Method for quantitatively detecting dexketoprofen trometamol injection and related substance levoketoprofen of dexketoprofen trometamol injection by high performance liquid chromatography

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for quantitatively detecting dexketoprofen trometamol injection and a related substance levoketoprofen of the dexketoprofen trometamol injection through high performance liquid chromatography. The method for quantitatively detecting the dexketoprofen trometamol injection and the related substance levoketoprofen of the dexketoprofen trometamol injection by the high performance liquid chromatography comprises the following steps: diluting the dexketoprofen trometamol injection with a solvent, and detecting by the high performance liquid chromatography, chromatographic conditions are as follows: a chromatographic column uses cellulose-tri-(4-methyl benzoate) as a filling agent; a mobile phase is an n-hexane-isopropanol-trifluoroacetic acid mixed solution; the flow velocity is 0.6 to 1.0 mL / min. The method for quantitatively detecting the dexketoprofen trometamol injection and the related substance levoketoprofen of the dexketoprofen trometamol injection through the high performance liquid chromatography, provided by the invention, is simple to operate and high in sensitivity, and has good specificity, system applicability, solution stability and chromatographic condition durability.

Description

Technical Field

[0001] The invention belongs to the technical field of drug detection, and particularly relates to a method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography. Background Art

[0002] Dexketoprofen tromethamine is the tromethamine salt of dexketoprofen, a non-steroidal anti-inflammatory drug, mainly used to relieve pain, reduce inflammation and reduce fever. It reduces the production of prostaglandins by inhibiting prostaglandin synthase (COX), thereby achieving the effect of relieving inflammation and pain. It can be used to treat a variety of diseases, such as arthritis, gout, dysmenorrhea, toothache, etc. It has injections, tablets, capsules, gels and other dosage forms, and is widely used in clinical practice. Dexketoprofen tromethamine injection is a non-steroidal anti-inflammatory drug.

[0003] Dexketoprofen tromethamine, the active ingredient of dexketoprofen tromethamine injection, is the dextrorotatory isomer of ketoprofen. Levoketoprofen will be present during the synthesis or purification of dexketoprofen, and levoketoprofen differs from dexketoprofen in efficacy and safety. Accurate detection and control of the levoketoprofen content in the injection is crucial to ensure drug quality and efficacy.

[0004] Currently, dexketoprofen tromethamine injection is not included in the Chinese Pharmacopoeia. Although the raw material dexketoprofen tromethamine is included, the recommended chromatographic column has been discontinued. Therefore, there is currently no method available for the detection of levoketoprofen in dexketoprofen tromethamine injection. A simple, efficient, sensitive and widely used method is urgently needed to detect levoketoprofen. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide a method for quantitatively detecting dexketoprofen trometamol injection and its related substance levoketoprofen by high performance liquid chromatography. The method is simple to operate, easy to control, highly sensitive, has good specificity, system applicability, solution stability and durability of chromatographic conditions, and provides an effective detection method for drug quality control.

[0006] The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography of the present invention is as follows: dexketoprofen tromethamine injection is diluted with a solvent and then detected by high performance liquid chromatography, and the chromatographic conditions are as follows: Chromatographic column: Cellulose-tri-(4-methylbenzoate) was used as filler; Mobile phase: a solution of n-hexane-isopropanol-trifluoroacetic acid in a volume ratio of 80-92:20-8:0.1, isocratic elution; Flow rate: 0.6~1.0mL / min; Column temperature: 23-30°C; Detection wavelength: 254~280nm.

[0007] Preferably, the dexketoprofen tromethamine injection is diluted with a solvent to a concentration of 0.5 mg / mL, and the solvent used is ethanol.

[0008] Preferably, the chromatographic column is a normal phase chiral chromatographic column.

[0009] Preferably, the mobile phase is a solution of n-hexane-isopropanol-trifluoroacetic acid mixed in a volume ratio of 90:10:0.1.

[0010] Preferably, the column temperature is 25°C.

[0011] Preferably, the detection wavelength is 270 nm.

[0012] The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography comprises the following steps: (1) Prepare the test solution; (2) Prepare control solution; (3) Prepare system suitability solution; (4) Detection: Take the test solution, control solution, and system suitability solution and inject them into the HPLC respectively. Record the chromatograms and separate and detect dexketoprofen trometamol and its related substance levoketoprofen by the self-control method.

