Method for detecting nitroso-(S)-(-)-N-(2, 6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride

Abstract

The invention discloses a method for detecting nitroso-(S)-(-)-N-(2, 6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, which comprises the following steps: weighing ropivacaine hydrochloride, and adding water for dissolving to obtain a test solution; the method comprises the following steps: weighing a nitroso-(S)-(-)-N-(2, 6-dimethylphenyl)-2-piperidine formamide reference substance, dissolving and diluting with methanol to obtain a reference substance solution; and measuring the test solution and the reference solution, injecting into a high performance liquid chromatography-tandem mass spectrometer, recording a chromatogram, and calculating by peak area according to an external standard method. According to the method, the content of nitroso-(S)-(-)-N-(2, 6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride is measured through a high performance liquid chromatography-tandem mass spectrometer method, and the method is simple, convenient, feasible, high in specificity, high in accuracy, good in repeatability and high in detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of anesthetic production, specifically to a method for detecting nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride. Background Technology

[0002] Ropivacaine hydrochloride is suitable for surgical anesthesia, such as epidural anesthesia (including cesarean section, subarachnoid anesthesia, and regional block), or for acute pain control, such as continuous epidural infusion or intermittent single-dose administration, like postoperative or vaginal delivery analgesia and regional block. During the synthesis of ropivacaine hydrochloride, nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide may be generated. This is a genotoxic impurity whose content must be strictly controlled; therefore, methods have been developed for its content determination.

[0003] Currently, there are no reported methods for detecting the content of nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride in pharmacopoeias or literature from various countries. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, which is highly sensitive and accurate.

[0005] This invention provides a method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, comprising the following steps: Weigh out ropivacaine hydrochloride and dissolve it in water to obtain the test solution; Weigh out N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard, dissolve and dilute it with methanol to obtain the reference standard solution; The test solution and the reference solution were injected into a high-performance liquid chromatography-tandem mass spectrometry instrument, the chromatograms were recorded, and the peak area was calculated using the external standard method.

[0006] The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, involves using an octadecylsilane-bonded silica gel column as the packing material, a mobile phase consisting of a 0.1% formic acid aqueous solution and a 0.1% formic acid methanol solution, a column temperature of 35°C, a flow rate of 0.5 ml per minute, and an injection volume of 10 µl.

[0007] The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, involves the following mobile phase elution method: The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, involves an analytical column with an inner diameter of 4.6 mm, a length of 100 mm, and a packing particle size of 3 μm.

[0008] The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, uses a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) instrument with a first-stage mass spectrometry selected ion of 262.3, a second-stage mass spectrometry selected ion of 140.9, an accelerating voltage of 75V, and a collision energy of 5V.

[0009] The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, uses the following ion source parameters for the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) instrument: Drying gas temperature: 250 ℃; gas flow rate: 5 L / min; atomizer pressure: 25 psi; sheath gas temperature: 150 ℃; sheath gas flow rate: 8 L / min; capillary voltage: 4000 V; nozzle voltage: 0 V.

[0010] The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, preferably, uses the following scanning parameters for the high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS): Polarity: positive ion mode; Scan type: multiple reaction monitoring (MRM); Scan time: 7.5 minutes; Collision cell acceleration voltage: 5 V.

[0011] Beneficial effects:

[0012] The present invention provides a method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride. This method utilizes high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) with secondary mass spectrometry. The method employs the secondary characteristic mass-to-charge ratio of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide as the detection ion, and uses gradient elution to reduce the competitive inhibition of the target analyte by the main component at the ion source. The method is simple, feasible, specific, accurate, reproducible, and highly sensitive, enabling accurate and sensitive detection of the N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide content in ropivacaine hydrochloride, thereby ensuring the quality of the ropivacaine raw material. Attached Figure Description

[0013] Figure 1 The chromatogram of the test sample solution; Figure 2 The chromatogram of the reference solution; Figure 3 This is a linear relationship diagram of the target object. Detailed Implementation

[0014] The invention will now be described more fully by way of examples. The invention can be embodied in many different forms and should not be construed as being limited to the exemplary embodiments described herein.

[0015] The molecular weight of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-pipecoloxilidide in ropivacaine hydrochloride of this invention is 261.32; the molecular formula is C 14 H 19 N3O2; The chemical structure is: Example 1

[0016] A method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-pipecoloxilidide in ropivacaine hydrochloride includes the following steps: S1: Weigh out ropivacaine hydrochloride and dissolve it in water to obtain the test solution.

