Method for determining related substances in loxoprofen sodium patch

Abstract

The invention discloses a method for determining related substances in a loxoprofen sodium patch in the technical field of drug detection, and aims to solve the technical blank existing in determination of related substances in a loxoprofen sodium patch in the prior art. The method comprises the following steps: respectively weighing a loxoprofen sodium reference substance and an impurity reference substance, and adding a solvent for dilution to prepare a reference substance solution; taking a loxoprofen sodium patch, stripping a protective layer, adding tetrahydrofuran and methanol, shaking to separate the paste from a backing layer, adding water, shaking again, adding a solvent for dilution, uniformly mixing, centrifuging, and taking supernate to obtain a test solution; injecting the prepared reference substance solution and the test solution into a high performance liquid chromatograph for determination to obtain peak areas of the reference substance solution and the test solution; and calculating the content of the related substances in the loxoprofen sodium patch by adopting an HPLC correction factor method according to the peak areas of the reference solution and the test solution. The method is suitable for measuring the content of the related substances in the loxoprofen sodium patch.

Description

Technical Field

[0001] This invention relates to the field of drug detection technology, and to a method for determining related substances in loxoprofen sodium patches, particularly a method for determining related substances in loxoprofen sodium patches based on HPLC correction factor method. Background Technology

[0002] Loxoprofen sodium patch is a topical heat-sealing patch formulation of a nonsteroidal anti-inflammatory drug (NSAID), primarily used for local anti-inflammatory and analgesic purposes. It is suitable for relieving mild to moderate pain in muscles, joints, or soft tissues (such as muscle strain, arthritis, sprains, etc.). It works by inhibiting prostaglandin synthesis, reducing inflammatory responses and pain signal transmission, and has a rapid onset of action. Topical application also reduces the risk of systemic side effects.

[0003] Currently, loxoprofen sodium patches are not included in the pharmacopoeias of China (Chp), England (EP), the United States (USP), and Japan (JP). Only the imported registration standard (JX20210065) introduced by Lead Chemical Co., Ltd. includes methods for detecting related substances in loxoprofen sodium patches. As a hot-melt patch product, its complex colloidal excipient matrix is ​​highly susceptible to interference during the detection of related substances, posing a challenge to the accuracy of impurity detection.

[0004] Chinese patent CN117805265A discloses an analytical method for related substances in hot-melt patches. In the preparation of the test sample solution, a suitable extraction solvent is selected to remove some of the colloids to reduce the interference of excipient blanks during the detection process. This method has good specificity and robustness when analyzing multiple impurities. However, whether the impurity results detected by this method can truly reflect the impurity level in the test sample has not been evaluated.

[0005] Chinese patent CN116500159B discloses a method for detecting related substances in loxoprofen sodium oral solution, and Chinese patent CN117805265A discloses an analytical method for genotoxic impurities in loxoprofen sodium and its preparations. Due to the large differences in excipient matrix between different dosage forms and the large differences in the structure and physicochemical properties of different impurities, the analytical methods are not applicable to patch products. Currently, there are no patent literature reports on the determination of related substances in loxoprofen sodium patches using the correction factor method. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining related substances in loxoprofen sodium patches, filling the technical gap in the prior art for determining related substances in loxoprofen sodium patches.

[0007] To achieve the above objectives, the present invention is implemented using the following technical solution:

[0008] This invention provides a method for determining related substances in loxoprofen sodium patches, comprising the following steps:

[0009] Weigh out loxoprofen sodium reference standard and impurity reference standard separately, and dilute them with solvent to prepare reference standard solution;

[0010] Take loxoprofen sodium patch, peel off the protective layer, add tetrahydrofuran and methanol, shake to separate the patch from the backing layer, add water, shake again, dilute with solvent, mix well, centrifuge, and take the supernatant to obtain the test solution.

[0011] The prepared reference solution and test solution were injected into a high-performance liquid chromatograph for determination, and the peak areas of the reference solution and test solution were obtained.

[0012] The content of related substances in loxoprofen sodium patch was calculated using the HPLC correction factor method based on the peak areas of the reference solution and the test solution.

[0013] The linear solution used in the HPLC correction factor method was prepared by mimicking the preparation method of the test sample solution using a loxoprofen sodium blank patch.

[0014] In the above technical solution, the correction factor obtained by simulating the preparation method of the test sample solution using a loxoprofen sodium blank patch eliminates the blank background interference caused by the excipient matrix in sample detection, enabling a true assessment of the impurity level in the test sample and achieving accurate quantification of related substances in the loxoprofen sodium patch. This method allows for qualitative and quantitative analysis of multiple impurities in the test sample, improving detection efficiency. The method is highly sensitive, specific, accurate, and reliable, providing technical support for the control and quality monitoring of the loxoprofen sodium patch production process and improving medication safety.

[0015] Furthermore, the determination of the correction factor in the HPLC correction factor method includes the following steps:

[0016] Preparation of linear solutions;

[0017] A series of linear solutions with varying concentration gradients were prepared and injected into a high-performance liquid chromatograph to determine the peak areas of the linear solutions with varying concentration gradients.

[0018] Linear regression was performed with concentration on the x-axis and the corresponding peak area on the y-axis.