[0013] Step (2) The steps for preparing the control solution are as follows: accurately measure an appropriate amount of the test solution, and quantitatively dilute it with ethanol to prepare a solution containing 0.1 μg to 7.5 μg of dexketoprofen per 1 mL.

[0014] Step (3) The steps for preparing the system suitability solution are as follows: take the ketoprofen reference substance, dissolve it in ethanol and dilute it to make a solution containing 0.1 μg to 1 mg per 1 mL.

[0015] Specifically, the method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography comprises the following steps: (1) Preparation of test solution: Dilute dexketoprofen tromethamine injection with ethanol to a concentration of 0.5 mg / mL.

[0016] (2) Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with ethanol to make a solution containing 0.1 μg to 7.5 μg of dexketoprofen per 1 mL.

[0017] (3) Prepare system suitability solution: Take ketoprofen reference substance, dissolve it in ethanol and dilute it to make a solution containing 0.1 μg~1 mg per 1 mL.

[0018] (4) Select chromatographic conditions: Chromatographic column: Cellulose-tri-(4-methylbenzoate) was used as filler; Mobile phase: a solution of n-hexane-isopropanol-trifluoroacetic acid in a volume ratio of 80-92:20-8:0.1, isocratic elution; Flow rate: 0.6~1.0mL / min; Column temperature: 23-30°C; Detection wavelength: 254~280nm; Injection volume: 20 μL.

[0019] (5) Detection: Take the test solution, control solution, and system suitability solution and inject them into the HPLC respectively. Record the chromatograms and calculate the content of levoketoprofen in dexketoprofen tromethamine injection by the self-control method.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The method described in the present invention can achieve effective separation of levoketoprofen and dexketoprofen in dexketoprofen tromethamine injection; and can accurately detect the amount of levoketoprofen in dexketoprofen tromethamine injection.

[0021] (2) The method for detecting levoketoprofen in dexketoprofen tromethamine injection described in the present invention is simple to operate, easy to control, highly sensitive, and has good specificity, system applicability, solution stability, and durability of chromatographic conditions, thereby providing an effective detection method for the formulation of quality standards for dexketoprofen tromethamine injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the chromatogram of the blank solvent of Example 1; Figure 2 is a chromatogram of the system suitability solution of Example 1; Figure 3 is the chromatogram of the test solution of Example 1; Figure 4 is the chromatogram of the control solution of Example 1; Figure 5 is a chromatogram of the system suitability solution of Example 2; Figure 6 is the chromatogram of the system suitability solution of Example 3; Figure 7 is a chromatogram of the system suitability solution of Example 4; Figure 8is the chromatogram of the system suitability solution of Example 5; Fig. 9 is the chromatogram of the system suitability solution of Example 6; Fig.10 is the chromatogram of the system suitability solution of Example 7; Fig.11 is the chromatogram of the system suitability solution of Example 8; Fig.12 is the chromatogram of the system suitability solution of Example 9; Fig.13 is the chromatogram of the quantitative limit solution of Example 10; Fig.14 It is the linear relationship diagram of dexketoprofen in Example 11. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments.

[0024] The raw materials and additives used in the following examples are all normal commercially available products.

[0025] Dexketoprofen tromethamine injection: produced by Shandong Qidu Pharmaceutical Co., Ltd.

[0026] Ketoprofen: purchased from China Food and Drug Inspection Institute.

[0027] The ethanol used in the present invention refers to anhydrous ethanol.

[0028] Example 1 The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography comprises the following steps: (1) Preparation of test solution: Dilute dexketoprofen tromethamine injection with ethanol to a concentration of 0.5 mg / mL.

[0029] (2) Preparation of control solution: Accurately measure an appropriate amount of the test solution and quantitatively dilute it with ethanol to produce a solution containing 5 μg of dexketoprofen per 1 mL.

[0030] (3) Prepare system suitability solution: Take ketoprofen reference substance, dissolve it in ethanol and dilute it to make a solution containing 1 mg per 1 mL.

[0031] (4) Select chromatographic conditions: Chromatographic column: Cellulose-tri-(4-methylbenzoate) as filler, Pheromone Chiral MJ (2), 250×4.6mm, 5μm; Mobile phase: a solution of n-hexane-isopropanol-trifluoroacetic acid in a volume ratio of 90:10:0.1, isocratic elution; Flow rate: 0.8 mL / min; Column temperature: 25°C; Detection wavelength: 270nm; Detector: UV detector; Injection volume: 20 μL; Blank solvent: ethanol.

