A method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology

Abstract

The present invention belongs to the technical field of drug analysis, and specifically relates to a method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations by using ESI-LC-MSMS technology. The present invention discloses an analytical method for simultaneously quantitatively determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride, including preparing an analytical solution; using an octadecylsilane bonded silica gel chromatographic column, using a mixed solution of an acid-organic phase as a mobile phase, and adopting an isocratic elution method; using an ESI source, a negative ion mode, and a multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer for detection; and measuring the configured analytical solution on a machine. The method of the present invention can effectively and accurately determine six genotoxic impurities in chloroprocaine hydrochloride, especially can effectively separate and accurately determine the isomeric impurities SM1 (4-nitro-2-chlorobenzoic acid) and SM1-B (4-nitro-3-chlorobenzoic acid), and provide a reliable quantitative method for the quality research of chloroprocaine hydrochloride or its preparations.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations by using ESI-LC-MSMS technology. Background Art

[0002] Chloroprocaine hydrochloride is a short-acting spinal anesthetic available in injectable form in Europe and the United States. Its structure is shown below:

[0003]

[0004] Chloroprocaine hydrochloride injection is suitable for local infiltration anesthesia, peripheral nerve block anesthesia, sacral and epidural anesthesia, and is strictly contraindicated for subarachnoid block anesthesia.

[0005] The genotoxic impurities contained in chloroprocaine hydrochloride or its preparations mainly come from the reaction reagents during the synthesis process, impurities introduced by the reaction raw materials, impurities introduced by the intermediates, by-product impurities and degradation impurities.

[0006] The invention aims at controlling genotoxic impurities introduced by reaction raw materials, and specifically relates to a method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations by using ESI-LC-MSMS technology.

[0007] Table 1 List of six benzoic acid genotoxic impurities in chloroprocaine hydrochloride

[0008]

[0009] There are many impurities with genotoxic warning structures involved in chloroprocaine hydrochloride, and there is no report in the existing literature on the method of simultaneously determining the above 6 genotoxic impurities. This patent uses ESI-LC-MSMS technology for the first time to simultaneously determine the above 6 genotoxic impurities.

[0010] In order to ensure the quality of drugs and the safety and effectiveness of clinical drugs, it is necessary to conduct in-depth quantitative research on genotoxic impurities in drugs. Since genotoxic impurities are highly toxic and have low limits, accurate quantitative determination is extremely important. Summary of the invention

[0011] The purpose of the present invention is to solve the problem of quantitatively determining genotoxic impurities in chloroprocaine hydrochloride and to provide an analytical method for quantitatively determining six genotoxic impurities in chloroprocaine hydrochloride, which is simple and easy to operate.

[0012] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0013] The present invention provides a method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations by using ESI-LC-MSMS technology. According to an embodiment of the present invention, high performance liquid chromatography tandem mass spectrometry is used, a reverse phase chromatographic column is adopted, an aqueous phase is used as a mobile phase A, an organic phase is used as a mobile phase B, a flow rate, a column temperature, an injection plate temperature and an injection volume, isocratic elution and mass spectrometry conditions are used.

[0014] According to an embodiment of the present invention, the present invention adopts a reverse phase chromatographic column that is a chromatographic column with octadecylsilane bonded silica gel as a filler (Phenomon Titank C18, specifications are 2.1mm×50mm, 3μm), a chromatographic column with octadecylsilane bonded silica gel as a filler (Phenomon Titank F5, specifications are 4.6mm×150mm, 3μm) and a chromatographic column with octadecylsilane bonded silica gel as a filler (ACE Excel C18-PFP, specifications are 4.6mm×150mm, 3μm).

[0015] Preferably, the chromatographic column is selected from a chromatographic column with octadecylsilane bonded silica gel as filler (ACE Excel C18-PFP, specification is 4.6mm×150mm, 3μm).

[0016] According to an embodiment of the present invention, the mobile phase A contains an acid selected from formic acid or acetic acid.

[0017] Preferably, the mobile phase A is selected from acetic acid-water, and the volume ratio is selected from 2:100.

[0018] According to an embodiment of the present invention, the mobile phase B is selected from acetonitrile or methanol.

[0019] Preferably, mobile phase B is selected from acetonitrile.

[0020] According to an embodiment of the present invention, in the liquid chromatography conditions disclosed in the present invention, the flow rate is selected from 0.45~0.65ml / min, the column temperature is selected from 28~32℃, the injection plate temperature is selected from 20~30℃, and the injection volume is selected from 5μL.

