μL;

[0075] Mobile phase: Acetonitrile-0.5% acetic acid solution (20:80).

[0076] 2) Sample preparation

[0077] Reference solution: Accurately weigh 10 mg of ethylparaben reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, allow to room temperature, dilute to the mark with methanol, and shake well; accurately measure 5 mL of this solution into a 25 mL volumetric flask, dilute to the mark with methanol, and the solution is ready. It contains 0.04 mg / mL of ethylparaben. Two parallel aliquots are designated RS-1 and RS-2.

[0078] Test solution: Take about 1g of the test sample and place it in a 50mL volumetric flask. Add an appropriate amount of methanol, heat and shake in a 65℃ water bath for 10 minutes, cool to room temperature, dilute with methanol to the mark, shake well, place in an ice-water bath to cool for more than 30 minutes, remove, shake well, and filter quickly. Discard 2mL of the initial filtrate and take the subsequent filtrate.

[0079] 3) System suitability test

[0080] For continuous injection of 5 doses of reference solution RS-1, the relative standard deviation (RSD) of the peak area of ​​5 doses of ethylparaben should not exceed 2.0%.

[0081] 4) Determination: Accurately measure 10 μL each of the test solution and the reference solution, inject them into the chromatograph, and record the chromatogram.

[0082] 5) Calculation

[0083] The content of ethylparaben in urea vitamin E cream was calculated based on peak area using the external standard method.

[0084]

[0085] In the formula:

[0086] -C X The concentration of ethylparaben in the test solution is in mg / mL;

[0087] -C R The concentration of ethylparaben in the reference solution is in mg / mL.

[0088] -A X The peak area of ​​ethylparaben in the chromatogram of the test solution;

[0089] -A R The peak area of ​​ethylparaben in the chromatogram of the reference solution;

[0090] -V represents the volume of the test solution, in mL;

[0091] -W represents the sample weight, in grams.

[0092] 1. Accuracy

[0093] Preparation of reference solution: Accurately weigh 10 mg of ethylparaben reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, let it cool to room temperature, dilute to the mark with methanol, and shake well; accurately measure 5 mL into a 25 mL volumetric flask, dilute to the mark with methanol, and the solution is ready.

[0094] Accuracy stock solution: Accurately weigh approximately 25 mg of ethylparaben, place it in a 25 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, dilute to the mark with methanol, and shake well. (Ethylparaben concentration is 1 mg / mL)

[0095] Recovery rate 1 (25%): Weigh 1 g of blank excipient and place it in a 50 mL volumetric flask. Add 0.5 mL of accuracy stock solution and 15 mL of methanol. Heat and shake in a 65 °C water bath for 10 minutes. Cool to room temperature, dilute to the mark with methanol, shake well, and place in an ice-water bath for at least 30 minutes. Remove, shake well, and filter quickly. Discard 2 mL of the initial filtrate and collect the subsequent filtrate. (3 replicates) (Ethylparaben is 0.01 mg / mL)

[0096] Recovery rate 2 (100%): Weigh 1 g of blank excipient and place it in a 50 mL volumetric flask. Add 2 mL of accuracy stock solution and 15 mL of methanol. Heat and shake in a 65°C water bath for 10 minutes. Cool to room temperature, dilute to the mark with methanol, shake well, and cool in an ice-water bath for at least 30 minutes. Remove the flask, discard 2 mL of the initial filtrate, and collect the subsequent filtrate. (Trial replicates) (Ethylparaben is 0.04 mg / mL)

[0097] Recovery rate 3 (150%): Weigh 1 g of blank excipient and place it in a 50 mL volumetric flask. Add 3 mL of accuracy stock solution and 15 mL of methanol. Heat and shake in a 65 °C water bath for 10 minutes. Cool to room temperature, dilute to the mark with methanol, shake well, and place in an ice-water bath for at least 30 minutes. Remove the flask, discard 2 mL of the initial filtrate, and collect the subsequent filtrate. (3 replicates) (Ethylparaben is 0.06 mg / mL)

[0098] Accurately measure 10 μL each of the above reference solution and recovery rate 1-3 solutions, inject them into the liquid chromatograph, record the peak area, calculate the recovery rate of ethylparaben at each concentration based on the theoretical amount added and the actual amount measured, and calculate the relative standard deviation (RSD) of the 9 recovery rates to examine the accuracy of the method.