[0017] Specifically, accurately weigh 20 mg of ropivacaine hydrochloride, place it in a 100 ml volumetric flask, add water to dissolve and quantitatively dilute to the mark, shake well, and the product is ready.

[0018] S2: Weigh N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard, dissolve and dilute it with methanol to obtain the reference standard solution.

[0019] Specifically, step S2 includes the following steps: S21: Accurately weigh 3.52 mg of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard and place it in a 100 ml volumetric flask. Dissolve it in methanol and dilute quantitatively to the mark. Shake well. S22: Accurately measure 1 ml of the solution obtained in step S21 into a 100 ml volumetric flask, dilute to the mark with methanol, shake well, and obtain the solution; S23: Accurately measure 1 ml of the solution obtained in step S22 into a 100 ml volumetric flask, dilute with methanol to the mark, shake well, and obtain the standard stock solution. S24: Accurately measure 2 ml of the reference stock solution obtained in step S23, place it in a 20 ml volumetric flask, dilute with water to the mark, and shake well to obtain the reference solution.

[0020] S3: Measure the test solution and the reference solution and inject them into the high performance liquid chromatography-tandem mass spectrometry instrument, record the chromatograms, and calculate the peak area according to the external standard method.

[0021] The analytical column of the high performance liquid chromatography-tandem mass spectrometry system is packed with octadecylsilane-bonded silica gel, the mobile phase is a combination of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution, the column temperature is 35℃, the flow rate is 0.5 ml per minute, and the injection volume is 10 µl.

[0022] The analytical column has an inner diameter of 4.6 mm, a length of 100 mm, and a particle size of 3 μm for the packing material.

[0023] The mobile phase elution method is as follows: Example 2

[0024] 1. Exclusivity Blank solution (diluent): Water.

[0025] Test sample stock solution: Weigh 103.13 mg of ropivacaine hydrochloride, place it in a 50 ml volumetric flask, add water to dissolve and quantitatively dilute to the mark, shake well, and the solution is ready.

[0026] Test solution: Accurately measure 2 ml of the test solution stock solution, place it in a 20 ml volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0027] Reference stock solution: Weigh 3.696 mg of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard into a 100 ml volumetric flask, dissolve in methanol and dilute quantitatively to the mark, and shake well; accurately measure 1 ml into a 100 ml volumetric flask, dilute to the mark with methanol, and shake well; accurately measure 1 ml into a 100 ml volumetric flask, dilute to the mark with methanol, and shake well to obtain the solution.

[0028] Positioning solution: i.e., reference standard stock solution.

[0029] Reference solution: Accurately measure 2 ml of the reference stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0030] Spiked solution for test sample: Accurately measure 2 ml of the test sample stock solution and place it in a 20 ml volumetric flask. Accurately add 2 ml of the reference stock solution, dilute with water to the mark, and shake well to obtain the solution.

[0031] Determination: Inject the above blank solution, positioning solution, reference solution, and spiked solution of the test sample into the sample for analysis.

[0032] like Figure 1 and Figure 2 As shown, the results indicate that no impurity peaks interfered with the determination of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in the test solution, demonstrating good method specificity.

[0033] Figure 1 The quantitative results are shown in Table 1. Figure 2 The quantitative results are shown in Table 2.

[0034] Table 1 Figure 1 Quantitative results Table 2 Figure 2 Quantitative results 2. Linear range Linear stock solution: Same as the reference stock solution under "Specificity".

[0035] Linear solutions: Prepare solutions for each linearity test point using diluent according to Table 3.

[0036] Table 3 Preparation of linear solutions Based on the results of the linearity and range tests, please refer to Table 4 for details. Figure 3The linear range of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide was confirmed to be within the concentration range of 0.035-0.702 ng / ml. The regression equation was y=10461.9315x+33.3359, and the correlation coefficient r=0.9999. The linearity and range met the requirements.

[0037] Table 4. Results of Linearity and Range Tests 3. Sensitivity Based on the results of the "Linearity and Range" test, the limit of quantitation of N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide was determined to be 0.035 ng / ml (S / N≥10), and the limit of detection was determined to be 0.018 ng / ml (S / N≥3).

[0038] 4. Accuracy and Precision Reference stock solution, reference solution, and test solution: same as under "Specificity".

[0039] 50% Accuracy Solution (API): Accurately measure 2 ml of the API stock solution and place it in a 20 ml volumetric flask. Accurately add 1 ml of the reference stock solution, dilute with water to the mark, and shake well. Prepare three parallel aliquots.

