Method for detecting content of Cibopadol in Cibopadol citrate

Abstract

The invention discloses a high performance liquid chromatography detection method for the content of cibopadol in cibopadol citrate. Chromatographic conditions are as follows: a reversed-phase chromatographic column is adopted, and a phosphate buffer solution and tetrahydrofuran-acetonitrile are taken as mobile phases. By detecting the content of the cibopadol in the cibopadol citrate, the quality of a cibopadol preparation product is favorably controlled, whether the content of the cibopadol meets the requirement or not is judged, and the stability of the cibopadol product is improved. The detection method disclosed by the invention is high in accuracy, good in separation degree, good in stability, simple and convenient to operate, good in repeatability, strong in pertinence and very good in application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting the content of Xibomopar in Xibomopar citrate. BACKGROUND

[0002] Xibomopar (trans-6-fluoro-4,9-dihydro-N,N-dimethyl-4-phenyl-spiro[cyclohexane-1,1(3H)-pyrano[3,4-b]indol]-4-amine) is an analgesic nociceptin / orphanin FQ peptide (NOP) and opioid receptor agonist. It is currently developed by German company Grunenthal GmbH and its partner DepoMed, a US pharmaceutical company, for the treatment of a variety of different acute and chronic pain states. Xibomopar shows highly potent analgesic and antihypertensive effects in various different pain animal models. Notably, it has also been found to be more effective in chronic neuropathic pain models than in acute nociceptive pain compared to selective mu-opioid receptor agonists. Tolerance to the analgesic effects of Xibomopar has been found to be delayed (26 days versus 11 days for complete tolerance) relative to morphine. Moreover, unlike morphine, Xibomopar has not been found to affect motor coordination or reduce animal respiration at or above analgesic dose ranges. Thus, it can have improved and prolonged effectiveness and greater tolerance compared to currently available opioid analgesics.

[0003] At present, there is no method for detecting the content of Xibomopar, and it is necessary to design a method for detecting the content of Xibomopar, which can help to accurately and effectively reflect the quality of Xibomopar in the raw material, so as to effectively control the quality of the subsequent prepared Xibomopar preparation. SUMMARY

[0004] In order to solve the above technical problems, the present application aims to provide a method for detecting the content of Xibomopar in Xibomopar citrate, which has good specificity, sensitivity and stability.

[0005] In one embodiment, the present application provides a method for detecting the content of Xibomopar in Xibomopar citrate, characterized in that high performance liquid chromatography is used, and the chromatographic conditions include: using a reversed-phase chromatographic column, and using phosphate buffer and tetrahydrofuran-acetonitrile as the mobile phase.

[0006] In one embodiment, the chromatographic column is a C18 chromatographic column.

[0007] In one embodiment, the C18 chromatographic column has a specification of 4.6*250mm, 5.0μm.

[0008] In one embodiment, the column temperature is 25-45℃, and preferably, the column temperature is 30-40℃.

[0009] In one embodiment, the mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is 0.005-0.09 mol / L phosphate buffer (pH 6.5-8.5), and mobile phase B is tetrahydrofuran-acetonitrile.

[0010] In one embodiment, mobile phase A is 0.02 mol / L phosphate buffer (pH 7.5), and tetrahydrofuran-acetonitrile is 1:5.

[0011] In one embodiment, the flow rate of the mobile phase is 0.8-1.2 mL / min, and the detection wavelength is 215-225 nm.

[0012] In one embodiment, the injection volume is 5-15 μL.

[0013] In one embodiment, the high performance liquid chromatography is used, and the chromatographic conditions comprise: using a C18 column; using an ultraviolet detector for detection, and the detection wavelength is 215-225 nm; mobile phase A is 0.02 mol / L phosphate buffer (pH 7.5), and mobile phase B is tetrahydrofuran-acetonitrile; the column temperature is 30-40 °C; the flow rate is 0.8-1.2 mL / min; and the isocratic elution is: mobile phase A: mobile phase B = 40:60.