[0032] (5) Detection: Take blank solvent, test solution, control solution, and system suitability solution and inject them into HPLC respectively. Record the chromatogram and calculate the content of levoketoprofen in dexketoprofen tromethamine injection by the self-control method.

[0033] like Figures 1 to 4 As shown, it can be seen that there is no interference in the blank, the left ketoprofen peak and the right ketoprofen peak of the system suitability solution are well separated, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected.

[0034] Example 2 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the mobile phase in the chromatographic conditions is changed to: a solution of n-hexane-isopropanol-trifluoroacetic acid mixed in a volume ratio of 80:20:0.1, and isocratic elution.

[0035] like Figure 5 As shown, it can be seen that the left ketoprofen peak and the right ketoprofen peak in the system suitability solution have good separation, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable under this mobile phase ratio.

[0036] Example 3 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the mobile phase in the chromatographic conditions is changed to: a solution of n-hexane-isopropanol-trifluoroacetic acid mixed in a volume ratio of 92:8:0.1, and isocratic elution.

[0037] like Figure 6 As shown, it can be seen that the left ketoprofen peak and the right ketoprofen peak in the system suitability solution have good separation, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable under this mobile phase ratio.

[0038] Example 4 The other conditions and steps of this embodiment are the same as those of embodiment 1, and a diode array detector is used to change the detection wavelength in the chromatographic conditions to 254 nm.

[0039] like Figure 7 As shown, it can be seen that the system suitability solution has good separation between the left ketoprofen peak and the right ketoprofen peak, and can accurately detect the amount of left ketoprofen in the right ketoprofen tromethamine injection. The method is applicable at this wavelength.

[0040] Example 5 The other conditions and steps of this embodiment are the same as those of embodiment 1, and the detection wavelength in the chromatographic condition is changed to 280 nm by using a diode array detector.

[0041] like Figure 8 As shown, it can be seen that the left ketoprofen peak and the right ketoprofen peak in the system suitability solution have good separation, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable at this wavelength.

[0042] Example 6 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the column temperature in the chromatographic condition is changed to 23°C.

[0043] like Fig. 9 As shown, it can be seen that the separation degree of the left ketoprofen peak and the right ketoprofen peak in the system suitability solution is good, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable at this column temperature.

[0044] Example 7 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the column temperature in the chromatographic condition is changed to 30°C.

[0045] like Fig.10 As shown, it can be seen that the separation degree of the left ketoprofen peak and the right ketoprofen peak in the system suitability solution is good, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable at this column temperature.

[0046] Example 8 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the flow rate in the chromatographic condition is changed to 0.6 mL / min.

[0047] like Fig.11 As shown, it can be seen that the left ketoprofen peak and the right ketoprofen peak in the system suitability solution have good separation, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable at this flow rate.

[0048] Example 9 The other conditions and steps of this embodiment are the same as those of embodiment 1, except that the flow rate in the chromatographic condition is changed to 1.0 mL / min.

[0049] like Fig.12 As shown, it can be seen that the left ketoprofen peak and the right ketoprofen peak in the system suitability solution have good separation, and the amount of left ketoprofen in the right ketoprofen tromethamine injection can be accurately detected. The method is applicable at this flow rate.

[0050] Example 10 The other conditions and steps of this example are the same as those of Example 1. A ketoprofen reference substance is taken, dissolved in ethanol and diluted to prepare a solution containing approximately 0.1 μg of dexketoprofen and 0.1 μg of levoketoprofen per mL. Six portions are prepared in parallel as the quantitative limit solutions.

[0051] like Fig.13 As shown, it can be seen that the separation degree of the left ketoprofen peak and the right ketoprofen peak of the quantitative limit solution is good, the signal-to-noise ratio of each component is greater than 10, and the precision RSD of each component after repeated determination for 6 times is less than 10. At this concentration, the amount of left ketoprofen in right ketoprofen tromethamine injection can be accurately detected. At this concentration, the method has good sensitivity and is applicable.

[0052] Embodiment 11 The other conditions and steps of this example are the same as those of Example 1. A ketoprofen reference substance is taken, dissolved in ethanol and diluted to prepare solutions containing approximately 0.1 μg, 1.25 μg, 2.5 μg, 5.0 μg, and 7.5 μg of dexketoprofen per 1 mL as a series of linear concentration solutions.

