A method for detecting related substances in loxoprofen sodium oral solution

Abstract

The invention provides a detection method for related substances in loxoprofen sodium oral solution. The adopted ternary mobile phase system solves the interference of auxiliary materials in the low absorption band on the detection of known impurities, thereby avoiding the problem of missed detection and insufficient detection of impurities, effectively solving the interference of auxiliary material methylparaben on the detection of impurities when the related substances are determined, and the types and numbers of impurities to be monitored are large, the separation between various impurities, between impurities and main components, and between impurities and auxiliary materials is good, the related substances in the loxoprofen sodium oral solution can be quickly and accurately monitored, and the method has important significance for the quality evaluation of the loxoprofen sodium oral solution.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical drug analysis, and particularly relates to a detection method for detecting related substances in loxoprofen sodium oral solution. Background Art

[0002] Loxoprofen sodium oral solution is a non-steroidal anti-inflammatory analgesic developed by NIK IKO Co., Ltd. It was launched in Japan in December 2012. Each package contains 68.1 mg of loxoprofen sodium hydrate (60 mg of anhydrous) and is manufactured by NIK IKO Co., Ltd.

[0003] Loxoprofen sodium, chemical name is 2-[4-(2-oxocyclopentylmethyl)phenyl]propionate sodium dihydrate, the structural formula is as follows:

[0004]

[0005] At present, there are few literature reports on the detection method of related substances in loxoprofen sodium oral solution, and the auxiliary materials and key and important degradation products are not analyzed and detected. Some impurities are easily interfered by the auxiliary material peaks, and there are certain risks in the evaluation of drug quality. Although there are reports, for example, patent CN 115236255 A discloses a method for detecting related substances in loxoprofen sodium, and the object of detection is loxoprofen sodium raw material drug, but because loxoprofen sodium oral solution contains a variety of auxiliary materials, the detection method of the patent is adopted, with phosphoric acid aqueous solution (with phosphoric acid adjusted to pH 2.3-2.7)-acetonitrile as the mixed mobile phase. No matter how the gradient elution procedure is adjusted, the problem of low separation between specific auxiliary materials and certain impurities in the detection process cannot be solved, and the interference of auxiliary materials on certain impurities cannot be eliminated, so it is not suitable for the detection of related substances in loxoprofen sodium oral solution.

[0006] In order to ensure the safety and effectiveness of loxoprofen sodium oral solution preparations, it is necessary to sort out the process by-products, degradation products of the raw materials, process products in the preparations and the excipients used, which is crucial for the quality comparison study of loxoprofen sodium oral solution preparations. Summary of the invention

[0007] The purpose of the present invention is to provide a method for detecting related substances in loxoprofen sodium oral solution on the basis of the prior art, which can detect more impurities, has high sensitivity and good specificity, has good separation between impurity peaks and between a main peak and its adjacent impurity peaks, effectively solves the interference of auxiliary material methylparaben on impurity detection when determining related substances, and can quickly, effectively and accurately monitor related substances in loxoprofen sodium oral solution.

[0008] The technical solution of the present invention is as follows:

[0009] A method for detecting related substances in loxoprofen sodium oral solution, wherein the method uses high performance liquid chromatography to quantitatively detect the related substances in loxoprofen sodium oral solution, and the high performance liquid chromatography conditions include:

[0010] The chromatographic column is octadecylsilane bonded silica gel, and gradient elution is performed with mobile phase A, mobile phase B and mobile phase C as mixed mobile phases, wherein mobile phase A is 0.05-0.15% phosphoric acid aqueous solution, and the pH value is adjusted to 2.0-4.0 with triethylamine; mobile phase B is acetonitrile, and mobile phase C is methanol. During the gradient elution process, the initial ratio of mobile phase A, mobile phase B and mobile phase C is 78-82:7-3:15. The specific gradient elution process is as follows: (1) in 0-10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 78-82:7-3:15 to 70:15:15 at a uniform speed; (2) Within 10-40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) within 40-60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) within 60-70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 78-82:7-3:15 at a constant speed; (5) within 70-80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 78-82:7-3:15.

[0011] At present, when detecting related substances in loxoprofen sodium raw materials, a methanol-free binary mobile phase system is generally selected, which can realize the determination of most related substances in the raw materials. However, for loxoprofen sodium oral solution containing other excipients, for example, the antibacterial agent methylparaben interferes with the detection of impurities 1 and impurity 6 in this application, resulting in the problem of missed detection of impurities. In this application, a ternary mobile phase system with mobile phase A, mobile phase B and mobile phase C as mixed mobile phases is adopted, and the ratio of time and mobile phase in the gradient elution process is optimized, so that more impurities are detected, the main components and each impurity have a strong retention ability in the detection method, the response is high, and the components are well separated. The interference of excipients on impurity detection during the determination of related substances is effectively solved, and the related substances in loxoprofen sodium oral solution can be quickly and accurately monitored, which is of great significance to the quality evaluation of loxoprofen sodium oral solution.