[0099]

[0100] Accuracy verification results:

[0101]

[0102] The recoveries of ethylparaben at 25%, 100%, and 150% concentrations ranged from 101.8% to 103.4%, with an average recovery of 102.3%. The relative standard deviation (RSD) for the nine recovery data was 1.0%. The method has good accuracy.

[0103] 2. Precision

[0104] 2.1 Repeatability

[0105] Reference solution: Prepared in the same way as under “2) Sample preparation”.

[0106] Test solution: Prepare 6 parallel solutions using the test sample, in the same manner as under "2) Sample Preparation".

[0107] Accurately measure 10 μL each of the above-mentioned reference standard and test solution, inject them into the liquid chromatograph, record the chromatograms, and have one tester perform the tests under identical conditions. Calculate the content and the relative standard deviation of the contents of the six test samples using the external standard method.

[0108] The results showed that the average content of ethylparaben in the 6 test solutions was 1.34 mg / g, with an RSD of 0.5%, which was less than 2%. The method had good repeatability. The results are detailed in the table below.

[0109] 1 2 3 4 5 6 Average (mg / g) RSD (%) Content (mg / g) 1.34 1.34 1.34 1.34 1.35 1.34 1.34 0.5

[0110] 2.2 Intermediate Precision

[0111] At different times, different testers prepared the reference solution and test solution according to the "2.1 Repeatability" section. 10 μL of each of the above reference solution and test solution was accurately measured and injected into the liquid chromatograph. The chromatograms were recorded. The content and the relative standard deviation of the content of 6 test samples were calculated by the external standard method. At the same time, the RSD of the 12 contents of the two testers (the current test and the repeatability test) were compared to examine the intermediate precision of this method.

[0112] The results showed that: (1) the average content of ethylparaben in the six test solutions of the two experimenters was 1.34 mg / g and 1.31 mg / g, respectively; the RSD was 0.5% and 1.3%, respectively, both less than 2.0%.

[0113] (2) The mean content of ethylparaben in the 12 test solutions was 1.33 mg / g, the RSD was 1.5%, which is less than 2.0%.

[0114] The intermediate precision of this method is good. See the table below for details.

[0115]

[0116] 3. Exclusivity

[0117] Blank interference

[0118] Blank solvent: methanol.

[0119] Preparation of blank excipient solution: Weigh 1g of blank excipient and place it in a 50mL volumetric flask. Add 15mL of methanol and heat in a 65℃ water bath for 10 minutes. Cool to room temperature and dilute to the mark with methanol. Place in an ice-water bath to cool for more than 30 minutes. Remove and filter quickly. Discard 2mL of the initial filtrate and collect the subsequent filtrate to obtain the blank excipient solution.

[0120] Preparation of the reference solution: Accurately weigh 10 mg of ethylparaben reference standard, place it in a 50 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, allow to room temperature, dilute to the mark with methanol, and shake well; accurately measure 5 mL into a 25 mL volumetric flask, dilute to the mark with methanol, and the solution is ready. (Contains 0.04 mg / mL ethylparaben, 1 part)

[0121] Preparation of the test solution: Take about 1g of the test sample and place it in a 50mL volumetric flask. Add an appropriate amount of methanol, heat and shake in a 65℃ water bath for 10 minutes, cool to room temperature, dilute to the mark with methanol, shake well, and place in an ice-water bath to cool for at least 30 minutes. Remove, shake well, and filter quickly. Discard 2mL of the initial filtrate and collect the subsequent filtrate. (1 serving)

[0122] Accurately measure 10 μL each of blank solvent, blank excipient solution, reference solution and test solution, inject them into the liquid chromatograph and record the chromatograms.