[0019] The correction factor was calculated using the linear slope of loxoprofen sodium reference standard and impurity reference standard;

[0020] The preparation steps of the linear solution are as follows:

[0021] Take a blank loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add tetrahydrofuran, then add loxoprofen sodium reference standard, impurity reference standard and methanol respectively, shake to separate the patch from the backing layer, add water, shake again, mix evenly, dilute with solvent, shake well, centrifuge, and take the supernatant to obtain the linear solution.

[0022] In the above technical solution, the linear solution is prepared by imitating the preparation method of the test solution using the blank patch. That is, the blank patch, loxoprofen sodium reference standard, and impurity reference standard are put into a stoppered conical flask and prepared using the test solution preparation method. Compared with directly using loxoprofen sodium reference standard and impurity reference standard to calculate the correction factor for linear determination, the correction factor calculated by the above technical solution can eliminate the background influence in the loxoprofen sodium patch and more accurately determine the content of related substances.

[0023] Furthermore, the conditions for both shaking and re-shaking are shaking at 200-250 rpm for 30-35 minutes;

[0024] The centrifugation conditions are 10000-12000 rpm for 5-10 minutes.

[0025] Furthermore, the series of concentration gradients for determining the correction factors of the impurities are the limit of quantitation concentration and 50%, 75%, 100%, 125%, and 150% of the limit concentration, respectively.

[0026] The correction factor for loxoprofen sodium was determined using a series of concentration gradients at the limit of quantitation concentration and at 50%, 75%, 100%, 125%, and 180% of the limit concentration.

[0027] The limits of quantitation (LOQs) for both loxoprofen sodium and impurities are 47.72–50.08 ng / ml.

[0028] The impurities include impurity D-1, impurity D-2 and impurity D-3;

[0029] The limit concentrations of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 are 1 μg / ml, 0.5 μg / ml, 0.4 μg / ml, and 1.2 μg / ml, respectively.

[0030] In the above technical solution, the response absorption of loxoprofen sodium is different from that of impurities D-1, D-2 and D-3. At the same concentration as the impurities, the response value of the upper limit of the concentration of a certain impurity may exceed the response value of the upper limit of the concentration of loxoprofen sodium. Therefore, the linear range of loxoprofen sodium will be set wider than that of the impurities.

[0031] Furthermore, the impurity D-1 is 2-[p-(5-carboxy-2-oxopentyl)phenyl]propionic acid;

[0032] Impurity D-2 is 2-{4-[(1-hydroxy-2-oxocyclopentyl)methyl]phenyl}propionic acid;

[0033] Impurity D-3 is ethyl 2-isopropyl-5-methylcyclohexyl-2-{4-[2-oxocyclopentyl)methyl]phenyl}propionate.

[0034] Furthermore, the solvent of the reference solution is 0.01% phosphoric acid:acetonitrile = 65:35.

[0035] Furthermore, the concentration of the four reference standards in the reference solution is 1 μg / ml.

[0036] Furthermore, the concentration of the test solution is 0.1 mg / ml.

[0037] Furthermore, the chromatographic conditions of the high-performance liquid chromatograph are as follows: YMC-Pack Pro C18 RS column, 100×4.6mm, 3μm; flow rate, 2.0ml / min; detection wavelength, 222nm; column temperature, 45℃; injection volume, 20μl; gradient elution using a mixture of mobile phases A and B; mobile phase A is 0.01% phosphoric acid:acetonitrile = 65:35, and mobile phase B is acetonitrile.

[0038] Furthermore, the elution program of the high performance liquid chromatograph is 0-1 minute, 0%-19%B; 1-8 minutes, 19%-72%B; 8-21 minutes, 72%-72%B; 21-23 minutes, 72%-0%B; 23-30 minutes, 0%-0%B.

[0039] Compared with the prior art, the beneficial effects achieved by the present invention are as follows:

[0040] By utilizing the correction factor obtained from the preparation method of the loxoprofen sodium blank patch to mimic the test sample solution, the blank background interference caused by the excipient matrix in sample detection is eliminated. This method can accurately assess the impurity level in the test sample and achieve accurate quantification of related substances in the loxoprofen sodium patch. Multiple impurities in the test sample can be qualitatively and quantitatively analyzed using this method, improving detection efficiency. The method is highly sensitive, specific, accurate, and reliable, providing technical support for the control and quality monitoring of the loxoprofen sodium patch production process and improving medication safety. Attached Figure Description

[0041] To more clearly illustrate the technical solutions in the embodiments of this disclosure or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this disclosure. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0042] Figure 1 This is a specific chromatogram overlay of Embodiment 2 of the present invention;

[0043] Figure 2 This is an undamaged overlay image of the forced degradation test in Example 2 of the present invention;

[0044] Figure 3 This is an overlay diagram of acid damage from the forced degradation test in Example 2 of the present invention;

[0045] Figure 4 This is a superimposed diagram of the alkali damage in the forced degradation test of Example 2 of the present invention;

[0046] Figure 5 This is a superimposed diagram of the oxidative damage from the forced degradation test in Example 2 of the present invention;

[0047] Figure 6 This is a superimposed diagram of the high-temperature damage during the forced degradation test of Embodiment 2 of the present invention;

[0048] Figure 7 This is a superimposed image of the light damage during the forced degradation test of Embodiment 2 of the present invention;

[0049] Figure 8 This is a linear graph of the linearity and range detection method 1 in Embodiment 2 of the present invention;

[0050] Figure 9 This is a linear graph of the linearity and range detection method 2 of Embodiment 2 of the present invention;

[0051] Figure 10 This is a typical chromatogram overlay of the blank solvent, reference solution, and test solution in Example 3 of the present invention. Detailed Implementation

[0052] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.