[0012] The detection method provided by the present invention uses mobile phase A, mobile phase B and mobile phase C as a mixed mobile phase for gradient elution. In chromatographic analysis, after selecting the chromatographic column and the mobile phase, it is necessary to determine whether the elution process is isocratic elution or gradient elution, and in the elution process, the ratio of mobile phase A, mobile phase B and mobile phase C will affect the response of the analyte on the chromatographic column, and whether the impurity peak after the main peak will interfere with the analysis of the main peak. For the present invention, the loxoprofen sodium oral solution, the excipients include methylparaben (antibacterial agent), anhydrous ethanol (antibacterial synergist), propylene glycol (antibacterial synergist), saccharin sodium (sweetener), sodium citrate (pH regulator), anhydrous citric acid (pH regulator) and flavor (flavor corrector), the amount of excipients is large and the types are many, the types of impurities that need to be detected are many, and the interfering factors are particularly many. The time of the gradient elution process and the ratio of the mobile phase cannot be randomly selected, and a large number of experiments and analyses are required to determine, otherwise, a large excipient peak will appear near the main peak, and the peaks of various impurities will be interfered by the excipient peaks. In the actual sample detection process, some impurities may be deducted as excipient peaks, resulting in serious consequences of misjudging the quality of the product.

[0013] In a preferred embodiment, the initial ratio of mobile phase A, mobile phase B and mobile phase C during gradient elution is 80:5:15. The specific gradient elution process is as follows: (1) in 0-10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) in 10-40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:2 5:15; (3) within 40-60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C gradually changes from 60:25:15 to 10:75:15; (4) within 60-70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C gradually changes from 10:75:15 to 80:5:15; (5) within 70-80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:15.

[0014] In the present invention, mobile phase A is a 0.05-0.15% phosphoric acid aqueous solution, and the pH value is adjusted to 2.0-4.0 with triethylamine; preferably, mobile phase A is a 0.1% phosphoric acid aqueous solution, and the pH value is adjusted to 2.3-2.7 with triethylamine; more preferably, mobile phase A is a 0.1% phosphoric acid aqueous solution, and the pH value is adjusted to 2.5 with triethylamine.

[0015] For the present invention, the solvent for dissolving the sample is a mixed solution composed of mobile phase A and mobile phase B; preferably, in the mixed solution, the volume ratio of mobile phase A and mobile phase B is 75-85:25-15, and can be but not limited to 75:25, 78:22, 80:20, 82:18 or 85:15; in order to obtain better results, in the mixed solution, the volume ratio of mobile phase A and mobile phase B is 80:20.

[0016] In a preferred embodiment, the chromatographic conditions include: the chromatographic column uses octadecylsilane bonded silica gel; the chromatographic column models are Inertsil ODS-3C18, InertSustain C18 and Wondasil C18; preferably InertSustain C18, the chromatographic column has a length of 250 mm, a diameter of 4.6 mm, and a filler particle size of 5 μm.

[0017] Furthermore, the detection wavelength of the detector is 220-224 nm, preferably 222 nm.

[0018] Furthermore, the column temperature is 20-30°C; preferably 25°C.

[0019] Furthermore, the flow rate is 0.5 to 1.5 ml / min, preferably 1.0 ml / min.

[0020] Furthermore, the injection volume is 10 to 50 μl, preferably 20 μl. For example, the injection volume can be 10 μl, 15 μl, 20 μl or 50 μl.

[0021] The present invention provides a method for detecting related substances in a loxoprofen sodium oral solution, wherein the related substances include the following substances:

[0022]

[0023]

[0024]

[0025] The detection method provided by the present invention can prepare the following solution. When preparing the following solution, the selected solvent is: a mixed solution of mobile phase A and mobile phase B in a volume ratio of 80:20.

[0026] Mixed impurity solution: Accurately weigh each impurity reference substance, first add acetonitrile to dissolve it, then add acetonitrile to make up the volume and dilute it to make a mixed solution of impurity reference substances containing approximately 12 μg of each impurity per 1 ml.

[0027] Sample plus mixed impurity solution: Accurately measure 2 ml of mixed impurity solution and 2 ml of loxoprofen sodium oral solution, place them in the same 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0028] Test solution: Accurately measure 2 ml of loxoprofen sodium oral solution, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well;

[0029] Accurately measure an appropriate amount of the test sample solution and dilute it with solvent to make a solution containing 12 μg per 1 ml as the control solution.