[0021] Preferably, the flow rate is selected from 0.5 ml / min, the column temperature is selected from 30° C., the injection plate temperature is selected from 25° C., and the injection volume is selected from 5 μL.

[0022] According to an embodiment of the present invention, in the liquid chromatography conditions disclosed in the present invention, isocratic elution is performed with 50% mobile phase B by volume, and the mobile phase ratio is maintained for 10 minutes.

[0023] According to an embodiment of the present invention, in the mass spectrometry conditions disclosed in the present invention, detection is performed using a multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization, and negative ion mode scanning; the curtain gas is 35 psi, the nebulizer 1 is 40 psi, the nebulizer 2 is 45 psi, the spray needle voltage is -4500 V, and the drying temperature is 550 ° C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0024]

[0025] The beneficial effect of the present invention is that the present invention can simultaneously and quantitatively determine the contents of six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations, and in particular can effectively separate and accurately determine the isomeric impurities SM1 (4-nitro-2-chlorobenzoic acid) and SM1-B (4-nitro-3-chlorobenzoic acid). The method is simple, accurate, highly sensitive, and highly specific, and provides a reliable quantitative method for the study of genotoxic impurities in chloroprocaine hydrochloride or its preparations, and has positive effects and practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Blank solvent SM1, SM1-C diagram;

[0027] Figure 2 Figures of blank solvent SM1-D and SM1-E;

[0028] Figure 3 Figures of blank solvent SM1-G and SM1-B;

[0029] Figure 4 Figure SM1 and SM1-C of reference solution;

[0030] Figure 5 Figures SM1-D and SM1-E of reference solution;

[0031] Figure 6 Figure SM1-G and SM1-B of the reference solution;

[0032] Figure 7 SM1 positioning map;

[0033] Figure 8 SM1-B positioning map;

[0034] Fig. 9 Figure SM1 and SM1-C of the test sample spiked solution;

[0035] Fig.10 Figures SM1-D and SM1-E of the test sample spiked solutions;

[0036] Fig.11Figure SM1-G and SM1-B of the test sample spiked solution;

[0037] Fig.12 Impurity SM1 linear graph;

[0038] Fig.13 Impurity SM1-B linear graph;

[0039] Fig.14 Impurity SM1-C linear graph;

[0040] Fig.15 Impurity SM1-D linear graph;

[0041] Fig.16 Impurity SM1-E linear graph;

[0042] Fig.17 Impurity SM1-G linear graph;

[0043] Fig.18 Figure SM1-C of impurities in the reference solution of Example 1;

[0044] Fig.19 Example 14 Impurities SM1-C in the reference solution;

[0045] Fig. 20 Example 15 Impurities SM1-C in the reference solution;

[0046] Fig.21 Example 16 Impurities SM1-C in the reference solution;

[0047] Fig. 22 Figure SM1 and SM1-B of impurities in methanol as mobile phase B reference solution;

[0048] Fig.23 Figure SM1-C and SM1-D of impurities in methanol as mobile phase B reference solution;

[0049] Fig.24 Figure SM1-E and SM1-G of impurities in methanol as mobile phase B reference solution;

[0050] Fig.25 TIC diagram of each impurity in the reference solution of Example 18;

[0051] Fig.26 XIC diagram of impurity SM1-C in the reference solution of Example 19. Specific implementation plan

[0052] The present invention discloses a method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology. Those skilled in the art can refer to the content of this article and appropriately improve the method parameters to achieve it. It is particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The detection method of the present invention has been described through preferred embodiments, and relevant personnel obviously modify or appropriately change and combine the method application described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0053] Example 1

[0054] (1) Sample preparation: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it. Dilute to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0055] (2) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0056] (3) Sensitivity solution: Accurately measure 1 ml of the reference solution (2) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0057] (4) Reference substance spiked solution: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of (2) reference substance solution to dissolve it. Dilute to the mark with (2) reference substance solution and shake well.