[0053] The experimental materials and instruments used in the following examples include the following:

[0054] 1. Medicines and reagents

[0055] Impurity D-1 reference standard (HST), Impurity D-2 reference standard (HST), Impurity D-3 reference standard (HST), Loxoprofen sodium reference standard (dihydrate, China National Institutes for Food and Drug Control), Loxoprofen sodium patch (Changsha Jingyi Pharmaceutical Technology Co., Ltd.), Blank patch - API-free (Changsha Jingyi Pharmaceutical Technology Co., Ltd., API is the active pharmaceutical ingredient), Methanol (HPLC grade, ACS), Phosphoric acid (AR grade, Sinopharm Chemical Reagent Co., Ltd.), Tetrahydrofuran (HPLC grade, ACS), Acetonitrile (HPLC grade, ACS).

[0056] Among them, impurity D-1 is 2-[p-(5-carboxy-2-oxopentyl)phenyl]propionic acid; impurity D-2 is 2-{4-[(1-hydroxy-2-oxocyclopentyl)methyl]phenyl}propionic acid; and impurity D-3 is ethyl 2-isopropyl-5-methylcyclohexyl-2-{4-[2-oxocyclopentyl)methyl]phenyl}propionate. The structural formulas of the impurities are shown below:

[0057] Impurity D-1:

[0058] Impurity D-2:

[0059] Impurity D-3:

[0060] 2. Instruments

[0061] MCA-6.6S-2CCN-M Electronic Balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0062] SQP / SECURA225D-1CN Electronic Balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0063] BCE95I-1OCN Electronic Balance: Sartorius Scientific Instruments (Beijing) Co., Ltd.

[0064] FA1004 Electronic Balance: Shanghai Sunny Hengping Scientific Instruments Co., Ltd.

[0065] 1260 Quat Pump / 1260 DAD WR High Performance Liquid Chromatograph: Agilent

[0066] LC 129 High Performance Liquid Chromatograph: Waters

[0067] SHA-B Oscillator: Changzhou Aohua Instrument Co., Ltd.

[0068] TGL 16 Centrifuge: Changsha Yingtai Instrument Co., Ltd.

[0069] Example 1

[0070] This embodiment provides a method for determining related substances in loxoprofen sodium patches.

[0071] 1.1 High Performance Liquid Chromatography Conditions

[0072] The chromatographic column was a YMC-Pack Pro C18 RS, 100×4.6mm, 3μm; the flow rate was 2.0ml / min; the detection wavelength was 222nm; the column temperature was 45℃; the injection volume was 20μl; gradient elution was performed using a mixture of mobile phases A and B; mobile phase A was 0.01% phosphoric acid:acetonitrile (65:35), and mobile phase B was acetonitrile.

[0073] The gradient elution program is as follows: 0-1 minute, 0%-19%B; 1-8 minutes, 19%-72%B; 8-21 minutes, 72%-72%B; 21-23 minutes, 72%-0%B; 23-30 minutes, 0%-0%B.

[0074] 1.2 Determination of related substances in loxoprofen sodium patches

[0075] 1.2.1 Solution Preparation

[0076] Preparation of reference solution: Weigh appropriate amounts of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately weigh them, and dissolve and dilute them with 0.01% phosphoric acid:acetonitrile = 65:35 solvent to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0077] Preparation of test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 min to separate the patch from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 min, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, mix well, centrifuge at 10000 rpm for 5 min, and take the supernatant. The test solution contains approximately 0.1 mg / ml of loxoprofen sodium.

[0078] The conditions for shaking and re-shaking can be set to any shaking speed and any time within the range of 200-250 rpm shaking for 30-35 minutes;

[0079] The centrifugation conditions can be set to any centrifugation speed and any time within the range of 10000-12000 rpm for 5-10 minutes.

[0080] 1.2.2 Inject the reference solution and the test solution into the high-performance liquid chromatograph and record the chromatograms.

[0081] 1.3 Determination of Correction Factor

[0082] 1.3.1 Determination of Limit of Quantitation and Limit of Detection

[0083] Take one blank patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran, add appropriate amounts of loxoprofen sodium reference standard and impurity reference standard, add 10 ml of methanol, shake at 200 rpm for 30 minutes, add 40 ml of water, shake at 200 rpm for 30 minutes, mix well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant to obtain the test solution.

[0084] When the S / N ≥ 10 and the solution concentration is not greater than the neglect limit (the neglect limit is 0.05%, i.e., the solution concentration is equivalent to 0.05% or less of the concentration of the test sample solution), it is used as the limit of quantitation solution; when the S / N ≥ 3, it is used as the limit of detection solution. In this embodiment, the limit of detection solution is diluted 2 times based on the limit of quantitation solution. Accurately measure the above solution, inject it into the liquid chromatograph, and record the chromatogram.

[0085] The results of the determination of the limit of quantitation and limit of detection are shown in Table 1 and Table 2.