[0030] Blank excipient solution: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor), place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0031] The present invention screens suitable chromatographic conditions, performs chromatographic detection on the loxoprofen sodium oral solution and the above-mentioned impurities, determines the detection method of the present invention, and verifies the specificity of the present invention through peak positioning tests on the impurities and loxoprofen sodium, interference tests and degradation tests of loxoprofen sodium.

[0032] Adopt the technical scheme of the present invention, the advantages are as follows:

[0033] (1) The method for detecting related substances in loxoprofen sodium oral solution provided by the present invention can monitor a large number of impurity types and numbers, and has good separation between each impurity, between impurities and main components, and between impurities and auxiliary materials, so that the related substances in loxoprofen sodium oral solution can be monitored quickly and accurately.

[0034] (2) The method for detecting related substances in loxoprofen sodium oral solution provided by the present invention adopts a ternary mobile phase system to solve the interference of auxiliary materials in the low absorption band on the detection of known impurities, thereby avoiding the problem of missed detection or insufficient detection of impurities, and effectively solves the interference of auxiliary material methylparaben on the detection of impurities during the determination of related substances. The related substances in loxoprofen sodium oral solution can be monitored quickly and accurately, which is of great significance to the quality evaluation of loxoprofen sodium oral solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is the HPLC chromatogram of the blank auxiliary material solution in Example 1;

[0036] Figure 2 is a high performance liquid chromatogram of the mixed impurity solution in Example 1;

[0037] Figure 3 is the HPLC chromatogram of the test solution in Example 1;

[0038] Figure 4 is a high performance liquid chromatogram of the sample plus mixed impurity solution in Example 1;

[0039] Figure 5 is a high performance liquid chromatogram of the sample in comparative example 1 plus a mixed impurity solution;

[0040] Figure 6 This is the HPLC chromatogram of the sample in Comparative Example 2 plus the mixed impurity solution. DETAILED DESCRIPTION

[0041] The following is a further detailed description of the method for detecting related substances in the loxoprofen sodium oral solution of the present invention in the form of embodiments, but this should not be understood as the scope of the above-mentioned subject matter of the present invention being limited to the following examples, and all technologies realized based on the above-mentioned contents of the present invention belong to the scope of the present invention.

[0042] Embodiment 1:

[0043] HPLC conditions:

[0044] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6 mm, 5 μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0 ml / min, detection wavelength of 222 nm, column temperature of 25°C, injection volume of 20 μl.

[0045] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 80:5:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:15.

[0046] The solvent is a mixed solution of mobile phase A and mobile phase B in a volume ratio of 80:20. The sample solution is prepared as follows:

[0047] Mixed impurity solution: Accurately weigh each impurity reference substance, first add acetonitrile to dissolve it, then add acetonitrile to make up the volume and dilute it to make a mixed solution of impurity reference substances containing approximately 12 μg of each impurity per 1 ml.

[0048] Sample plus mixed impurity solution: Accurately measure 2 ml of mixed impurity solution and 2 ml of loxoprofen sodium oral solution, place them in the same 10 ml volumetric flask, add solvent to dilute to the scale, and shake well.

[0049] Test solution: Accurately measure 2 ml of loxoprofen sodium oral solution, place it in a 10 ml volumetric flask, add solvent to dilute to the scale, and shake well;

[0050] Accurately measure an appropriate amount of the test sample solution and dilute it with solvent to make a solution containing 12 μg per 1 ml as the control solution.

[0051] Blank excipient solution: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor), place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, and shake well.

[0052] Take 20 μl of each of the above solutions, inject and analyze, and record the chromatogram. For specific spectra, see Figure 1 , Figure 2 , Figure 3 and Figure 4 .

[0053] Depend on Figure 1 to Figure 4 It can be seen that the baseline is stable and the blank excipient does not interfere with the determination of each component; in the test solution, mixed impurity solution and sample plus mixed impurity solution, the separation between the main component and impurities, and between impurities is good.

[0054] In the chromatogram of the test solution, only impurities 1 and 10 were detected. The test results are shown in Table 1:

[0055] Table 1 Test results of the test products

[0056] Impurity No. content / % RSD / % 1 0.036 0.87 10 0.019 2.39

[0057] Verify the detection methods of relevant substances as follows:

[0058] 1. Exclusivity

[0059] Take 20 μl of each of the blank excipient, each single impurity reference solution, test solution, mixed impurity solution and sample plus mixed impurity solution, inject and analyze, record the chromatogram, and examine the retention time, separation degree and theoretical plate number of each component. The results are shown in Table 2.