[0058] (5) SM1 positioning solution: Take 2.4 mg of impurity SM1 reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place it in a 25 ml volumetric flask, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0059] (6) SM1-B positioning solution: Take 2.4 mg of the impurity SM1-B reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place it in a 20 ml volumetric flask, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place it in a 25 ml volumetric flask, dilute it to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0060] (7) Brand and model of HPLC: Agilent 1260;

[0061] (8) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0062] (9) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0063] (10) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min];

[0064] (11) Flow rate is 0.5 ml per minute;

[0065] (12) Column temperature is 30°C;

[0066] (13) The sample tray temperature is 25°C;

[0067] (14) Injection volume: 5 μl;

[0068] (15) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering potential (DP), and collision potential (CE) of each impurity are shown in the following table:

[0069]

[0070] (16) Take blank solvent, sensitivity solution, reference solution, test sample spiked solution, SM1 positioning solution and SM1-B positioning solution for injection;

[0071] (17) Figure 1 , Figure 2 and Figure 3 It is a blank solvent map and does not interfere with the detection; Figure 4 , Figure 5 and Figure 6 This is the reference solution diagram; Figure 7 This is the SM1 positioning solution map, and the peak retention time of the impurity SM1 positioning solution is 5.099min; Figure 8 This is the SM1-B localization solution diagram, and the peak retention time of the impurity SM1-B localization solution is 6.965min; Fig. 9 , Fig.10 and Fig.11 The retention time of each impurity in the spiked solution of the test sample was consistent with that of the reference solution, indicating that other impurities in chloroprocaine hydrochloride did not interfere with the detection of the target peak, and the isomers SM1 and SM1-B could be effectively separated, with the elution order being SM1 and SM1-B.

[0072] Example 2

[0073] (1) SM1 stock solution: Take 2.4 mg of impurity SM1 reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0074] (2) SM1-B stock solution: Take 2.4 mg of impurity SM1-B reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0075] (3) SM1-C stock solution: Take 2.4 mg of the impurity SM1-C reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0076] (4) SM1-D stock solution: Take 2.4 mg of the impurity SM1-D reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0077] (5) SM1-E stock solution: Take 2.4 mg of the impurity SM1-E reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0078] (6) SM1-G stock solution: Take 2.4 mg of the impurity SM1-G reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0079] (7) Reference substance stock solution: Accurately measure 1 ml of each of (1) to (6) and place in a 100 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0080] (8) Linear solution 1: Accurately measure 2 ml of impurity SM1 stock solution, 1 ml of SM1-B stock solution, 2.5 ml of SM1-C stock solution, 3 ml of SM1-D stock solution, 0.5 ml of SM1-E stock solution and 3 ml of SM1-G stock solution, respectively, place in a 100 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0081] (9) Linear solution 2: Accurately measure 1 ml of the reference stock solution (7) and place it in a 100 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0082] (10) Linear solution 3: Accurately measure 3 ml of the reference stock solution (7) and place it in a 100 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0083] (11) Linear solution 4: Accurately measure 1 ml of the reference stock solution (7) and place it in a 20 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0084] (12) Linear solution 5: Accurately measure 2 ml of the reference stock solution (7) and place it in a 20 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0085] (13) Linear solution 6: Accurately measure 3 ml of the reference stock solution (7) and place it in a 20 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0086] (14) Linear solution 7: Accurately measure 4 ml of the reference stock solution (7) and place it in a 20 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0087] (15) Brand and model of HPLC: Agilent 1260;

[0088] (16) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0089] (17) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0090] (18) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0091] (19) Flow rate is 0.5 ml per minute;

[0092] (20) Column temperature is 30°C;

[0093] (21) The sample tray temperature is 25°C;

[0094] (22) Injection volume 5 μl;

[0095] (23) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0096]

[0097] (24) Take linear solution 1, linear solution 2, linear solution 3, linear solution 4, linear solution 5, linear solution 6 and linear solution 7 for injection.

[0098] (25) Fig.12 Impurity SM1 linear, Fig.13 Impurity SM1-B linear, Fig.14 Impurity SM1-C linear, Fig.15 Impurity SM1-D linear, Fig.16 Impurity SM1-E linear, Fig.17 Impurity SM1-G is linear. As can be seen from the figure, the linear correlation coefficient R² of each impurity is greater than 0.990, which meets the requirements of the Chinese Pharmacopoeia.

[0099] Example 3

[0100] (1) SM1 stock solution: Take 2.4 mg of impurity SM1 reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0101] (2) SM1-B stock solution: Take 2.4 mg of impurity SM1-B reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0102] (3) SM1-C stock solution: Take 2.4 mg of the impurity SM1-C reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0103] (4) SM1-D stock solution: Take 2.4 mg of the impurity SM1-D reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0104] (5) SM1-E stock solution: Take 2.4 mg of the impurity SM1-E reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0105] (6) SM1-G stock solution: Take 2.4 mg of the impurity SM1-G reference substance, accurately weigh it, place it in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0106] (7) Quantitative limit solution: Accurately measure 2 ml of impurity SM1 stock solution, 1 ml of SM1-B stock solution, 2.5 ml of SM1-C stock solution, 3 ml of SM1-D stock solution, 0.5 ml of SM1-E stock solution and 3 ml of SM1-G stock solution, respectively, place in a 100 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0107] (8) Detection limit solution: Accurately measure 3 ml of the quantification limit solution (7) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0108] (9) Brand and model of HPLC: Agilent 1260;

[0109] (10) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0110] (11) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0111] (12) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min.