[0086] Table 1: Results of Limit of Quantitation and Limit of Detection

[0087]

[0088] Table 2: Repeatability results at the limit of quantitation

[0089]

[0090] The results above show that in the limit of quantitation (LOQ) solution, the lowest S / N ratio between loxoprofen sodium and each impurity peak is 19.4, with a concentration level equivalent to 0.048%~0.050% of the test sample concentration. This meets the requirement of S / N≥10 and the solution concentration not exceeding the negligible limit. Therefore, a concentration of 47.72~50.02 ng / ml is proposed as the LQ concentration. In the limit of detection (LOD) solution, the lowest S / N ratio between loxoprofen sodium and each impurity peak is 20.6, which meets the requirement of S / N≥3. This concentration is equivalent to 0.024%~0.025% of the test sample concentration. Therefore, a concentration of 23.86~25.01 ng / ml is proposed as the LOD concentration. In the LQ solution, the highest RSD of the peak area of ​​the main component loxoprofen sodium and each impurity in the six needles is 6.84%, which meets the determination standard for the LQ.

[0091] 1.3.2 Linearity and Range Determination

[0092] Take one blank label, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran, add appropriate amounts of the main component and each impurity, 10 ml of methanol, shake at 200 rpm for 30 minutes, add 40 ml of water, shake at 200 rpm for another 30 minutes, mix well, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant to obtain the linear solution.

[0093] Prepare a series of linear solutions with varying concentration gradients, and perform linear regression with concentration on the x-axis and peak area on the y-axis.

[0094] The concentration gradients of the impurity solution series are 50%, 75%, 100%, 125%, and 150% of the limit concentration, and the concentration gradients of the loxoprofen sodium solution series are 50%, 75%, 100%, 125%, and 180% of the limit concentration.

[0095] The limit of quantitation concentration is the limit of quantitation concentration measured in 1.3.1;

[0096] The limit concentrations are based on the limits specified for loxoprofen sodium patches in the National Medical Products Administration's Import Drug Registration Standard (JX20210062): In the chromatogram of the test solution, if impurity peaks are present, the peak area multiplied by a correction factor is compared with the main peak area of ​​the related substance reference solution. Impurity D-1 must not exceed 0.5%, impurity D-2 must not exceed 0.4%, impurity D-3 must not exceed 1.2%, other single unknown impurities must not exceed 0.2%, and total impurities must not exceed 2.5%. The linear solution is prepared using the same method as the test sample. The test solution contains approximately 0.1 mg / ml of loxoprofen sodium. Therefore, the limit concentrations for impurities D-1, D-2, and D-3 are 0.5 μg / ml, 0.4 μg / ml, and 1.2 μg / ml, respectively. The proposed concentration of the loxoprofen sodium reference standard is 1 μg / ml.

[0097] The results of the linearity and range determination are shown in Table 3.

[0098] Table 3: Results of Linearity and Range Measurements

[0099]

[0100] The results showed that impurity D-1 exhibited good linearity in the concentration ranges of 0.05 μg / ml to 0.75 μg / ml, impurity D-2 in the range of 0.05 μg / ml to 0.57 μg / ml, impurity D-3 in the range of 0.05 μg / ml to 1.80 μg / ml, and loxoprofen sodium in the range of 0.05 μg / ml to 1.83 μg / ml, with correlation coefficients of 1.000 for all three.

[0101] 1.3.3 Correction Factor

[0102] As per section 1.3.2, the correction factor was calculated using the linear slope of each component, and the results of the correction factor are shown in Table 4.

[0103] The formula is: Correction factor = Loxoprofen sodium slope / Impurity slope

[0104] Table 4: Correction Factor Results

[0105]

[0106] The results showed that when the blank patch was used to simulate the linear solution prepared by the test sample, loxoprofen sodium and each impurity showed a good linear relationship with the peak area within the test concentration range, with a correlation coefficient of 1.000 for all impurities. Taking two significant figures, the correction factors for impurities D-1, D-2 and D-3 were 1.2, 1.0 and 2.0, respectively.

[0107] 1.4 Method Establishment

[0108] The above results confirm that the method for determining related substances in loxoprofen sodium patches is as follows:

[0109] High performance liquid chromatography conditions

[0110] The chromatographic column was a YMC-Pack Pro C18 RS, 100×4.6mm, 3μm; the flow rate was 2.0ml / min; the detection wavelength was 222nm; the column temperature was 45℃; the injection volume was 20μl; gradient elution was performed using a mixture of mobile phases A and B; mobile phase A was 0.01% phosphoric acid:acetonitrile (65:35), and mobile phase B was acetonitrile.

[0111] The gradient elution program is as follows: 0-1 minute, 0%-19%B; 1-8 minutes, 19%-72%B; 8-21 minutes, 72%-72%B; 21-23 minutes, 72%-0%B; 23-30 minutes, 0%-0%B.

[0112] Preparation of reference solution: Weigh appropriate amounts of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately weigh them, and dissolve and dilute them with 0.01% phosphoric acid:acetonitrile = 65:35 solvent to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0113] Preparation of test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 min to separate the patch from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 min, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, mix well, centrifuge at 10000 rpm for 5 min, and take the supernatant. The test solution contains approximately 0.1 mg / ml of loxoprofen sodium.

[0114] The reference standard and test sample solutions were injected into a liquid chromatograph for determination. The peak areas of the reference standard and test sample were obtained. The content was calculated using the HPLC correction factor method. The correction factors for impurities D-1, D-2, and D-3 were 1.2, 1.0, and 2.0, respectively.

[0115] The conditions for shaking and re-shaking can be set to any shaking speed and any time, such as shaking at 200-250 rpm for 30-35 minutes.