[0060] Table 2 Specificity test results

[0061] Retention time (min) Separation Theoretical plate number Tailing Factor Attribution 16.348 / 43752 1.16 unknown 17.713 4.2 45586 1.10 Impurity 13 20.888 8.4 34746 1.04 Impurity 8 28.300 13.1 27629 1.18 Impurity 6 29.618 2.2 54876 1.10 Impurity 1 34.917 10.2 68826 1.13 Impurity 9 35.941 1.8 58305 1.08 Impurity 12 39.979 6.8 72157 1.11 unknown 46.508 12.4 166363 1.37 Impurity 4 47.884 3.4 280647 1.24 principal component 50.355 7.9 575925 1.12 Impurity 11 53.132 7.7 604163 0.79 unknown 57.034 15.6 1002293 1.11 Impurity 10 58.339 5.6 967120 0.69 unknown

[0062] The results showed that under the chromatographic conditions, the baseline was stable and the blank excipients did not interfere with the determination of the components; the separation between the components in the mixed impurity solution was good, the theoretical plate number was high, and the purity factor was high; the separation between the impurities in the test solution was good.

[0063] 2. Destructive test

[0064] Take an appropriate amount of this product and conduct destructive tests under various harsh conditions to investigate the source of degradation products, the separation of degradation products and main components, and material balance. Use the high performance liquid chromatography conditions in Example 1 to determine the relevant substances. The specific method is as follows:

[0065] Solvent: A mixed solution of mobile phase A and mobile phase B in a volume ratio of 80:20.

[0066] Undamaged: Accurately measure 2 ml of the solution into a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0067] Photodestruction: Accurately measure 2 ml of the solution into a 10 ml transparent volumetric bottle, dilute with an appropriate amount of solvent, place at 5000 Lx for 8 hours, dilute to the scale with solvent, and shake well.

[0068] Photodestruction of blank excipients: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor) into a 10 ml transparent volumetric bottle, dissolve and dilute with an appropriate amount of solvent, place at 5000Lx for 8 hours, dilute to the scale with solvent, and shake well.

[0069] Acid destruction: Accurately measure 2 ml of the solution into a 10 ml volumetric flask, add 1 ml of 2M hydrochloric acid solution, leave at room temperature for 4 hours, then add 1 ml of 2M sodium hydroxide solution, neutralize, first add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0070] Acid destruction of blank excipients: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor) into a 10 ml volumetric flask, add 1 ml of 2M hydrochloric acid solution, leave it at room temperature for 4 hours, then add 1 ml of 2M sodium hydroxide solution, neutralize it, first add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0071] Alkali destruction: Accurately measure 2 ml of the solution into a 10 ml volumetric flask, add 1 ml of 2M sodium hydroxide solution, leave it at room temperature for 4 hours, then add 1 ml of 2M hydrochloric acid solution, neutralize it, first add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0072] Alkali destruction of blank excipients: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor) into a 10 ml volumetric flask, add 1 ml of 2M sodium hydroxide solution, leave it at room temperature for 4 hours, then add 1 ml of 2M hydrochloric acid solution, neutralize it, first add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0073] Oxidative damage: Accurately measure 2 ml of this product into a 10 ml volumetric flask, add 1 ml of 30% hydrogen peroxide, mix well, and leave at room temperature for 4 hours. First add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0074] Oxidative destruction of blank excipients: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor) into a 10 ml volumetric flask, add 1 ml of 30% hydrogen peroxide, mix well, and place at room temperature for 4 h. First add 2 ml of acetonitrile, then dilute to the scale with solvent and shake well.

[0075] High temperature destruction: Accurately measure 2 ml of this product into a 10 ml volumetric flask, dilute with an appropriate amount of solvent, destroy in a 100°C water bath for 1 hour, and dilute to the scale with solvent.

[0076] High-temperature destruction of blank excipients: Weigh the excipients required to prepare 2 ml of loxoprofen sodium oral solution (including methylparaben, anhydrous ethanol, propylene glycol antibacterial enhancer, saccharin sodium, sodium citrate, anhydrous citric acid and flavor) into a 10 ml volumetric flask, dissolve and dilute with an appropriate amount of solvent, destroy in a 100°C water bath for 1 h, and dilute to the scale with solvent.

[0077] Take 20 μl of the sample under each destruction condition, inject and analyze, and record the chromatogram.

[0078] The results showed that this product was relatively stable, the main impurity produced by each degradation condition was impurity 1, and the separation between each degradation product and the main peak was good. The peak purity of the main peak was high (the peak purity values ​​were all greater than the threshold value of 990), indicating that the main peak did not contain impurities or degradation products that could not be separated, that is, the proposed chromatographic conditions for related substances were suitable for the detection of related substances of this product.

[0079] The results of material balance investigation are shown in Table 3.