[0014] The present application provides a method for detecting the content of sibopirdine in sibopirdine citrate, comprising the following steps:

[0015] (1) preparing a test sample solution and a control sample solution;

[0016] (2) detection: detecting according to the above detection method;

[0017] (3) calculating the content of sibopirdine in the detection spectrum of step (2).

[0018] In one embodiment, the concentration of the test sample solution in step (1) is 0.1-2 mg / mL.

[0019] In one embodiment, the content of sibopirdine in step (3) is calculated according to the external standard method.

[0020] The present application establishes a detection method for the content of sibopirdine in sibopirdine citrate, which can accurately and effectively reflect the quality of sibopirdine in the raw material, thereby effectively controlling the quality of the subsequent prepared sibopirdine preparation. The detection method of the present application can accurately detect the content of sibopirdine in sibopirdine citrate, and has good specificity, sensitivity, stability, standard curve, accuracy, repeatability, and durability. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 HPLC chromatogram of the test sample;

[0022] Figure 2 HPLC chromatogram of the reference sample. DETAILED DESCRIPTION

[0023] Specificity experiment of Example 1

[0024] 1. Preparation of reference sample and test sample solution

[0025] The specificity experiment needs to verify that the blank solution and each impurity do not interfere with the main peak retention time in the test sample and reference sample solutions.

[0026] The following is the preparation method of each impurity and main component:

[0027] Test sample solution preparation method: take about 13 mg of the product, accurately weigh, place in a 100 mL volumetric flask, add about 50 mL of tetrahydrofuran and ultrasonically dissolve, dilute to the mark with solvent, shake well, and use as the test sample solution.

[0028] Reference sample solution preparation method: take about 13 mg of the reference sample, accurately weigh, place in a 100 mL volumetric flask, add about 50 mL of tetrahydrofuran and ultrasonically dissolve, dilute to the mark with solvent, shake well, and use as the reference sample solution.

[0029] 2. Detection chromatographic conditions

[0030] Chromatographic column: C18 (Thermo Hypersil BDS-C18 250mm x 4.6mm, 5μm);

[0031] Detector: ultraviolet detector;

[0032] Detection wavelength: 220nm;

[0033] Mobile phase: mobile phase A and mobile phase B; wherein, mobile phase A is 0.02 mol / L phosphate buffer (pH 7.5 adjusted with phosphoric acid), and mobile phase B is tetrahydrofuran-acetonitrile (10:50);

[0034] Use mobile phase A and mobile phase B for isocratic gradient elution; A-B 40:60;

[0035] Flow rate: 1.0 mL / min;

[0036] Column temperature: 35℃;

[0037] Diluent: 50% acetonitrile;

[0038] Injection volume: 10μL.

[0039] Refer to the above detection chromatographic conditions, respectively, 10 μL of the above solution, injection liquid chromatograph, record chromatogram Figure 1 and chromatography Figure 2 . According to Figure 1 and Figure 2 The results show that the blank solution has no interference with the Xibopa peak, indicating that the detection method of the application meets the requirements of specificity.

[0040] Example 2 Sensitivity test

[0041] Xibopa control solution: take Xibopa control product, accurately weighed, add solvent, dissolve with solvent, dilute to appropriate multiple, with signal-to-noise ratio ≥10:1 solution as the limit of quantification solution; with signal-to-noise ratio ≥3:1 solution as the limit of detection solution.

[0042] Refer to the detection chromatographic conditions of Example 1, respectively, 10 μL of the above solution, injection liquid chromatograph, record chromatogram, the results are shown in Table 1.

[0043] Table 1 limit of detection and limit of quantification results

[0044]

[0045]

[0046] As can be seen from Table 1, the detection limit of the detection method of the application is 0.05%, the quantification limit is 0.16%, which shows that the sensitivity of the detection method of the application meets the requirements.

[0047] Example 3 Solution stability test

[0048] Test solution preparation method: take about 13 mg of the product, accurately weighed, placed in a 100 mL volumetric flask, added about 50 mL of tetrahydrofuran, ultrasonic dissolved, diluted to the mark with solvent, shaken, as test solution.