[0123] The results showed that the blank (solvent, excipient) had no interference with the determination of the main peak. (See attached table). Figure 9 .

[0124] 4. Linear

[0125] Linear stock solution: Weigh approximately 25 mg of ethylparaben reference standard into a 50 mL volumetric flask, add an appropriate amount of methanol, sonicate to dissolve, allow to room temperature, and dilute to the mark with methanol. (Ethylparaben is 0.5 mg / mL)

[0126] Linear Solution 1 (25%): Measure 1 mL of the linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and mix well. (Ethylparaben is 0.01 mg / mL)

[0127] Linear Solution 2 (50%): Measure 2 mL of the linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and mix well. (Ethylparaben is 0.02 mg / mL)

[0128] Linear solution 3 (75%): Measure 3 mL of the linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and mix well. (Ethylparaben is 0.03 mg / mL)

[0129] Linear solution 4 (100%): Measure 4 mL of the linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and mix well. (Ethylparaben is 0.04 mg / mL)

[0130] Linear solution 5 (125%): Measure 5 mL of the linear stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, and shake well. (Ethylparaben is 0.05 mg / mL)

[0131] Linear solution 6 (150%): Measure 6 mL of the linear stock solution and place it in a 50 mL volumetric flask. Dilute to the mark with methanol and mix well. (Ethylparaben is 0.06 mg / mL)

[0132] Accurately measure 10 μL of each of the above linear solutions 1–6 and inject them into the liquid chromatograph, recording the chromatograms. Perform linear regression with concentration as the abscissa (X) and peak area as the ordinate (Y) to obtain the linear regression equation. Calculate the correlation coefficient (r) of the regression line, the RSD% of the response factor, and the ratio of the Y-intercept to the 100% response value.

[0133] The results showed that, within the concentration range of 0.01006 mg / mL to 0.06038 mg / mL, the linear regression equation for ethylparaben was y = 5.2356 × 10⁻⁶. 4x + 3.7102, the correlation regression coefficient r is 0.9999, the Y-intercept is 0.14% of the 100% response value (less than 2%), and the relative standard deviation (RSD) of the response factor (A / C) is 0.3% (less than 2%). Within the above linear range, this method exhibits good linearity. See the table below for detailed results. Figure 10 .

[0134]

[0135] 5. Durability

[0136] 5.1 Solution stability

[0137] Reference solution and test solution: Prepare as described under “3. Specificity”.

[0138] Take the reference solution and the test solution, let them stand at room temperature for 24 hours, and accurately measure 10 μL at different time points after preparation, inject them into the liquid chromatograph, and record the chromatograms.

[0139] The results showed that the RSDs of the peak areas at each time point and at 0 hour for both the test solution and the reference solution were 0.5% and 0.3%, respectively, both less than 2%, indicating good solution stability. Detailed results are shown in the table below:

[0140] Solution stability results

[0141]

[0142] 5.2 Chromatographic condition robustness

[0143] The effects of variations in chromatographic column, column temperature (±2℃), detection wavelength (±2nm), mobile phase organic phase (±2%), and flow rate (±0.1mL / min) were investigated. The system suitability under each condition should meet the requirements, and the RSD of the content determined under each condition compared to the content determined under the initial conditions should not exceed 2.0%.

[0144]

[0145] (1) Blank solvent: methanol

[0146] (2) Reference solution and test solution: Same as under “2) Sample preparation”, prepare 2 copies of each.

[0147] The results show that (1) the system suitability meets the requirements under all conditions; and (2) the RSD of the ethylparaben content measured under all conditions is 1.4%, which is less than 2.0%. Detailed results are shown in the table below:

[0148]

[0149] illustrate:

[0150] * indicates the conditions in “1) Detection Method” above, namely: chromatographic column: Agilent Eclipse XDB-C18 (4.6mm×150mm, 5μm), Waters XBridge Shield RP18 (4.6mm×150mm, 5μm); column temperature: 30℃; flow rate: 1.0mL / min; detection wavelength: 254nm; injection volume: 10μL; mobile phase: acetonitrile-0.5% acetic acid solution (20:80).