[0080] Table 3 Material balance investigation

[0081] sample f / f not damaged Total impurities (%) Unbroken / 0.243 Acid damage 100.6 0.206 Alkali damage 100.3 1.443 High temperature damage 100.6 0.223 Oxidative damage 100.7 0.583 Light damage 100.4 0.238

[0082] The material balance investigation data showed that the sample materials were basically conserved under each destruction condition, and the ratio of the total peak area of ​​each destroyed solution to the total peak area of ​​the undestroyed solution was between 90% and 110%; the main peak purity of each destruction condition was good (the peak purity value was greater than the set threshold of 990), and the material was balanced.

[0083] 3. Quantitative limit and detection limit

[0084] 50 μl of the mixed impurity solution of loxoprofen sodium oral solution was injected for analysis, and the chromatogram was recorded. The detection limit and quantification limit were determined at a signal-to-noise ratio of S / N≈3 and S / N≈10, respectively. The results are shown in Table 4.

[0085] Table 4 Detection limit results

[0086]

[0087]

[0088] It can be seen from Table 4 that the detection limits of loxoprofen sodium oral solution and each impurity are relatively small, which fully verifies that the detection method of the present invention has high detection sensitivity.

[0089] 4. Solution stability

[0090] Under room temperature conditions within 50 hours, the RSD of the peak area of ​​each component in the reference mixed solution was less than 2.0%, and the reference solution had good stability within 50 hours; the number of impurities in the test solution did not change, the RSD of the peak area of ​​each impurity was less than 5.0%, and the RSD of the peak area of ​​the main component was less than 2.0%, and the sample solution had good stability.

[0091] 5. Linear

[0092] Take about 13.6 mg of loxoprofen sodium reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add water to dissolve it and quantitatively dilute it to make a solution containing about 1.2 mg of loxoprofen sodium (calculated as anhydrous substance) per 1 ml, which is used as the loxoprofen sodium reference substance mother solution.

[0093] Take appropriate amount of impurity 1, impurity 4, impurity 6, and impurities 8-13 reference substances, weigh accurately, add acetonitrile to dissolve and dilute to prepare impurity reference substance mother solutions containing approximately 0.24 mg of impurities per 1 ml.

[0094] Accurately measure 1 ml of loxoprofen sodium reference substance mother solution and each impurity reference substance mother solution, place them in the same 20 ml volumetric flask, and quantitatively dilute them with solvent to prepare a reference substance mixed mother solution containing approximately 60 μg of loxoprofen sodium (calculated as anhydrous substance) and approximately 12 μg of each impurity per 1 ml.

[0095] Take the mixed solution of the quantitative limit concentration of each component as the linear minimum concentration point; at the same time, accurately measure 0.5ml, 1ml, 1.6ml, 2ml, 2.4ml and 3ml of the mixed mother solution of the reference substance, respectively, into a 10ml volumetric flask, dilute to the scale with solvent, shake well, and obtain a series of concentration solutions. Accurately pipette 20μl of each of the above series of gradient concentration solutions and quantitative limit concentration solutions, and analyze them in sequence from low concentration to high concentration, and record the chromatogram. With the concentration C (μg / ml) of the reference substance solution as the horizontal axis and the peak area of ​​the reference substance as the vertical axis, perform linear regression and obtain the regression equation. The results are shown in Table 5.

[0096] Table 5 Linear relationship investigation results

[0097]

[0098]

[0099]

[0100] It can be seen from Table 5 that the loxoprofen sodium oral solution and its impurities have a good linear relationship within the linear range.

[0101] 6. Determination of correction factor

[0102] On two liquid chromatographs, three chromatographic columns were used for a total of four times. Reference solutions of impurities and main components were prepared at the quantitative limit concentration of each impurity and main component, 50%, 80%, 100% (limit concentration), 120% and 150% of the specified limit concentration (0.2%) of the impurity, respectively. The peak area was regressed with mass concentration, and the correction factor of the impurity relative to loxoprofen sodium was calculated. The determination results are shown in Table 6.

[0103] Table 6 Correction factor determination results

[0104]

[0105]

[0106]

[0107] It can be seen from Table 6 that the correction factors of impurities 1, 6, 8, 10 and 11 are all between 0.9 and 1.1; impurities 4, 9, 12 and 13 need to be calculated after peak area correction.

[0108] 7. Accuracy

[0109] Accurately weigh 9 samples of loxoprofen sodium oral solution, add 80%, 100%, and 120% of the impurity limit of the impurity reference solution, respectively, add solvent to dissolve and dilute to the scale, accurately measure 50 μl, inject into liquid chromatograph, record the chromatogram, and calculate the recovery rate of each impurity as [(measured amount-background amount) / added amount]. The results are shown in Table 7.