[0049] Control solution preparation method: take about 13 mg of the control product, accurately weighed, placed in a 100 mL volumetric flask, added about 50 mL of tetrahydrofuran, ultrasonic dissolved, diluted to the mark with solvent, shaken, as test solution.

[0050] Refer to the detection chromatographic conditions of Example 1, respectively, 10 μL of the above solution, injection liquid chromatograph, record chromatogram, the results are shown in Table 2.

[0051] Table 2 solution stability test results

[0052] Name 0h 2h 4h 6h 8h 10h 20 24 36 RSD % Control solution 4404175 4381991 4394198 4406919 4416577 4432412 4445104 4519709 4583847 1.50 Test solution 4533570 4524684 4536808 4546351 4557106 4562806 4572072 4620032 4673635 1.05

[0053] From Table 2, the peak area of the test solution is <3% within 36 hours at room temperature, and the peak area of the control solution is <3% within 36 hours, indicating that the stability of the detection method meets the requirements.

[0054] Example 4 Standard curve test

[0055] Preparation of standard curve stock solution:

[0056] 1. 50% linear solution: Take about 6.5 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 50% linear solution.

[0057] 2. 80% linear solution: Take about 10.4 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 80% linear solution.

[0058] 3. 90% linear solution: Take about 11.7 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 90% linear solution.

[0059] 4. 100% linear solution: Take about 13 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 100% linear solution.

[0060] 5. 110% linear solution: Take about 14.3 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 110% linear solution.

[0061] 6. 120% linear solution: Take about 15.6 mg of the test sample, accurately weigh, and place it in a 100 mL volumetric flask. Add about 50 mL of tetrahydrofuran and ultrasonically dissolve. Dilute to the mark with solvent, shake well, and use as the 120% linear solution.

[0062] Accurately take 10 μL of each of the different concentrations of the curve solution, inject it into the liquid chromatograph, and record the chromatogram. The results are shown in Table 3.

[0063] Table 3 Standard curve test results

[0064]

[0065] As can be seen from Table 3, the peak area of Xibopa shows a good linear relationship in the concentration range of 2.08-31.27 μg / mL. This indicates that the standard curve of the detection method meets the requirements.

[0066] Repeatability test of example 5

[0067] Preparation of test sample solution: about 13 mg of test sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added to dissolve the sample under ultrasonic, and then the solvent was added to the mark, and the mixture was shaken to prepare a test sample solution. Six samples were prepared in parallel.

[0068] Preparation of control sample solution: about 13 mg of control sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added to dissolve the sample under ultrasonic, and then the solvent was added to the mark, and the mixture was shaken to prepare a control sample solution. Two samples were prepared in parallel.

[0069] The above solutions were accurately measured at 10 μL each, injected into a liquid chromatograph, and the chromatogram was recorded under the detection chromatographic conditions of reference example 1. The results are shown in Table 4.

[0070] Table 4 Repeatability test results

[0071] Name 1 2 3 4 5 6 AVE RSD % Content 79.43 79.35 78.94 79.49 78.77 79.32 79.2 0.37

[0072] As can be seen from Table 4, the content of sibopirdine in the six sibopirdine samples was in the range of 78% to 82%, with an average content of 79.2% and an RSD of 0.37 < 2.0%. This indicates that the repeatability of the detection method meets the requirements.

[0073] Accuracy test of example 6

[0074] Preparation of control sample solution: about 13 mg of control sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added to dissolve the sample under ultrasonic, and then the solvent was added to the mark, and the mixture was shaken to prepare a control sample solution. Two samples were prepared in parallel.

[0075] Preparation of 80% test sample solution: about 10.4 mg of test sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added to dissolve the sample under ultrasonic, and then the solvent was added to the mark, and the mixture was shaken to prepare a 80% test sample solution. Three samples were prepared in parallel.

[0076] Preparation of 100% test sample solution: about 13 mg of test sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added to dissolve the sample under ultrasonic, and then the solvent was added to the mark, and the mixture was shaken to prepare a 100% test sample solution. Three samples were prepared in parallel.

