Method for detecting nitroso compound serving as raw material medicine in riptacaine emulsifiable paste

Abstract

The invention belongs to the technical field of medicine detection, and particularly relates to a method for detecting a nitroso compound in a raw material medicine in a riptacaine emulsifiable paste, which comprises the following steps: preparing a test sample: taking the riptacaine emulsifiable paste, adding a 5mol / L sodium hydroxide solution, uniformly mixing, then adding a 5mol / L hydrochloric acid solution, and uniformly mixing; diluting with a solvent with the pH value of 7.20 + / -0.02, and then centrifugally filtering to obtain a test sample; wherein the solvent is a mixed solvent composed of monopotassium phosphate and acetonitrile; liquid chromatography separation: the tested sample is separated by liquid chromatography, and a mobile phase A is a 0.2% formic acid solution; the mobile phase B is methanol; the chromatographic column is SB-C18, 2.1 * 100 mm, 1.7 [mu] m; and mass spectrum detection: detecting the substance separated by the liquid chromatography by using a mass spectrometer to obtain the content of the nitroso compound. The detection method is efficient, sensitive and excellent in reproducibility.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection, and specifically relates to a method for detecting nitroso compounds of a raw material drug in a leprocaine cream, and in particular to a method for detecting nitrosamine impurities of lidocaine and nitrosamine impurities of prilocaine in the leprocaine cream. Background Art

[0002] In the pharmaceutical field, drug safety and efficacy are always of primary concern. As a commonly used local anesthetic, quality control of leuprocaine cream is crucial. In recent years, with increased awareness of potential impurities in pharmaceuticals, nitrosamine impurities have garnered significant attention due to their potential carcinogenicity. Leuprocaine cream is a combination of lidocaine and prilocaine, both amide local anesthetics. Both stabilize nerve cell membranes by blocking the ion flow required for the generation and conduction of nerve impulses, thereby producing a local anesthetic effect. It is primarily used for local anesthesia of normal, intact skin and as pretreatment for superficial genital mucosal surgery and infiltration anesthesia. Compared to traditional anesthetics, leuprocaine cream offers advantages such as convenience, high safety, and lack of dependence. It is widely used clinically in departments such as anesthesiology, dermatology, plastic surgery, general surgery, anorectal surgery, and urology.

[0003] There are secondary or tertiary amine groups in the structural formula of prilocaine and lidocaine. During the production and storage process, prilocaine and lidocaine may directly or indirectly produce nitrosamine impurities. The specific structure is shown in Figure 1 According to the "Recommended Acceptable Intake Limits for Nitrosamine Drug Substance-Related Impurities (NDSRIs)" issued by the FDA and the "Questions and Answers to Marketing Authorization Holders / Applicants on the CHMP Opinion on Article 5(3) of Regulation (EC) No. 726 / 2004 on Nitrosamine Impurities in Products for Human Medicinal Use," the recommended daily acceptable intake (AI) for nitrosamine impurities in prilocaine is 1500 ng / day, and the AI for nitrosamine impurities in lidocaine is 100 ng / day. Based on the maximum daily dose of lidocaine cream, the limit of nitrosamine impurities in prilocaine is calculated to be 1 ppm, and the limit of nitrosamine impurities in lidocaine is calculated to be 0.066 ppm.

[0004] Currently, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) are widely used internationally as the primary means of detecting nitrosamine impurities. Based on the characteristics of leprocaine cream and its low detection limit requirements, we have developed a highly efficient and accurate LC-MS / MS method through in-depth research and practice, capable of simultaneously detecting two nitrosamine impurities in the cream. Summary of the Invention

[0005] The present invention aims to provide a method for detecting nitroso compounds in a raw material drug of leuprocaine cream. By liquid chromatography separation and mass spectrometry detection, the content of nitrosamine impurities in leuprocaine cream can be efficiently and sensitively detected, thereby helping to ensure the quality and safety of leuprocaine cream.