[0110] Table 7 Impurity recovery determination results

[0111] Impurity No. Recovery rate results (%) Average recovery rate (%) RSD(%) 1 100.6～102.9 101.5 0.76 4 100.7～102.6 101.5 0.67 6 96.8～100.7 98.5 1.60 8 99.9～103.6 101.3 1.59 9 101.5～105.3 102.6 1.28 10 98.4～99.8 99.2 0.53 11 102.2～104.7 103.1 0.91 12 99.5～100.9 100.2 0.53 13 98.4～99.5 99.0 0.40

[0112] It can be seen from Table 7 that the recovery rates of various impurities are all between 92% and 105%, and the RSDs are all less than 5.0%, indicating that the recovery rates are good, indicating that the method of the present invention is suitable for the determination of related substances of this product.

[0113] 8. Repeatability

[0114] Under the test conditions, the same homogeneous sample was used and the same analyst took samples for a series of tests. The test results of known impurities 1 and 10 were compared using the known impurity external standard method and the self-control method with correction factors. The results are shown in Table 8.

[0115] Table 8 Impurity 1 and impurity 10 repeatability test results (external standard method and self-control method with correction factor)

[0116]

[0117] As can be seen from Table 8, there is no difference in the results of the known impurities calculated using the external standard method and the self-control method with correction factors, and the RSDs are all within 10.0%, indicating that the intermediate precision of the determination method of the present invention is good.

[0118] 9. Intermediate precision

[0119] Under the test conditions, the same homogeneous test product was used at different times, with different instruments, and by different analysts for a series of tests. The test results of known impurities 1 and 10 were calculated by the self-control method with correction factors, and the intermediate precision was examined. The results are shown in Table 9.

[0120] Table 9 Results of intermediate precision tests for impurities 1 and 10

[0121]

[0122] As can be seen from Table 9, the known impurities were calculated using the self-control method with a correction factor, and the results were the same, with RSDs within 10.0%, indicating that the intermediate precision of the determination method of the present invention was good.

[0123] Embodiment 2:

[0124] HPLC conditions:

[0125] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6mm, 5μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0ml / min, detection wavelengths of 220nm and 224nm, column temperature of 25°C, injection volume of 20μl.

[0126] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 80:5:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:15.

[0127] Preparation of sample plus mixed impurity solution and test sample solution: same as in Example 1.

[0128] Take the sample and add 20 μl of the mixed impurity solution and the test solution, inject and analyze, and record the chromatogram.

[0129] In this example, the wavelength was adjusted to 220nm and 224nm to investigate the separation between the peaks of loxoprofen sodium oral solution and the peaks of impurities and between impurities, as well as the detection of impurities in the sample. The results showed that the separation between the components was good, and the detection effect was the same as in Example 1.

[0130] In summary, the detection effect is good when the wavelength is in the range of 220nm to 224nm.

[0131] Embodiment 3:

[0132] HPLC conditions:

[0133] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6mm, 5μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0ml / min, detection wavelength of 222nm, column temperatures of 20°C and 30°C, respectively, and injection volume of 20μl.

[0134] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 80:5:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:15.

[0135] Preparation of sample plus mixed impurity solution and test sample solution: same as in Example 1.

[0136] Take the sample and add 20 μl of the mixed impurity solution and the test solution, inject and analyze, and record the chromatogram.

[0137] In this example, the column temperature was adjusted to 20°C and 30°C to investigate the separation between the peaks of loxoprofen sodium oral solution and the peaks of impurities and between impurities, as well as the detection of impurities in the sample. The results showed that the separation between the components was good, and the detection effect was the same as in Example 1.

[0138] In summary, the detection effect is good when the column temperature is in the range of 20℃~30℃.

[0139] Embodiment 4:

[0140] HPLC conditions:

[0141] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6mm, 5μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.3 and 2.7 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0ml / min, detection wavelength of 222nm, column temperature of 25°C, injection volume of 20μl.

[0142] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 80:5:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:15.

[0143] Preparation of sample plus mixed impurity solution and test sample solution: same as in Example 1.

[0144] Take the sample and add 20 μl of the mixed impurity solution and the test solution, inject and analyze, and record the chromatogram.

[0145] The pH value of mobile phase A in this example was adjusted to 2.3 and 2.7, and the separation between the peaks of loxoprofen sodium oral solution and the peaks of impurities and between impurities, as well as the detection of impurities in the sample were investigated. The results showed that the separation between the components was good, and the detection effect was the same as in Example 1.

[0146] In summary, the detection effect was good when the pH value of mobile phase A was in the range of 2.2 to 2.7.

[0147] Embodiment 5:

[0148] HPLC conditions:

[0149] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6 mm, 5 μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0 ml / min, detection wavelength of 222 nm, column temperature of 25°C, and injection volume of 20 μl.

[0150] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 82:3:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 82:3:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 82:3:15.

[0151] Preparation of sample plus mixed impurity solution and test sample solution: same as in Example 1.