[0151] a: The other test conditions in the table above are determined by keeping all other factors in the conditions unchanged, only changing the corresponding factors in the list to the conditions in the corresponding list.

[0152] 5.3 Membrane Adsorption

[0153] (1) Test solution

[0154] Test solution to be filtered: The test solution before filtration (1 part) under "3. Specificity".

[0155] Unfiltered test solution: Take an appropriate amount of the test solution to be filtered, centrifuge at 10000RPM for 10 min at 2-10℃, and take the supernatant.

[0156] Filter membrane adsorption of test solution: Take an appropriate amount of the test solution to be filtered, filter it through a nylon membrane and a PES (polyethersulfone filter membrane), and take 0 mL, 2 mL, 4 mL, 6 mL and 8 mL of the subsequent filtrate respectively to obtain the final product.

[0157] (2) Reference solution

[0158] Reference solution: Use the reference solution (1 part) under "3. Specificity".

[0159] Membrane adsorption of reference solution: Take an appropriate amount of reference solution and filter it through a nylon membrane and a PES (polyethersulfone membrane). Take 0 mL, 2 mL, 4 mL, 6 mL and 8 mL of the subsequent filtrate and discard the initial filtrate to obtain the final solution.

[0160] Accurately measure 10 μL of each of the above-mentioned types of test solutions and reference solutions, inject them into the liquid chromatograph, record the chromatograms, and examine the changing trend of the peak area of ​​the test sample adsorbed by the filter membrane after discarding different volumes of initial filtrate. Take the peak area of ​​the unfiltered test solution as 0 adsorption, and calculate the adsorption amount of other solutions after discarding different volumes.

[0161]

[0162] In the formula:

[0163] A nThe peak areas of the test solution and the reference solution adsorbed by the filter membranes with different volumes of initial filtrate were determined.

[0164] A0 represents the peak area of ​​the unfiltered test solution / reference solution.

[0165] The results showed that the adsorption rates of both the reference solution and the test solution using PES and nylon membranes were no greater than 2.0% (according to the "Standard Operating Procedures for Drug Inspection in China", when the recovery rate of the filter membrane adsorption is between 98% and 102%, that is, when the adsorption rate and the introduced interference do not exceed 2%, the adsorption effect of the filter membrane can be ignored). The nylon membrane was superior to the PES membrane. Therefore, the nylon membrane was selected for sample filtration in the experiment. The results are detailed in the table below.

[0166] Adsorption results of reference solution filter membrane

[0167]

[0168] Adsorption results of the test solution through the filter membrane

[0169]

[0170] 6. Sample testing

[0171] Three batches of the test sample were taken and tested according to the aforementioned content determination method. The results are detailed in the table below.

[0172] Sample test results

[0173] Batch No. Content (mg / g) 20231123-1 2.01 20231122-1 1.99 20231121-1 2.00

[0174] The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for determining the content of hydroxyphenyl ethyl ester in a urea vitamin E cream, characterized in that, Prepare the test sample solution, and detect the test sample solution by HPLC, and the HPLC detection conditions include: the chromatographic column is octadecylsilane bonded silica gel as the filler, and acetonitrile-0.5% acetic acid solution is used as the mobile phase. Preferably, the mobile phase is a solution of acetonitrile and 0.5% acetic acid in a volume ratio of (18:82) to (30:70); Preferably, the mobile phase is a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 30:70, or a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 22:78, or a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 20:80, or a solution of acetonitrile and 0.5% acetic acid in a volume ratio of 18:

82.

2. The assay method of claim 1, wherein The chromatographic column is Agilent Eclipse XDB-C18 or Waters XBridge Shield RP18.