[0112] (13) Flow rate is 0.5 ml per minute;

[0113] (14) Column temperature is 30°C;

[0114] (15) The temperature of the sample tray is 25°C;

[0115] (16) Injection volume 5 μl;

[0116] (17) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering potential (DP), and collision potential (CE) of each impurity are shown in the following table:

[0117]

[0118] (18) Take samples of the detection limit and quantification limit solutions and inject them.

[0119] (19) Table 2 Results of detection limits and quantification limits. As can be seen from Table 2, the detection limits and quantification limits of each impurity meet the sensitivity requirements for impurity detection.

[0120] Table 2 Results of detection limit and quantification limit

[0121]

[0122] Example 4

[0123] (1) Sample preparation: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it. Dilute to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0124] (2) Reference substance stock solution: Take 2.4 mg of impurity SM1 reference substance, 2.4 mg of SM1-B reference substance, 2.4 mg of SM1-C reference substance, 2.4 mg of SM1-D reference substance, 2.4 mg of SM1-E reference substance and 2.4 mg of SM1-G reference substance, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), shake well. Accurately measure 1 ml, place in a 100 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), shake well.

[0125] (3) Reference substance solution: Accurately measure 1 ml of the reference substance stock solution (2) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0126] (4) 30% recovery limit solution: Take 20 mg of chloroprocaine hydrochloride, accurately weigh it, and place it in a 20 ml volumetric flask. At the same time, accurately measure 0.6 ml of (2) reference substance stock solution, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0127] (5) Solution for limiting 100% recovery rate: Take 20 mg of chloroprocaine hydrochloride, accurately weigh it, and place it in a 20 ml volumetric flask. At the same time, accurately measure 2 ml of (2) reference substance stock solution, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute it to the scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0128] (6) Solution with a recovery limit of 150%: accurately weigh 20 mg of chloroprocaine hydrochloride and place it in a 20 ml volumetric flask. At the same time, accurately measure 3 ml of the reference substance stock solution (2), add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve it, dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0129] (7) Brand and model of HPLC: Agilent 1260;

[0130] (8) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0131] (9) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0132] (10) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0133] (11) Flow rate is 0.5 ml per minute;

[0134] (12) Column temperature is 30°C;

[0135] (13) The sample tray temperature is 25°C;

[0136] (14) Injection volume: 5 μl;

[0137] (15) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering potential (DP), and collision potential (CE) of each impurity are shown in the following table:

[0138]

[0139] (16) Inject blank solvent, sample solution, reference solution, 30% recovery limit solution, 100% recovery limit solution, and 150% recovery limit solution.

[0140] (17) Table 3 shows the recovery and precision results. It can be seen from Table 3 that the recovery of each impurity is between 80% and 120%, and the precision is less than 10%, which meets the requirements of the Chinese Pharmacopoeia.

[0141] Table 3 Recovery and precision results

[0142]

[0143] Note: The limits of the base toxic impurities SM1, SM1-B, SM1-C, SM1-D, SM1-E and SM1-G of chloroprocaine hydrochloride calculated according to ICH guideline M7 are all 120ppm.

[0144] Example 5

[0145] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0146] (2) Reference substance spiked solution: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of (2) reference substance solution to dissolve it. Dilute to the mark with (2) reference substance solution and shake well.

[0147] (3) Brand and model of HPLC: Agilent 1260;

[0148] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0149] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0150] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0151] (7) Flow rate is 0.5 ml per minute;

[0152] (8) Column temperature is 30°C;

[0153] (9) The temperature of the sample tray is 8°C;

[0154] (10) Injection volume: 5 μl;

[0155] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0156]

[0157] (12) Take the reference solution and the test sample spiked solution and inject them at 0 hours, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours respectively.

[0158] Example 6

[0159] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0160] (2) Reference substance spiked solution: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of (2) reference substance solution to dissolve it. Dilute to the mark with (2) reference substance solution and shake well.