[0116] The centrifugation conditions can be set to any centrifugation speed and any time, such as 10000-12000 rpm for 5-10 min.

[0117] Example 2

[0118] This embodiment provides the methodology validation of the high performance liquid chromatography method used in Example 1, including the following:

[0119] 2.1 System Adaptability

[0120] 2.1.1 Solution Preparation

[0121] Blank solvent: 0.01% phosphoric acid: acetonitrile = 65:35

[0122] Reference solution: Weigh appropriate amounts of loxoprofen sodium reference standard, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately weigh them, dissolve and dilute them with solvent to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0123] Sensitivity solution: Accurately measure 2 ml of the reference solution, place it in a 20 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0124] 2.1.2 High Performance Liquid Chromatography (HPLC) Determination

[0125] Inject the above solution into a high-performance liquid chromatograph and record the chromatogram. The system suitability is shown in Table 5.

[0126] Table 5: System Applicability Results

[0127]

[0128] The results showed that the blank solvent did not interfere with the detection of the main peak (loxoprofen sodium peak) or any impurity peaks. In the chromatogram of the reference solution, impurities D-1, D-2, loxoprofen sodium, and D-3 eluted sequentially, with the lowest resolution (2.27) between impurities D-1 and D-2. After five injections of the reference solution, the RSD of the main peak area was 0.16%, and the main peak area in the sensitivity solution chromatogram was 9.9% of the average main peak area in the five injections of the related substance reference solution chromatograms. The system suitability met the requirements.

[0129] 2.2 Specificity

[0130] 2.2.1 Solution Preparation

[0131] Blank patch solution: Take one blank patch, peel off the protective layer, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 min to separate the paste from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 min, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute with solvent to the mark, mix well, centrifuge at 10000 rpm for 5 min, and take the supernatant.

[0132] Test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 min to separate the patch from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 min, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, mix well, centrifuge at 10000 rpm for 5 min, and take the supernatant. The test solution contains approximately 0.1 mg / ml of loxoprofen sodium.

[0133] Reference solution: Weigh appropriate amounts of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately, and dissolve and dilute them with 0.01% phosphoric acid:acetonitrile = 65:35 to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0134] Limit concentration spiked test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, add appropriate amounts of impurities D-1, D-2, and D-3 respectively, shake at 200 rpm for 30 minutes to allow the paste to detach from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant to obtain the limit concentration spiked test solution, which contains approximately 0.1 mg / ml loxoprofen sodium, approximately 0.5 μg / ml impurity D-1, approximately 0.4 μg / ml impurity D-2, and approximately 1.2 μg / ml impurity D-3.

[0135] Single impurity positioning solution: Weigh appropriate amounts of impurity D-1, impurity D-2, and impurity D-3 reference standards, dissolve and dilute them with solvents to prepare solutions of a certain concentration, and mix well.

[0136] 2.2.2 High Performance Liquid Chromatography (HPLC) Determination

[0137] Accurately measure the above solution and inject it into the high-performance liquid chromatograph (HPLC), then record the chromatogram. Specificity results are shown in Table 6, and related chromatograms are shown in [reference needed]. Figure 1 .

[0138] Table 6: Specificity Results

[0139]

[0140] The results showed that the blank solvent and blank patch solution did not interfere with any of the impurities or the main peak. In the chromatogram of the reference solution, impurities D-1, D-2, loxoprofen sodium, and D-3 eluted sequentially, with a resolution of 2.31 between impurities D-1 and D-2. In the spiked test solution at the limit concentration, the minimum resolution between the main peak and each known impurity peak and adjacent peaks was 2.21, and the minimum peak purity was 993. The results met the requirements.

[0141] 2.3 Forced Degradation Test

[0142] Loxoprofen sodium patches were subjected to forced degradation under harsh conditions such as high temperature, acid, alkali, oxidation, and light. The degradation conditions of the forced degradation test are shown in Table 7.

[0143] X: Volume under failure conditions

[0144] Z: Time under failure conditions

[0145] Table 7: Destruction conditions of forced degradation test

[0146]

[0147] 2.3.1 Solution Preparation

[0148] Preparation of acid and alkali destructive solution: Take one blank patch or one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 20 ml of tetrahydrofuran, shake at 200 rpm for 30 minutes, add X ml of destructive solution, place under destructive conditions for Z hours, add X ml of neutralization solution, then add 30 ml of tetrahydrofuran, 10 ml of methanol, and (40-2X) ml of water, shake at 200 rpm for 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant.

[0149] Preparation of oxidative degradation solution: Take one blank patch or one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 20 ml of tetrahydrofuran, shake at 200 rpm for 30 minutes, add X ml of degradation solution, place under degradation conditions for Z hours, then add 30 ml of tetrahydrofuran, 10 ml of methanol, and (40-X) ml of water, shake at 200 rpm for 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant.

[0150] Preparation of high temperature and light-induced damage solution: Take one blank patch or one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, place it under the damage conditions for Z days, cool to room temperature, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 minutes, add 40 ml of water, shake at 200 rpm for 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and collect the supernatant.

[0151] 2.3.2 High Performance Liquid Chromatography (HPLC) Determination

[0152] Accurately measure the above solution and inject it into a high-performance liquid chromatograph (HPLC), recording the chromatogram. The results of the forced degradation test are shown in Tables 8 and 9, and the relevant chromatograms are shown in... Figures 2-7 .