[0006] To achieve the above object, the present invention provides a method for detecting nitroso compounds in a raw material drug of ripracaine cream, comprising the following steps:

[0007] Preparation of test samples: Take levobivacaine cream, add 5 mol / L sodium hydroxide solution, mix well, then add 5 mol / L hydrochloric acid solution, mix well; dilute with a solvent with a pH of 7.20±0.02, and then centrifuge and filter to obtain a test sample; wherein the solvent is a mixed solvent of potassium dihydrogen phosphate and acetonitrile;

[0008] Liquid chromatography separation: The test sample was separated by liquid chromatography, wherein the mobile phase A was 0.2% formic acid solution; the mobile phase B was methanol; the chromatographic column was SB-C18, 2.1×100 mm, 1.7 μm;

[0009] Mass spectrometry: The substances separated by liquid chromatography were detected using a mass spectrometer to obtain the content of nitroso compounds in the raw material drug of levobuccaine cream; the ion source of the mass spectrometer was an electrospray ion source with a capillary voltage of 3500 V; the nebulizer pressure was 35 psi; the dryer temperature was 300°C; the dryer flow rate was 7 L / min; the sheath gas temperature was 350°C; and the sheath gas flow rate was 11 L / min.

[0010] Furthermore, the nitroso compounds include lidocaine nitrosamine impurities and prilocaine nitrosamine impurities, and their structural formulas are as follows:

[0011]

[0012] Furthermore, the gradient elution procedure of the liquid chromatography is:

[0013]

[0014] Furthermore, the solvent was prepared by weighing 2.73 g of potassium dihydrogen phosphate, adding 630 mL of ultrapure water to dissolve it, adjusting the pH to 7.20±0.02 with 5 mol / L sodium hydroxide solution, and then diluting it to 1000 mL with acetonitrile and shaking it well.

[0015] Furthermore, the preparation of the test sample includes: taking 0.5g of ripracaine cream, adding 2mL of 5mol / L sodium hydroxide solution, mixing, then adding 2mL of 5mol / L hydrochloric acid solution, mixing; diluting to 10mL with a solvent with a pH of 7.20±0.02, and then centrifuging to obtain a test sample.

[0016] Furthermore, the column temperature of the liquid chromatography is 60-65° C., the flow rate is 0.18-0.22 ml / min, and the injection volume is 10 μL.

[0017] Furthermore, the detection method also includes drawing a standard curve and calculating a regression equation based on the concentration and the corresponding peak area; if there is a peak in the chromatogram of the test solution that is consistent with the retention time of each nitrosamine impurity, the content is calculated according to the standard curve method.

[0018] The method for obtaining the standard curve includes:

[0019] Preparation of standard samples: using the solvent (containing the test sample matrix) to dilute the nitroso compound into standard samples of different concentrations for later use;

[0020] Liquid chromatography separation: The standard sample was separated by liquid chromatography, wherein the mobile phase A was 0.2% formic acid solution; the mobile phase B was methanol; the chromatographic column was SB-C18, 2.1×100 mm, 1.7 μm;

[0021] Mass spectrometry detection: Use a mass spectrometer to detect substances separated by high performance liquid chromatography. Utilize the multiple reaction monitoring mode of triple quadrupole mass spectrometry to specifically detect the content of nitroso compounds. Calculate the limit of quantification and detection based on the signal-to-noise ratio. Use the concentration and corresponding peak area to calculate the regression equation. If there are peaks in the chromatogram of the test solution that coincide with the retention time of each nitrosamine impurity, calculate the content using the standard curve method.

[0022] Furthermore, the concentration ranges of the standard samples of different concentrations are: a series of standard curve solutions containing lidocaine nitrosamine impurities 0 ng, 0.025 ng, 0.042 ng, 0.084 ng, 0.126 ng, and 0.168 ng, and prilocaine nitrosamine impurities 0 ng, 0.375 ng, 0.625 ng, 1.250 ng, 1.875 ng, and 2.500 ng per 1 ml.

[0023] In general, the above technical solutions conceived by the present invention have the following technical advantages compared with the existing technology:

[0024] 1. The present invention provides a method for detecting nitroso compounds in the raw material drug of lidocaine cream. Matrix interference is a major problem currently existing in the detection of lidocaine cream. The main matrix interfering components in lidocaine cream are carbomer and polyoxyethylene hydrogenated castor oil. Compared with existing detection methods, the detection method of the present invention uses a smaller amount of lidocaine cream for detection, greatly reducing the enrichment of interfering components during the detection process, thereby reducing the matrix effect during the detection of lidocaine nitrosamine impurities and prilocaine nitrosamine impurities, making the detection of lidocaine nitrosamine impurities and prilocaine nitrosamine impurities more sensitive and accurate, and greatly improving the extraction effect of lidocaine nitrosamine impurities and prilocaine nitrosamine impurities.