[0040] 100% Accuracy Solution (API): Accurately measure 2 ml of the API stock solution and place it in a 20 ml volumetric flask. Accurately add 2 ml of the reference stock solution, dilute with water to the mark, and shake well. Prepare three parallel solutions.

[0041] 150% Accuracy Solution (API): Accurately measure 2 ml of the API stock solution and place it in a 20 ml volumetric flask. Accurately add 3 ml of the reference stock solution, dilute with water to the mark, and shake well. Prepare three parallel aliquots.

[0042] The above solutions were analyzed by injection, and the results are detailed in Table 5. It is shown that when nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide was added to the test sample at 50%-150% of the limit, the recovery rate was calculated. The average recovery rate of nitrogen-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide was 89.1%, and the RSD (n=9) was 1.31%. The method has good accuracy and precision.

[0043] Table 5. Results of Accuracy and Precision Tests 5. Durability Robustness condition 1: Mobile phase A is a 0.09% formic acid aqueous solution; Mobile phase B is a 0.09% formic acid methanol solution; Elution was performed according to Table 6, with a column temperature of 30℃, a flow rate of 0.45 ml per minute, and an injection volume of 10 µl. Table 6. Elution methods of mobile phase under durability condition 1 Robustness condition 2: Mobile phase A is a 0.11% formic acid aqueous solution; Mobile phase B is a 0.11% formic acid methanol solution; Elution was performed according to Table 7, with a column temperature of 40℃, a flow rate of 0.55 ml per minute, and an injection volume of 10 µl. Table 7. Elution methods of mobile phase under durability condition 2 The durability tests are shown in Table 8. The reference solution and the spiked solution of the test sample were prepared using the preparation methods under the specificity section.

[0044] Table 8 Durability Test Results Statistics show that the recovery rate of the target substance under various durability conditions ranges from 80.39% to 89.50%, with an RSD (n=9) of 5.53%.

[0045] In summary, Example 2 investigated the specificity, linear range, limit of detection and limit of quantitation, accuracy and precision, and robustness. The results show that the proposed method has good specificity, high accuracy, and good robustness.

[0046] The above examples are only for illustrating the present invention. In addition, there are many other different implementations, which can be conceived by those skilled in the art after understanding the concept of the present invention. Therefore, they will not be listed one by one here.

Claims

1. A method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride, characterized in that, Includes the following steps: Weigh out ropivacaine hydrochloride and dissolve it in water to obtain the test solution; Weigh out N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard, dissolve and dilute it with methanol to obtain the reference standard solution; Measure the test solution and the reference solution and inject them into the high performance liquid chromatography-tandem mass spectrometry instrument, record the chromatograms, and calculate the peak area according to the external standard method; The analytical concentration was: 0.352 ng / ml of nitrogen-containing nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide reference standard.

2. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 1, characterized in that, The analytical column of the high performance liquid chromatography-tandem mass spectrometry system is packed with octadecylsilane-bonded silica gel, the mobile phase is a combination of 0.1% formic acid aqueous solution and 0.1% formic acid methanol solution, the column temperature is 35℃, the flow rate is 0.5 ml per minute, and the injection volume is 10 µl.

3. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 2, characterized in that, The mobile phase elution method is as follows: 。 4. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 3, characterized in that, The analytical column has an inner diameter of 4.6 mm, a length of 100 mm, and a particle size of 3 μm for the packing material.

5. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 4, characterized in that, The high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) instrument has a first-stage mass spectrometry selected ion of 262.3, a second-stage mass spectrometry selected ion of 140.9, an accelerating voltage of 75V, and a collision energy of 5V.

6. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 5, characterized in that, The ion source parameters of the high-performance liquid chromatography-tandem mass spectrometry instrument are as follows: Drying gas temperature: 250 ℃; gas flow rate: 5 L / min; atomizer pressure: 25 psi; sheath gas temperature: 150 ℃; sheath gas flow rate: 8 L / min; capillary voltage: 4000 V; nozzle voltage: 0 V.

7. The method for detecting N-nitroso-(S)-(-)-N-(2,6-dimethylphenyl)-2-piperidinecarboxamide in ropivacaine hydrochloride according to claim 6, characterized in that, The scanning parameters of the high-performance liquid chromatography-tandem mass spectrometry instrument are as follows: Polarity: positive ion mode; Scan type: multiple reaction monitoring (MRM); Scan time: 7.5 minutes; Collision cell acceleration voltage: 5 V.

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