[0153] Table 8: Forced Degradation Experiment-1

[0154]

[0155] Table 9: Forced Degradation Experiment-2

[0156]

[0157] The results showed that loxoprofen sodium patches degraded to varying degrees under high temperature, acid, alkali, oxidation, and light conditions. Under alkaline conditions, the degradation rate of the main peak was 18.2%. The patches were relatively stable under high temperature, acid, oxidation, and light conditions. The degradation products produced under each degradation condition were detectable. The peak purity angles of the main component and each known impurity peak were all less than the purity threshold. The separation degree between the main peak and each known impurity peak and the adjacent peak was greater than 1.2. The material balance was within the range of 90% to 110%, indicating material conservation.

[0158] 2.4 Limit of Quantitation and Limit of Detection

[0159] The determination of the limit of quantitation and the limit of detection were performed using the method and results described in section 1.3.1 of Example 1.

[0160] 2.5 Solution stability

[0161] 2.5.1 Solution Preparation

[0162] Limit concentration spiking test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, add appropriate amounts of impurities D-1, D-2, and D-3 respectively, shake at 200 rpm for 30 minutes to allow the paste to detach from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant to obtain the limit concentration spiking test solution, which contains approximately 0.1 mg / ml of loxoprofen sodium, approximately 0.5 μg / ml of impurity D-1, approximately 0.4 μg / ml of impurity D-2, and approximately 1.2 μg / ml of impurity D-3.

[0163] Reference solution: Weigh appropriate amounts of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately, and dissolve and dilute them with 0.01% phosphoric acid:acetonitrile = 65:35 to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0164] 2.5.2 Stability Conditions

[0165] The reference solution was incubated at room temperature for 0, 15, 26.5, 35, 45.5, 55.5, 57.5, and 59.5 hours, respectively. The spiked test solution at the limit concentration was incubated at room temperature for 0, 8.5, 17, 27.5, 37.5, 39.5, 41.5, 50.5, 59, and 67.5 hours, respectively.

[0166] 2.5.3 High Performance Liquid Chromatography (HPLC) Determination

[0167] Accurately measure the above solution, inject it into the high-performance liquid chromatograph, and record the chromatogram. The solution stability results are shown in Tables 10 and 11.

[0168] Table 10: Solution stability results of the reference solution (room temperature)

[0169]

[0170] Table 11: Solution stability results of spiked test solutions at limit concentrations (room temperature)

[0171]

[0172] The results showed that the reference solution was stable at room temperature for 59.5 h, and the spiked test solution at the limit concentration was stable at room temperature for 67.5 h.

[0173] 2.6 Linearity and Range

[0174] 2.6.1 Solution Preparation

[0175] Method 1: The linearity and range determination method and results were obtained using the method described in section 1.3.2 of Example 1.

[0176] Method 2 (Comparative Experiment): Accurately weigh appropriate amounts of loxoprofen sodium and impurity D-1 and impurity D-3 reference standards, dissolve and dilute them with solvent to prepare a stock solution of a certain concentration, accurately measure an appropriate amount of the stock solution, and prepare a series of linear solutions with concentration gradients, namely solutions with the limit of quantitation concentration and solutions with the limit concentrations of 20%, 50%, 80%, 100%, 120%, and 150%.

[0177] According to Method 1, it has been confirmed that the upper limit response value of 150% of the concentration of loxoprofen sodium can meet the upper limit response requirements of impurities D-1 and D-3. Therefore, in Method 2, the upper limit concentration of the linear solution of loxoprofen sodium is set to 150% of the limit concentration.

[0178] Based on previous research results, the blank excipients in loxoprofen sodium patches have little interference with the detection of impurity D-2. The main impurity affected is D-3. The blank excipients have a slight impact on impurity D-2. For comparative experiments, impurities with greater impact, namely impurity D-1 and impurity D-3, were selected for result comparison.

[0179] 2.6.2 High Performance Liquid Chromatography (HPLC) Determination

[0180] Accurately measure each of the above solutions and inject them into the high-performance liquid chromatograph (HPLC) and record the chromatograms. Perform linear regression with concentration on the x-axis and peak area on the y-axis. The linearity and range results are shown in Tables 3 and 12, and the relevant chromatograms are shown in [reference needed]. Figures 8-9 .

[0181] Table 12: Linearity and Range Results for Method 2 Preparation

[0182]

[0183] 2.7 Correction Factor

[0184] The linear equations obtained from the linearity and range under section 2.6 are used to calculate the correction factors using the linear slopes of each component. The comparison of the correction factor results is shown in Table 13.

[0185] The formula is: Correction factor = Loxoprofen sodium slope / Impurity slope

[0186] Table 13: Comparison of Correction Factor Results

[0187]

[0188] The results showed that both the linear solutions prepared using blank patches to simulate the sample preparation process and the linear solutions prepared directly with blank solvents exhibited good linear relationships between the main components and peak areas within the test concentration range, with correlation coefficients of 1.000 for both methods. However, the linearity results for the two preparation methods showed that the correction factors for impurity D-1 were relatively close, while the correction factors for impurity D-3 differed significantly. The correction factor for method 2 was significantly lower than that for method 1, indicating that the participation of blank patches in the solution preparation process affected the detection level of impurity D-3. By simulating the sample preparation process and using the correction factor obtained from the linear preparation of the blank patch solution for the correction calculation of the detection results, the background interference from the excipient matrix can be effectively eliminated, truly reflecting the impurity level in the sample and reducing product risk.