[0025] 2. After the cream sample is extracted with a conventional organic solvent, it will be turbid or difficult to pass through the membrane. The present invention first uses a 5 mol / L sodium hydroxide solution to cause saponification reaction, then neutralizes and breaks the emulsion with a 5 mol / L hydrochloric acid solution, and then dissolves it with a solvent. After high-speed refrigerated centrifugation, it is tested on a machine, which can fully extract the nitroso compounds and is convenient for testing.

[0026] 3. The present invention has a low limit and requires the use of a liquid chromatography-mass spectrometer for analysis and testing. Compared with previous detection methods, the detection process is simpler, uses fewer reagents and consumables, especially organic reagents, and is less environmentally polluting. In addition, the method has excellent reproducibility, can achieve good recovery effects, and extracts more fully.

[0027] 4. The present invention establishes a method for detecting lidocaine nitrosamine impurities and prilocaine nitrosamine impurities generated by the reaction of its API with nitrite in leprocaine cream (semi-solid preparation). The method is efficient and sensitive and facilitates the quality control of leprocaine drugs, thereby ensuring the quality and safety of the drugs and thus protecting life safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The composition and structural formula of the ingredients in leprocaine cream;

[0029] Figure 2 It is the liquid phase mass spectrometry specific spectrum;

[0030] Figure 3 is the mass spectrum of the reference solution;

[0031] Figure 4 is the mass spectrum of blank solution;

[0032] Figure 5 is the mass spectrum of the test solution;

[0033] Figure 6 is the liquid chromatography-mass spectrometry of the spiked test solution;

[0034] Figure 7 is the standard curve of nitrosamine impurities in lidocaine;

[0035] Figure 8 This is the standard curve of nitrosamine impurities in prilocaine. DETAILED DESCRIPTION

[0036] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are only intended to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0037] 1. Reagents and Consumables

[0038] Unless otherwise specified, all reagents used were of analytical grade and the water used was first-grade water.

[0039] 1.1 Acetonitrile: mass spectrometry grade.

[0040] 1.2 Formic acid: mass spectrometry grade.

[0041] 1.3 Methanol: mass spectrometry grade.

[0042] 1.4 Sodium hydroxide: analytical grade.

[0043] 1.5 Hydrochloric acid: analytical grade.

[0044] 1.6 Potassium dihydrogen phosphate: analytical grade.

[0045] 1.7 5 mol / L sodium hydroxide solution: Weigh 20 g of sodium hydroxide into a beaker, stir to dissolve, transfer to a 100 ml volumetric flask, dilute to the mark with ultrapure water, and shake well.

[0046] 1.8 5 mol / L hydrochloric acid solution: Measure 42 ml of hydrochloric acid, place it in a 100 ml volumetric flask, add ultrapure water to dilute to the scale, and shake well.

[0047] 1.9 Solvent: Weigh 2.73 g of potassium dihydrogen phosphate, add 630 mL of ultrapure water to dissolve, adjust the pH to 7.20 ± 0.02 with 5 mol / L sodium hydroxide solution, dilute to 1000 mL with acetonitrile, and shake well.

[0048] 1.10 Lidocaine nitrosamine impurity (LNI) standard substance: purity not less than 98.0%.

[0049] 1.11 Prilocaine nitrosamine impurity (PNI) standard substance: purity not less than 98.0%.

[0050] 1.12 Lidocaine nitrosamine impurity and prilocaine nitrosamine impurity standard working solutions: dilute with solvent to 1 μg / mL and 10 μg / mL respectively, store below refrigeration, and prepare freshly before use.

[0051] 1.13 Test solution: Accurately weigh 0.5 g of ripracaine cream and place it in a volumetric flask. Add 2 mL of 5 mol / L sodium hydroxide solution and mix thoroughly. Add 2 mL of 5 mol / L hydrochloric acid solution and mix thoroughly. Dilute to the 10 mL mark with a solvent having a pH of 7.20 ± 0.02, shake well, and centrifuge for 10 min (11,000 rpm, 0°C). Filter the supernatant through a 0.22 μm polytetrafluoroethylene membrane filter to obtain the filtrate.