3. The assay method according to claim 1 or 2, characterized in that, The chromatographic conditions further include: column temperature: 28-32°C; flow rate: 0.9-1.1 mL / min; detection wavelength: 252-256 nm; Preferably, the column temperature is 30°C, the flow rate is 1.0 mL / min, and the detection wavelength is 254 nm.

4. Assay according to any one of claims 1 to 3, characterized in that The preparation method of the test sample solution is as follows: the test sample urea and vitamin E cream is taken, a diluent is added to obtain a final concentration of 15-25 mg / mL, heated in a 60-70°C water bath and shaken for 9-16 minutes, cooled to room temperature, filtered, and the filtrate or the supernatant after centrifugation is taken as the test sample solution; the diluent is methanol or ethanol; Preferably, the cooled solution is further cooled in an ice water bath for 30-60 minutes, and then filtered or centrifuged; Preferably, a nylon filter membrane is used to obtain the final test sample solution by filtration; Preferably, after filtration, the initial filtrate of 3-20% of the volume is discarded, and the subsequent filtrate of 80-97% of the volume is used as the final test sample solution for determination.

5. Assay according to any one of claims 1 to 4, characterized in that A control sample solution is also prepared: hydroxyphenyl ethyl ester control sample is weighed, and a diluent is used to prepare a solution with a final concentration of 0.01-0.06 mg / mL, and the diluent is methanol or ethanol; preferably, the final concentration of the control sample solution is 0.04 mg / mL.

6. Assay according to any one of claims 1 to 5, characterized in that The content of hydroxyphenyl ethyl ester in the urea and vitamin E cream is calculated by peak area according to the external standard method, and the calculation formula is: where C X is the concentration of hydroxyphenethyl ester in the test solution, mg / mL; C R is the concentration of hydroxyphenethyl ester in the control solution, mg / mL; A X is the peak area of the hydroxyphenethyl ester peak in the chromatogram of the test solution; A R is the peak area of the hydroxyphenethyl ester peak in the chromatogram of the control solution; V is the volume of the test solution, mL; and W is the sample weight of the test, g.

7. The assay method of claim 1 wherein, including: (1) chromatographic conditions: chromatographic column: octadecylsilane bonded silica gel as the filler; mobile phase: acetonitrile-0.5% acetic acid solution in a volume ratio of 20:80; column temperature: 30°C; flow rate: 1 mL / min; wavelength: 254 nm; injection volume: 10 μL; (2) sample preparation, Preparation of the control sample solution: accurately weigh 10 mg of hydroxyphenyl ethyl ester control sample into a 50 mL volumetric flask, add an appropriate amount of diluent, ultrasonically dissolve, and let it stand to room temperature, dilute to the mark with the diluent, and shake well; accurately take 5 mL into a 25 mL volumetric flask, and dilute to the mark with the diluent; Preparation of test solution: 1 g of the test sample was placed in a 50 mL flask, and an appropriate amount of diluent was added. The mixture was heated in a 65 °C water bath for 10 min, and then cooled to room temperature. The solution was diluted to the mark with diluent, shaken well, and cooled in an ice water bath for more than 30 min. The solution was filtered, and the first 2 mL of the filtrate was discarded. The remaining filtrate was collected. (3) Determination: 10 μL of the above-mentioned control solution and test solution was precisely measured, respectively, and injected into the liquid chromatograph. The chromatogram was recorded, and the content of hydroxyphenyl ethyl ester was calculated by external standard method. The calculation formula was: In the formula: -C X Concentration of hydroxyphenethyl ester in the test solution, mg / mL; -0.04 is the concentration of hydroxyphenyl ethyl ester in the control solution, 0.04 mg / mL; -A X the peak area of the peak of hydroxyphenyl ethyl ester in the chromatogram of the test solution; -A R The peak area of the peak of hydroxyphenyl ethyl ester in the chromatogram of the control solution; -50 is the volume of the test solution, 50 mL; -1 is the sample weight of the test sample, 1 g.

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