[0189] 2.8 Precision

[0190] 2.8.1 Solution Preparation

[0191] Limit concentration spiking test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, add appropriate amounts of impurities D-1, D-2, and D-3 respectively, shake at 200 rpm for 30 minutes to allow the paste to detach from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 minutes, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge at 10000 rpm for 5 minutes, and take the supernatant to obtain the limit concentration spiking test solution, which contains approximately 0.1 mg / ml of loxoprofen sodium, approximately 0.5 μg / ml of impurity D-1, approximately 0.4 μg / ml of impurity D-2, and approximately 1.2 μg / ml of impurity D-3.

[0192] 2.8.2 High Performance Liquid Chromatography (HPLC) Determination

[0193] Repeatability: Prepare six parallel spiked test solutions at the limit concentration, and perform a series of tests multiple times by the same analyst to evaluate the dispersion of the results.

[0194] Intermediate precision: Repeatability testing of six limit concentration spiked test solutions performed by different analysts using different instruments at different times.

[0195] Accurately measure the above solution, inject it into the high performance liquid chromatograph, record the chromatogram, and the repeatability and intermediate precision results are shown in Table 14.

[0196] Table 14: Repeatability and Intermediate Precision Results

[0197]

[0198] RRT: Relative Retention Time.

[0199] The results show that the method has good repeatability and intermediate precision.

[0200] 2.9 Accuracy

[0201] 2.9.1 Solution Preparation

[0202] Quantitation limit, 100%, and 120% accuracy test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran, appropriate amounts of each impurity, and 10 ml of methanol, shake for 30 minutes (200 rpm), add 40 ml of water, shake for another 30 minutes (200 rpm), accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, shake well, centrifuge (10000 rpm for 5 min), and collect the supernatant. The amounts of each impurity added based on the test solution are shown in Table 16. Prepare three parallel aliquots for each concentration.

[0203] Test solution: Take one loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add 50 ml of tetrahydrofuran and 10 ml of methanol, shake at 200 rpm for 30 min to separate the patch from the backing layer, add 40 ml of water, shake at 200 rpm for another 30 min, accurately measure 2 ml, place it in a 20 ml volumetric flask, dilute to the mark with solvent, mix well, centrifuge at 10000 rpm for 5 min, and take the supernatant. The test solution contains approximately 0.1 mg / ml of loxoprofen sodium.

[0204] Reference solution: Weigh appropriate amounts of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 reference standards, accurately, and dissolve and dilute them with 0.01% phosphoric acid:acetonitrile = 65:35 to prepare a solution containing 1 μg / ml of loxoprofen sodium and each impurity.

[0205] 2.9.2 High Performance Liquid Chromatography (HPLC) Determination

[0206] Accurately measure each of the above solutions, inject them into the high-performance liquid chromatograph, and record the chromatograms. The accuracy results are shown in Table 15.

[0207] Table 15: Accuracy Results

[0208]

[0209] The results showed that the recoveries at the limits of quantitation (LOQ) ranged from 87.8% to 108.9%, with a maximum RSD of 5.79%. The average recovery rate of the LOQ for impurity D-1 was 92.4%, with an RSD of 1.56%; the average recovery rate of the LOQ for impurity D-2 was 105.8%, with an RSD of 1.53%; and the average recovery rate of the LOQ for impurity D-3 was 87.8%, with an RSD of 5.79%. The average recoveries of impurity D-1 at 100% and 120% concentrations were 103.6% and 104.4%, with RSDs of 0.94% and 0.53%, respectively; the average recoveries of impurity D-2 at 100% and 120% concentrations were 108.4% and 108.9%, with RSDs of 0.28% and 0.49%, respectively; and the average recoveries of impurity D-3 at 100% and 120% concentrations were 103.0% and 105.7%, with RSDs of 0.43% and 0.18%, respectively. The method demonstrated good accuracy.

[0210] 2.10 Durability

[0211] To examine the tolerance of this method to minor changes in conditions, a robustness test was conducted. The robustness parameters of the high performance liquid chromatography (HPLC) conditions are shown in Table 16, and the robustness results are shown in Table 17.

[0212] Table 16: Parameters for variation of robust chromatographic conditions

[0213]

[0214] Table 17: Durability Results

[0215]

[0216] The results showed that the method had good durability at column temperatures of 40℃~50℃, mobile phase A ratio of 0.01% phosphoric acid solution-acetonitrile (63∶37)~0.01% phosphoric acid solution-acetonitrile (67∶33), different instruments (Agilent, Waters), and different batches of the same model of chromatographic column.

[0217] The system suitability, specificity, solution stability, limit of quantitation and limit of detection, linearity and range, correction factor, precision, accuracy and robustness of this validation all meet the validation requirements, indicating that this method is suitable for the detection of related substances in loxoprofen sodium patches.

[0218] Example 3

[0219] This embodiment uses the determination method of Example 1 to detect related substances in loxoprofen sodium patches.

[0220] Three batches of loxoprofen sodium patches were collected, and reference solutions and test solutions were prepared according to the determination method in section 1.2. The content of related substances in the three batches of loxoprofen sodium patches was calculated using the principal component external standard method with correction factors, and the correction factors in section 1.3.3 were used. The results are shown in Table 18. A typical chromatogram of the test solution is shown in [the table / reference needed]. Figure 10 .