[0052] 1.14 Organic filter membrane (polytetrafluoroethylene filter membrane): 0.22μm.

[0053] 2. Instruments and Equipment

[0054] 2.1 Liquid chromatography tandem mass spectrometer: equipped with an electrospray ionization (ESI) source.

[0055] 2.2 Oscillator.

[0056] 2.3 Vortex instrument.

[0057] 2.4 Centrifuge: The speed should be no less than 11,000 r / min, equipped with refrigeration function, must reach 0°C, and equipped with a 50mL plastic centrifuge.

[0058] 2.5 pH meter.

[0059] 3. Measurement steps

[0060] 3.1 Take 0.5g of levocaine cream, accurately weigh it, and place it in a volumetric flask. Add 2mL of 5mol / L sodium hydroxide solution and mix well. Add 2mL of 5mol / L hydrochloric acid solution and mix well. Dilute to the 10mL mark with a solvent with a pH of 7.20±0.02, shake well, and centrifuge for 10min (11000r / min, 0℃). Filter the supernatant (0.22μm polytetrafluoroethylene membrane) and obtain the filtrate.

[0061] 4. Determination

[0062] 4.1 Liquid chromatography conditions

[0063] 4.1.1 Chromatographic column: SB-C18, 2.1 × 100 mm, 1.7 μm or equivalent.

[0064] 4.1.2 Column temperature: 60-65℃.

[0065] 4.1.3 Flow rate: 0.18-0.22 ml / min.

[0066] 4.1.4 Injection volume: 10 μL, which can be adjusted appropriately as needed.

[0067] 4.1.5 Mobile phase: Mobile phase A: 0.2% formic acid solution (volume ratio of formic acid and water); Mobile phase B: methanol.

[0068] The liquid chromatography mobile phase gradient program is shown in Table 1.

[0069] Table 1 Gradient elution program

[0070]

[0071]

[0072] Liquid chromatography mass spectrometry specificity spectrum Figure 2 As shown, the retention time of the nitrosamine impurity of lidocaine is 9.724 min, and the retention time of the nitrosamine impurity of prilocaine is 12.482 min.

[0073] Then, a mass spectrometer is used to detect the substances separated by liquid chromatography to obtain the content of nitroso compounds in the raw material drug of ripracaine cream.

[0074] Among them, the multiple reaction monitoring mode in the triple quadrupole mass spectrometer is used to specifically detect the content of nitroso compounds, the quantification limit and detection limit are calculated based on the signal-to-noise ratio, and the regression equation is calculated using the concentration and the corresponding peak area; if there are peaks in the chromatogram of the test solution that are consistent with the retention time of each nitrosamine impurity, the content is calculated according to the standard curve method.

[0075] 4.2 Mass spectrometry conditions

[0076] 4.2.1 Ion source: electrospray ionization (ESI).

[0077] 4.2.2 Scanning mode: positive ion mode.

[0078] 4.2.3 Detection method: Multiple reaction monitoring (MRM).

[0079] 4.2.4 Capillary voltage: 3500 V; nebulizer pressure: 35 psi; dryer temperature: 300°C; dryer flow rate: 7 L / min; sheath gas temperature: 350°C; sheath gas flow rate: 11 L / min.

[0080] 4.2.5 Qualitative ion pairs, quantitative ion pairs, collision energies, and fragmentor voltages are shown in Table 2.

[0081] Table 2 Qualitative ion pairs, quantitative ion pairs, collision energy, and fragmentor voltage of lidocaine nitrosamine impurities and prilocaine nitrosamine impurities

[0082]

[0083] The standard curve of lidocaine nitrosamine impurities is as follows Figure 7 , the fitting equation is: y = 10004.52x + 34.80, R 2 =0.9996; the standard curve of prilocaine nitrosamine impurity is as follows Figure 8 , the fitting equation is: y = 3734.24x + 483.57, R 2 =0.9999.