[0152] Take the sample and add 20 μl of the mixed impurity solution and the test solution, inject and analyze, and record the chromatogram.

[0153] In this example, the volume ratio of the initial ratio of mobile phase A, mobile phase B and mobile phase C in the gradient elution program was adjusted from 80:5:15 to 82:3:15, and the termination ratio of the elution program was adjusted accordingly to investigate the separation between the peaks of loxoprofen sodium oral solution and the impurity peaks and between the impurities, as well as the detection of impurities in the sample. The results showed that the separation between the components was good, and the detection effect was the same as in Example 1.

[0154] Embodiment 6:

[0155] HPLC conditions:

[0156] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6 mm, 5 μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0 ml / min, detection wavelength of 222 nm, column temperature of 25°C, and injection volume of 20 μl.

[0157] The gradient elution program is as follows: (1) from 0 to 10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 78:7:15 to 70:15:15 at a constant speed; (2) from 10 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) from 40 to 60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) from 60 to 70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 78:7:15 at a constant speed; (5) from 70 to 80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is kept unchanged at 78:7:15.

[0158] Preparation of sample plus mixed impurity solution and test sample solution: same as in Example 1.

[0159] Take the sample and add 20 μl of the mixed impurity solution and the test solution, inject and analyze, and record the chromatogram.

[0160] In this example, the volume ratio of the initial ratio of mobile phase A, mobile phase B and mobile phase C in the gradient elution program was adjusted from 80:5:15 to 82:3:15, and the termination ratio of the elution program was adjusted accordingly to investigate the separation between the peaks of loxoprofen sodium oral solution and the impurity peaks and between the impurities, as well as the detection of impurities in the sample. The results showed that the separation between the components was good, and the detection effect was the same as in Example 1.

[0161] In summary, the gradient elution steps are as follows: (1) in 0-10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 78-82:7-3:15 to 70:15:15 at a constant speed; (2) in 10-40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) in 40-60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; The volume ratio of mobile phase B and mobile phase C was changed from 60:25:15 to 10:75:15 at a constant speed; (4) within 60-70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C was changed from 10:75:15 to 78-82:7-3:15 at a constant speed; (5) within 70-80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remained unchanged at 78-82:7-3:15. When the initial elution ratio and the final elution ratio were changed, the detection effect was good.

[0162] Comparative Example 1:

[0163] HPLC conditions:

[0164] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6 mm, 5 μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, methanol as mobile phase C, gradient elution, flow rate of 1.0 ml / min, detection wavelength of 222 nm, column temperature of 25°C, injection volume of 20 μl.

[0165] The gradient elution program was as follows: (1) from 0 to 5 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remained constant at 75:20:5; (2) from 5 to 40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C gradually changed from 75:20:5 to 30:65:5; (3) from 40 to 41 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C gradually changed from 30:65:5 to 75:20:5; (4) from 41 to 48 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remained constant at 75:20:5.

[0166] Preparation of sample plus mixed impurity solution: same as Example 1.

[0167] Take the sample and add 20 μl of the mixed impurity solution, inject and analyze, and record the chromatogram, such as Figure 5 shown.

[0168] like Figure 5 As shown, the peak time of impurity 6 is 21.052min, the peak time of methylparaben is 21.494min, and the peak time of impurity 1 is 22.335min. Impurity 6 and methylparaben are partially overlapped. Methylparaben interferes with impurity 6, affecting the accurate quantification of impurity 6. The peak of impurity 10 is 46.097min, which partially overlaps with the unknown impurity 45.769min, affecting the accurate quantification of impurity 10. The peaks of the excipients saccharin sodium and sodium citrate completely overlap.

[0169] Comparative Example 2:

[0170] HPLC conditions:

[0171] The chromatographic column was an octadecylsilane bonded silica column, model InertSustain C18 (250×4.6 mm, 5 μm), with 0.1% phosphoric acid aqueous solution (adjusted to pH 2.5 with triethylamine) as mobile phase A, acetonitrile as mobile phase B, gradient elution, flow rate of 1.0 ml / min, detection wavelength of 222 nm, column temperature of 25°C, and injection volume of 20 μl.

[0172] The gradient elution program was as follows: (1) from 0 to 15 minutes, the volume ratio of mobile phase A to mobile phase B was changed from 80:20 to 72:28 at a constant speed; (2) from 15 to 24 minutes, the volume ratio of mobile phase A to mobile phase B was changed from 72:28 to 35:65 at a constant speed; (3) from 24 to 30 minutes, the volume ratio of mobile phase A to mobile phase B was kept constant at 35:65; (4) from 30 to 34 minutes, the volume ratio of mobile phase A to mobile phase B was changed from 35:65 to 80:20 at a constant speed; (5) from 34 to 40 minutes, the volume ratio of mobile phase A to mobile phase B was kept constant at 80:20.