[0077] The above solutions were accurately measured at 10 μL each, injected into a liquid chromatograph, and the chromatogram was recorded under the detection chromatographic conditions of reference example 1. The results are shown in Table 5.

[0078] Table 5 Accuracy test results

[0079]

[0080] From Table 5, the content of Ceftobiprole is 78.54%~79.71%, the average content is 79.2%, RSD is 0.56<2.0%, which shows that the accuracy of the detection method meets the requirements.

[0081] Example 7 Durability test

[0082] The content of Ceftobiprole in Ceftobiprole citrate was determined by changing the proportion of mobile phase, wavelength, column temperature, flow rate, different concentrations of phosphate solution and pH value.

[0083] Preparation of test sample solution: about 13 mg of test sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added and dissolved by ultrasonic, and then diluted to the mark with solvent, shaken well, and used as the test sample solution.

[0084] Preparation of control sample solution: about 13 mg of control sample was accurately weighed into a 100 mL volumetric flask, about 50 mL of tetrahydrofuran was added and dissolved by ultrasonic, and then diluted to the mark with solvent, shaken well, and used as the control sample solution, and two samples were prepared in parallel.

[0085] Referring to the detection chromatographic conditions of Example 1, 10 μL of each of the above solutions was accurately measured and injected into the liquid chromatograph, and the chromatogram was recorded, and the results are shown in Tables 6-11.

[0086] Table 6 Durability test-different wavelength experimental results

[0087]

[0088] Table 7 Durability test-different column temperature experimental results

[0089]

[0090]

[0091] Table 8 Durability test-different flow rate experimental results

[0092]

[0093] Table 9 Durability test-different flow phosphate concentration experimental results

[0094]

[0095] Table 10 Durability test-different pH experimental results

[0096]

[0097] Table 11 Durability test results

[0098]

[0099] As can be seen from Tables 6-11, the content of Xibomaba meets the requirements and the RSD% is less than or equal to 10% under different chromatographic conditions, indicating that the robustness of the detection method meets the requirements.

Claims

1. A method for detecting the cibopardo content in cibopardo citrate, characterized in that, High performance liquid chromatography was used, and the chromatographic conditions included: a reversed-phase column, and phosphate buffer and tetrahydrofuran-acetonitrile as the mobile phase.

2. The detection method according to claim 1, characterized in that, The chromatographic column is a C18 column.

3. The detection method according to claim 2, characterized in that, The C18 chromatographic column has dimensions of 4.6*250mm and a diameter of 5.0μm.

4. The detection method according to claim 1, characterized in that, The column temperature is 25–45°C, preferably 30–40°C.

5. The detection method according to claim 1, characterized in that, The mobile phase comprises mobile phase A and mobile phase B, wherein mobile phase A is 0.005-0.09 mol / L phosphate buffer (pH 6.5-8.5) and mobile phase B is tetrahydrofuran-acetonitrile.

6. The detection method according to claim 5, characterized in that, Mobile phase A was 0.02 mol / L phosphate buffer (pH 7.5), with a tetrahydrofuran-acetonitrile ratio of 1:

5.

7. The detection method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8–1.2 mL / min, and it is detected using an ultraviolet detector at a wavelength of 215–225 nm.

8. The detection method according to claim 1, characterized in that, The injection volume is 5-15 μL.

9. The detection method according to any one of claims 1-8, characterized in that, High-performance liquid chromatography (HPLC) was used, and the chromatographic conditions included: a C18 column; UV detection at a wavelength of 215-225 nm; mobile phase A of 0.02 mol / L phosphate buffer (pH 7.5) and mobile phase B of tetrahydrofuran-acetonitrile; column temperature of 30–40 °C; flow rate of 0.8–1.2 mL / min; and isogradient elution with mobile phase A:mobile phase B = 40:

60.

10. A method for detecting the cibopardo content in cibopardo citrate, characterized in that, Includes the following steps: (1) Prepare the test sample and reference solution; (2) Detection: Detection according to the detection method of any one of claims 1-9; (3) Calculate the content of cibopado in the detection spectrum of step (2).

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