[0161] (3) Brand and model of HPLC: Agilent 1260;

[0162] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0163] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0164] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0165] (7) Flow rate is 0.5 ml per minute;

[0166] (8) Column temperature is 30°C;

[0167] (9) The temperature of the sample tray is 20°C;

[0168] (10) Injection volume: 5 μl;

[0169] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0170]

[0171] (12) Take the reference solution and the test sample spiked solution and inject them at 0 hours, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours respectively.

[0172] Example 7

[0173] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0174] (2) Reference substance spiked solution: Take 10 mg of chloroprocaine hydrochloride and place it in a 10 ml volumetric flask. Add about 6 ml of (2) reference substance solution to dissolve it. Dilute to the mark with (2) reference substance solution and shake well.

[0175] (3) Brand and model of HPLC: Agilent 1260;

[0176] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0177] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0178] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0179] (7) Flow rate is 0.5 ml per minute;

[0180] (8) Column temperature is 30°C;

[0181] (9) The sample tray temperature is 30°C;

[0182] (10) Injection volume: 5 μl;

[0183] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0184]

[0185] (12) Take the reference solution and the test sample spiked solution and inject them at 0 hours, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours respectively.

[0186] (13) Table 4 shows the stability results of the solutions under various conditions in Example 1, Example 5, Example 6 and Example 7. It can be seen from Table 4 that when the injection plate temperature is controlled at 20~30℃, the stability of the reference solution and the test solution is up to 12 hours.

[0187] Table 4 Signal-to-noise ratio results of each impurity

[0188]

[0189] Example 8

[0190] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0191] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0192] (3) Brand and model of HPLC: Agilent 1260;

[0193] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0194] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0195] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0196] (7) Flow rate is 0.5 ml per minute;

[0197] (8) Column temperature is 28°C;

[0198] (9) The sample tray temperature is 25°C;

[0199] (10) Injection volume: 5 μl;

[0200] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0201]

[0202] (12) Take a sample of the sensitivity solution.

[0203] Example 9

[0204] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0205] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0206] (3) Brand and model of HPLC: Agilent 1260;

[0207] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0208] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0209] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0210] (7) Flow rate is 0.5 ml per minute;

[0211] (8) Column temperature is 32°C;

[0212] (9) The sample tray temperature is 25°C;

[0213] (10) Injection volume: 5 μl;

[0214] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0215]

[0216] (12) Take a sample of the sensitivity solution.

[0217] Example 10

[0218] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0219] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0220] (3) Brand and model of HPLC: Agilent 1260;

[0221] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0222] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0223] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0224] (7) Flow rate is 0.45 ml per minute;

[0225] (8) Column temperature is 30°C;

[0226] (9) The sample tray temperature is 25°C;

[0227] (10) Injection volume: 5 μl;

[0228] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0229]

[0230] (12) Take a sample of the sensitivity solution.

[0231] Embodiment 11

[0232] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0233] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0234] (3) Brand and model of HPLC: Agilent 1260;

[0235] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0236] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0237] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:50:50), run for 10 min].

[0238] (7) Flow rate is 0.55 ml per minute;

[0239] (8) Column temperature is 30°C;

[0240] (9) The sample tray temperature is 25°C;

[0241] (10) Injection volume: 5 μl;

[0242] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0243]

[0244] (12) Take a sample of the sensitivity solution.

[0245] Example 12

[0246] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0247] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0248] (3) Brand and model of HPLC: Agilent 1260;

[0249] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0250] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0251] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 45% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:55:45), run for 10 min].

[0252] (7) Flow rate is 0.5 ml per minute;

[0253] (8) Column temperature is 30°C;

[0254] (9) The sample tray temperature is 25°C;

[0255] (10) Injection volume: 5 μl;

[0256] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0257]

[0258] (12) Take a sample of the sensitivity solution.

[0259] Example 13

[0260] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0261] (2) Sensitivity solution: Accurately measure 1 ml of the reference solution (1) and place it in a 10 ml volumetric flask. Dilute to the mark with 2% acetic acid (v / v) solution-acetonitrile (50:50) and shake well.

[0262] (3) Brand and model of HPLC: Agilent 1260;

[0263] (4) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0264] (5) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0265] (6) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 55% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase: acetic acid-water-acetonitrile (1:45:55), run for 10 min].

[0266] (7) Flow rate is 0.5 ml per minute;

[0267] (8) Column temperature is 30°C;

[0268] (9) The sample tray temperature is 25°C;

[0269] (10) Injection volume: 5 μl;

[0270] (11) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0271]

[0272] (12) Take a sample of the sensitivity solution.