[0173] Preparation of sample plus mixed impurity solution: same as Example 1.

[0174] Take the sample and add 20 μl of the mixed impurity solution, inject and analyze, and record the chromatogram, such as Figure 6 shown.

[0175] like Figure 6 As shown, the peak time of impurity 6 is 13.326min; the peak time of impurity 1 is 13.778min, and the peak time of methylparaben is 14.140min. Impurity 1, impurity 6 and methylparaben partially overlap and interfere with each other, affecting the accurate quantification of impurities 1 and 6. The peak of impurity 4 is 24.857min, which partially overlaps with the unknown impurity 24.432min, affecting the accurate quantification of impurity 4.

[0176] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that it is still possible to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on some of the technical features therein. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting related substances in loxoprofen sodium oral solution, It is characterized in that The detection method uses high performance liquid chromatography to quantitatively detect the relevant substances in loxoprofen sodium oral solution, and the high performance liquid chromatography conditions include: The chromatographic column is octadecylsilane bonded silica gel, and gradient elution is performed with mobile phase A, mobile phase B and mobile phase C as mixed mobile phases, wherein the mobile phase A is a 0.05-0.15% phosphoric acid aqueous solution, and the pH value is adjusted to 2.0-4.0 with triethylamine; the mobile phase B is acetonitrile, and the mobile phase C is methanol. During the gradient elution process, the initial ratio of the mobile phase A, mobile phase B and mobile phase C is 78-82:7-3:

15. The specific gradient elution process is as follows: (1) in 0-10 minutes, the volume ratio of the mobile phase A, mobile phase B and mobile phase C is gradually changed from 78-82:7-3:15 to 70:15:15 at a uniform speed; (2) in 1 Within 0-40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:15 at a constant speed; (3) within 40-60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant speed; (4) within 60-70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 78-82:7-3:15 at a constant speed; (5) within 70-80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 78-82:7-3:15; the related substances are as follows:

2. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The initial ratio of mobile phase A, mobile phase B and mobile phase C in the gradient elution process is 80:5:

15. The specific gradient elution process is as follows: (1) in 0-10 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 80:5:15 to 70:15:15 at a constant speed; (2) in 10-40 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 70:15:15 to 60:25:1 5; (3) Within 40-60 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 60:25:15 to 10:75:15 at a constant rate; (4) Within 60-70 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C is gradually changed from 10:75:15 to 80:5:15 at a constant rate; (5) Within 70-80 minutes, the volume ratio of mobile phase A, mobile phase B and mobile phase C remains unchanged at 80:5:

15.

3. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The solvent for dissolving the sample is a mixed solution composed of mobile phase A and mobile phase B.

4. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 3, It is characterized in that In the mixed solution, the volume ratio of mobile phase A to mobile phase B is 75-85:25-15.

5. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 4, It is characterized in that In the mixed solution, the volume ratio of mobile phase A to mobile phase B was 80:

20.

6. A method for detecting related substances in the loxoprofen sodium oral solution according to any one of claims 1 to 5, It is characterized in that The mobile phase A is a 0.05-0.15% phosphoric acid aqueous solution, and the pH value is adjusted to 2.3-2.7 with triethylamine.

7. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 6, It is characterized in that The mobile phase A is a 0.1% phosphoric acid aqueous solution, and the pH value is adjusted to 2.3-2.7 with triethylamine.

8. According to the detection method of related substances in the loxoprofen sodium oral solution described in claim 7, It is characterized in that The pH was adjusted to 2.5 with triethylamine.

9. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The models of the chromatographic columns are Inertsil ODS-3C18, InertSustain C18 and Wondasil C18.

10. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 9, It is characterized in that The model of the chromatographic column is InertSustain C18.

11. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 10, It is characterized in that The length of the chromatographic column is 250 mm, the diameter is 4.6 mm, and the particle size of the filler is 5 μm.

12. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The chromatographic conditions include: a detection wavelength of 220 to 224 nm.

13. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 12, It is characterized in that The chromatographic conditions include: a detection wavelength of 222 nm.

14. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The chromatographic conditions include: a column temperature of 20 to 30°C.

15. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 14, It is characterized in that The chromatographic conditions include: a column temperature of 25°C.

16. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The chromatographic conditions include: a flow rate of 0.5 to 1.5 mL / min.

17. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 16, It is characterized in that The chromatographic conditions include: a flow rate of 1.0 mL / min.

18. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 1, It is characterized in that The chromatographic conditions include: the injection volume is 10 to 50 μl.

19. The method for detecting related substances in the loxoprofen sodium oral solution according to claim 18, It is characterized in that The chromatographic conditions include: the injection volume is 20 μl.

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