[0053] like Fig.14 As shown, it can be seen that within the concentration range of 0.1 μg / mL to 7.5 μg / mL, the linear relationship is good, and the amount of left ketoprofen in right ketoprofen tromethamine injection can be accurately detected within this concentration range. The method is applicable within this concentration range.

[0054] The method of quantitatively detecting dexketoprofen trometamol injection and its related substance levoketoprofen by high performance liquid chromatography of the present invention is subjected to the following verifications: specificity verification, system suitability verification, verification of quantitative limit and detection limit, verification of precision, verification of solution stability, and verification of durability.

[0055] a) Specificity verification Blank solvent: ethanol.

[0056] Auxiliary material solution: 10wt% ethanol, 0.4wt% sodium chloride, and 89.6wt% water were prepared according to the mass percentage to prepare the auxiliary material solution.

[0057] Blank excipient: Accurately measure 2 mL of excipient solution, place in a 100 mL volumetric flask, dilute to the mark with ethanol, and shake well.

[0058] Test solution: Accurately measure 2 mL of the product, place it in a 100 mL volumetric flask, dilute to the mark with ethanol, and shake well.

[0059] Control solution: Accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, dilute to the mark with ethanol, and shake well.

[0060] System suitability solution: Take 10.24 mg of levoketoprofen reference substance, accurately weigh it, place it in a 10 mL volumetric flask, add ethanol to dissolve and dilute to the scale, and shake well.

[0061] The above solutions were taken and injected into liquid chromatographs respectively, and measured according to the chromatographic conditions of the above Example 1, and the chromatograms were recorded.

[0062] The chromatogram of the blank solvent tested is as follows: Figure 1 The chromatogram of the system suitability solution is shown in Figure 2 The chromatogram of the test solution is shown in Figure 3 The chromatogram of the control solution is shown in Figure 4 The specificity test results are shown in Table 1.

[0063] Table 1 Specificity test results

[0064] From the above Figure 1 , Figure 2 and Figure 3 As can be seen from Table 1, the blank solvent does not interfere with the detection of levoketoprofen, and the separation degree between levoketoprofen and dextroketoprofen is greater than 1.5, and the separation effect is good.

[0065] b) System suitability verification Take the system suitability solution under the specificity item, inject it 6 times continuously according to the above method, and record the chromatogram. The test results are shown in Table 2.

[0066] Table 2 System suitability test results

[0067] It can be seen from Table 2 that the RSD of the peak area of ​​each component of the system suitability solution after 6 consecutive injections was less than 2%, and the RSD of the retention time was less than 1%. In the chromatogram of the system suitability solution, the left ketoprofen peak and the right ketoprofen peak appeared in sequence, the separation between the two peaks was greater than 1.5, and the theoretical plate number calculated based on the right ketoprofen peak was not less than 2000, indicating that the method system had good applicability.

[0068] c) Quantification limit and detection limit verification Take ketoprofen reference substance, dissolve it in ethanol and dilute it to obtain the corresponding limit of quantification and limit of detection concentration. When the signal-to-noise ratio S / N is not less than 10 and the RSD of the precision of repeated injections 6 times is ≤10%, this concentration is determined as LOQ. The LOQ solution is diluted 3 times to determine the LOD. In addition, the LOD of the system is determined based on S / N≈3. The test results are shown in Table 3.

[0069] Table 3 Results of limit of quantitation and limit of detection tests

[0070] It can be seen from Table 3 above that the quantification limit concentration of left-ketoprofen is 0.1257μg / mL, the signal-to-noise ratio is greater than 10, the RSD of the peak area of ​​the quantification limit precision is 4.1%, and the detection limit concentration is 0.0377μg / mL; the quantification limit concentration of right-ketoprofen is 0.1268μg / mL, the signal-to-noise ratio is greater than 10, the RSD of the peak area of ​​the quantification limit precision is 5.5%, and the detection limit concentration is 0.0380μg / mL.

[0071] d) Precision verification Prepare 6 test sample solutions respectively, inject them according to the above method, and record the chromatograms. Another experimenter sets up the system separately, re-prepare 6 identical test sample solutions for testing, and use different instruments to conduct intermediate precision tests. The verification results are shown in Table 4.

[0072] Table 4 Precision test results

[0073] It can be seen from Table 4 above that the average content of the 12 determination results is 0.23%, the RSD is 0%, and the method precision is good.