[0221] The sensitivity test before sample determination was performed according to the system adaptability determination method in section 2.1 of Example 2.

[0222] The detection limits for each impurity are met: impurity D-1 not exceeding 0.5%, impurity D-2 not exceeding 0.4%, impurity D-3 not exceeding 1.2%, other single unknown impurities not exceeding 0.2%, and total impurities not exceeding 2.5%.

[0223] Table 18: Content of related substances in 3 batches of loxoprofen sodium patches

[0224]

[0225] 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 and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for determining related substances in loxoprofen sodium patches, characterized in that, Includes the following steps: Weigh out loxoprofen sodium reference standard and impurity reference standard separately, and dilute them with solvent to prepare reference standard solution; Take loxoprofen sodium patch, peel off the protective layer, place it in a stoppered container, add tetrahydrofuran and methanol, shake to separate the patch from the backing layer, add water, shake again, dilute with solvent, mix well, centrifuge, and take the supernatant to obtain the test solution. The prepared reference solution and test solution were injected into a high-performance liquid chromatograph for determination, and the peak areas of the reference solution and test solution were obtained. The content of related substances in loxoprofen sodium patch was calculated using the HPLC correction factor method based on the peak areas of the reference solution and the test solution. The linear solution used in the HPLC correction factor method was prepared by mimicking the preparation method of the test sample solution using a loxoprofen sodium blank patch.

2. The method for determining related substances in loxoprofen sodium patch according to claim 1, characterized in that, The determination of the correction factor in the HPLC correction factor method includes the following steps: Preparation of linear solutions; A series of linear solutions with varying concentration gradients were prepared and injected into a high-performance liquid chromatograph to determine the peak areas of the linear solutions with varying concentration gradients. Linear regression was performed with concentration on the x-axis and the corresponding peak area on the y-axis. The correction factor was calculated using the linear slope of loxoprofen sodium reference standard and impurity reference standard; The preparation steps of the linear solution are as follows: Take a blank loxoprofen sodium patch, peel off the protective layer, place it in a stoppered conical flask, add tetrahydrofuran, then add loxoprofen sodium reference standard, impurity reference standard and methanol respectively, shake to separate the patch from the backing layer, add water, shake again, mix evenly, dilute with solvent, shake well, centrifuge, and take the supernatant to obtain the linear solution.

3. The method for determining related substances in loxoprofen sodium patch according to claim 2, characterized in that, The conditions for both shaking and re-shaking are 200-250 rpm shaking for 30-35 minutes; The centrifugation conditions are 10000-12000 rpm for 5-10 minutes.

4. The method for determining related substances in loxoprofen sodium patch according to claim 2, characterized in that, The correction factors for the impurities were determined using a series of concentration gradients, namely the limit of quantitation concentration and 50%, 75%, 100%, 125%, and 150% of the limit concentration. The correction factor for loxoprofen sodium was determined using a series of concentration gradients at the limit of quantitation concentration and at 50%, 75%, 100%, 125%, and 180% of the limit concentration. The limits of quantitation (LOQs) for both loxoprofen sodium and impurities are 47.72–50.08 ng / ml. The impurities include impurity D-1, impurity D-2 and impurity D-3; The limit concentrations of loxoprofen sodium, impurity D-1, impurity D-2, and impurity D-3 are 1 μg / ml, 0.5 μg / ml, 0.4 μg / ml, and 1.2 μg / ml, respectively.

5. The method for determining related substances in loxoprofen sodium patch according to claim 4, characterized in that, The impurity D-1 is 2-[p-(5-carboxy-2-oxopentyl)phenyl]propionic acid; Impurity D-2 is 2-{4-[(1-hydroxy-2-oxocyclopentyl)methyl]phenyl}propionic acid; Impurity D-3 is ethyl 2-isopropyl-5-methylcyclohexyl-2-{4-[2-oxocyclopentyl)methyl]phenyl}propionate.

6. The method for determining related substances in loxoprofen sodium patch according to claim 1, characterized in that, The solvent for the reference solution is 0.01% phosphoric acid:acetonitrile = 65:

35.

7. The method for determining related substances in loxoprofen sodium patch according to claim 1, characterized in that, The concentration of each reference standard in the reference solution was 1 μg / ml.

8. The method for determining related substances in loxoprofen sodium patch according to claim 1, characterized in that, The concentration of loxoprofen sodium in the test solution was 0.1 mg / ml.

9. The method for determining related substances in loxoprofen sodium patch according to any one of claims 1-8, characterized in that, The chromatographic conditions of the high-performance liquid chromatograph were as follows: YMC-Pack Pro C18 RS column, 100×4.6mm, 3μm; flow rate, 2.0ml / min; detection wavelength, 222nm; column temperature, 45℃; injection volume, 20μl; gradient elution using a mixture of mobile phases A and B; mobile phase A was 0.01% phosphoric acid:acetonitrile = 65:35, and mobile phase B was acetonitrile.

10. The method for determining related substances in loxoprofen sodium patch according to any one of claims 1-8, characterized in that, The elution program of the high performance liquid chromatograph is as follows: 0-1 minute, 0%-19%B; 1-8 minutes, 19%-72%B; 8-21 minutes, 72%-72%B; 21-23 minutes, 72%-0%B; 23-30 minutes, 0%-0%B.

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