[0084] It will be easily understood by those skilled in the art that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for detecting nitroso compounds in a raw material drug of levulinic acid cream, characterized in that: The following steps are involved: Preparation of test samples: Take levobivacaine cream, add 5 mol / L sodium hydroxide solution, mix well, then add 5 mol / L hydrochloric acid solution, mix well; dilute with a solvent with a pH of 7.20±0.02, and then centrifuge and filter to obtain a test sample; wherein the solvent is a mixed solvent of potassium dihydrogen phosphate and acetonitrile; Liquid chromatography separation: The test sample was separated by liquid chromatography, wherein the mobile phase A was 0.2% formic acid solution; the mobile phase B was methanol; the chromatographic column was SB-C18, 2.1×100 mm, 1.7 μm; Mass spectrometry: The substances separated by liquid chromatography were detected using a mass spectrometer to obtain the content of nitroso compounds in the raw material drug of levobuccaine cream; the ion source of the mass spectrometer was an electrospray ion source with a capillary voltage of 3500 V; the nebulizer pressure was 35 psi; the dryer temperature was 300°C; the dryer flow rate was 7 L / min; the sheath gas temperature was 350°C; and the sheath gas flow rate was 11 L / min.

2. The method for detecting the nitroso compounds of the raw material drug in the levulinic acid cream according to claim 1, wherein The nitroso compounds include lidocaine nitrosamine impurities and prilocaine nitrosamine impurities, and their structural formulas are as follows:

3. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 1, wherein The gradient elution procedure of the liquid chromatography is: Gradient time / minMobile phase A volume percentage / %Mobile phase B volume percentage / % 4. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 1, wherein The solvent was prepared by weighing 2.73 g of potassium dihydrogen phosphate, adding 630 mL of ultrapure water to dissolve the solution, adjusting the pH to 7.20±0.02 with a 5 mol / L sodium hydroxide solution, and then diluting the solution to 1000 mL with acetonitrile and shaking well.

5. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 1, wherein The test sample preparation includes: taking 0.5g of ripracaine cream, adding 2mL of 5mol / L sodium hydroxide solution, mixing, then adding 2mL of 5mol / L hydrochloric acid solution, mixing; diluting to 10mL with a solvent with a pH of 7.20±0.02, and then centrifuging to obtain a test sample.

6. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 1, wherein The column temperature of the liquid chromatography was 60-65° C., the flow rate was 0.18-0.22 ml / min, and the injection volume was 10 μL.

7. The method for detecting the nitroso compounds of the raw material drug in the levulinic acid cream according to any one of claims 1 to 6, wherein: The detection method further comprises drawing a standard curve, precisely measuring the standard curve solution and the test solution, injecting them into a liquid chromatography-mass spectrometer respectively, recording a chromatogram, and calculating a regression equation based on the concentration and the corresponding peak area.

8. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 7, wherein The method for obtaining the standard curve includes: Preparation of standard samples: using the solvent containing the test sample matrix to dilute the nitroso compound into standard samples of different concentrations for later use; Liquid chromatography separation: The standard sample was separated by liquid chromatography, wherein the mobile phase A was 0.2% formic acid solution; the mobile phase B was methanol; the chromatographic column was SB-C18, 2.1×100 mm, 1.7 μm; Mass spectrometry detection: Use a mass spectrometer to detect substances separated by high performance liquid chromatography. Utilize the multiple reaction monitoring mode of triple quadrupole mass spectrometry to specifically detect the content of nitroso compounds. Calculate the limit of quantification and detection based on the signal-to-noise ratio. Use the concentration and corresponding peak area to calculate the regression equation to obtain a standard curve. If there are peaks in the chromatogram of the test solution that coincide with the retention time of each nitrosamine impurity, calculate the content according to the standard curve method.

9. The method for detecting the nitroso compounds of the raw material medicine in the levulinic acid cream according to claim 8, wherein The concentration range of the standard samples of different concentrations is: per 1 ml, a series of standard curve solutions containing 0 ng, 0.025 ng, 0.042 ng, 0.084 ng, 0.126 ng, and 0.168 ng of lidocaine nitrosamine impurities, and 0 ng, 0.375 ng, 0.625 ng, 1.250 ng, 1.875 ng, and 2.500 ng of prilocaine nitrosamine impurities.

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