[0074] e) Solution stability verification Take the test solution and the control solution under the specificity item and inject them at different times for determination, and record the chromatogram. The verification results are shown in Table 5.

[0075] Table 5 Solution stability test results

[0076] It can be seen from Table 5 above that the RSD of the peak area of ​​the test solution and the control solution within 44 hours was less than 2%, and the solution stability was good.

[0077] f) Durability verification Changing the flow rate: Keeping other test conditions unchanged, change the flow rate by ±0.2mL / min, i.e. 0.6mL / min and 1.0mL / min.

[0078] Changing column temperature: While keeping other test conditions unchanged, change the column temperature by ±2°C, i.e. 23°C and 27°C.

[0079] Changing the ratio of mobile phase: While keeping other conditions unchanged, the ratio of n-hexane and isopropanol in the mobile phase was changed by ±2, i.e., n-hexane-isopropanol-trifluoroacetic acid (88:12:0.1) and n-hexane-isopropanol-trifluoroacetic acid (92:8:0.1).

[0080] Take the blank solution, system suitability solution, test solution and control solution under the specificity item and measure them according to the above method. The verification results are shown in Table 6.

[0081] Table 6 Durability test results

[0082] It can be seen from Table 6 above that when the flow rate of the chromatographic condition parameters is changed by ±0.2 mL / min, the column temperature is changed by ±2°C, and the ratio of n-hexane and isopropanol in the mobile phase is changed by ±2, the slight changes in different chromatographic parameters have almost no effect on the detection of levoketoprofen, and the chromatographic conditions have good durability.

[0083] In summary, it can be seen that the detection method of left ketoprofen in the right ketoprofen tromethamine injection of the present invention has high sensitivity, good separation, strong specificity, good durability, and accurate method, which is of great significance to the quality control of the right ketoprofen tromethamine injection.

Claims

1. A method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography, characterized in that: After dexketoprofen tromethamine injection was diluted with a solvent, it was detected by high performance liquid chromatography. The chromatographic conditions were as follows: Chromatographic column: Cellulose-tri-(4-methylbenzoate) was used as filler; Mobile phase: a solution of n-hexane-isopropanol-trifluoroacetic acid in a volume ratio of 80-92:20-8:0.1, isocratic elution; Flow rate: 0.6~1.0mL / min; Column temperature: 23-30°C; Detection wavelength: 254~280nm.

2. the method for quantitatively detecting dexketoprofen tromethamine injection and related substance levoketoprofen thereof by high performance liquid chromatography according to claim 1, is characterized in that: The solvent is ethanol.

3. The method for quantitatively detecting dexketoprofen tromethamine injection and related substance levoketoprofen thereof by high performance liquid chromatography according to claim 1, characterized in that: The chromatographic column is a normal phase chiral chromatographic column.

4. The method for quantitatively detecting dexketoprofen tromethamine injection and related substance levoketoprofen thereof by high performance liquid chromatography according to claim 1, characterized in that: The mobile phase is a solution of n-hexane-isopropanol-trifluoroacetic acid mixed in a volume ratio of 90:10:0.

1.

5. The method for quantitatively detecting dexketoprofen tromethamine injection and related substance levoketoprofen thereof by high performance liquid chromatography according to claim 1, characterized in that: The column temperature is 25°C.

6. The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography according to claim 1, characterized in that: The detection wavelength is 270nm.

7. The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography according to claim 1, characterized in that: The following steps are involved: (1) Prepare the test solution; (2) Prepare control solution; (3) Prepare system suitability solution; (4) Detection: Take the test solution, control solution, and system suitability solution and inject them into the HPLC respectively. Record the chromatograms and separate and detect dexketoprofen trometamol and its related substance levoketoprofen by the self-control method.

8. The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography according to claim 7, characterized in that: The steps of preparing the control solution in step (2) are as follows: accurately measure the test solution, and quantitatively dilute it with ethanol to prepare a solution containing 0.1 μg to 7.5 μg of dexketoprofen per 1 mL.

9. The method for quantitatively detecting dexketoprofen tromethamine injection and its related substance levoketoprofen by high performance liquid chromatography according to claim 7, characterized in that: Step (3) The steps for preparing the system suitability solution are as follows: take the ketoprofen reference substance, dissolve it in ethanol and dilute it to make a solution containing 0.1 μg to 1 mg per 1 mL.

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