[0273] (13) Table 5 shows the signal-to-noise ratio results of each impurity in Example 1, Example 8, Example 9, Example 10, Example 11, Example 12 and Example 13. It can be seen from Table 5 that when the flow rate is 0.45-0.55 ml / min, the column temperature is 28-32 °C, and the proportion of mobile phase B is 45%-55%, the signal-to-noise ratio of each impurity in the sensitivity solution of this method is greater than 10, which meets the requirements of the Chinese Pharmacopoeia.

[0274] Table 5 Signal-to-noise ratio results of each impurity

[0275]

[0276] Embodiment 14

[0277] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0278] (2) Brand and model of HPLC: Agilent 1260;

[0279] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0280] (4) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0281] (5) Mobile phase A: water, mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min.

[0282] (6) Flow rate is 0.5 ml per minute;

[0283] (7) Column temperature is 30°C;

[0284] (8) The sample tray temperature is 25°C;

[0285] (9) Injection volume: 5 μl;

[0286] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0287]

[0288] (11) Take a sample of the reference solution and inject it.

[0289] Embodiment 15

[0290] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0291] (2) Brand and model of HPLC: Agilent 1260;

[0292] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0293] (4) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0294] (5) Mobile phase A: formic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, mobile phase is formic acid-water-acetonitrile (1:50:50), run for 10 min].

[0295] (6) Flow rate is 0.5 ml per minute;

[0296] (7) Column temperature is 30°C;

[0297] (8) The sample tray temperature is 25°C;

[0298] (9) Injection volume: 5 μl;

[0299] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0300]

[0301] (11) Take a sample of the reference solution and inject it.

[0302] Example 16

[0303] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0304] (2) Brand and model of HPLC: Agilent 1260;

[0305] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0306] (4) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0307] (5) Mobile phase A: acetic acid-water (5:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min [or, the mobile phase is acetic acid-water-acetonitrile (2.5:50:50), run for 10 min].

[0308] (6) Flow rate is 0.5 ml per minute;

[0309] (7) Column temperature is 30°C;

[0310] (8) The sample tray temperature is 25°C;

[0311] (9) Injection volume: 5 μl;

[0312] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0313]

[0314] (11) Take a sample of the reference solution and inject it.

[0315] (12) Fig.18 This is the impurity SM1-C diagram in the reference solution of Example 1; Fig.19 This is a graph of impurities SM1-C in the reference solution of Example 14; Fig. 20 This is the impurity SM1-C diagram in the reference solution of Example 15; Fig.21 The figure is for the impurity SM1-C in the reference solution of Example 16. By comparing Example 1, Example 14, Example 15 with Example 16, taking the peak of impurity SM1-C as an example, it is shown that 2% formic acid and 2% to 5% acetic acid are suitable as mobile phase A.

[0316] Embodiment 17

[0317] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0318] (2) Brand and model of HPLC: Agilent 1260;

[0319] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0320] (4) Chromatographic column: ACE Excel C18-PFP, 4.6×150 mm, 3 μm;

[0321] (5) Mobile phase A: acetic acid-water (2:100), mobile phase B: methanol; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min.

[0322] (6) Flow rate is 0.5 ml per minute;

[0323] (7) Column temperature is 30°C;

[0324] (8) The sample tray temperature is 25°C;

[0325] (9) Injection volume: 5 μl;

[0326] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0327]

[0328] (11) Take a sample of the reference solution and inject it.

[0329] (12) Fig. 22 , Fig.23 and Fig.24The figure shows the impurities in the reference solution. By comparing Example 1 with Example 17, it is shown that acetonitrile is suitable as mobile phase B.

[0330] Embodiment 18

[0331] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0332] (2) Brand and model of HPLC: Agilent 1260;

[0333] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0334] (4) Chromatographic column: Pheromones Titank C18, specifications: 2.1 mm × 50 mm, 3 μm;

[0335] (5) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 10 min.

[0336] (6) Flow rate is 0.2 ml per minute;

[0337] (7) Column temperature is 30°C;

[0338] (8) The sample tray temperature is 25°C;

[0339] (9) Injection volume: 5 μl;

[0340] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0341]

[0342] (11) Take a sample of the reference solution and inject it.

[0343] (12) Fig.25 This is the TIC diagram of each impurity in the reference solution.

[0344] Embodiment 19

[0345] (1) Reference solution: Take 2.4 mg of impurity SM1 reference, 2.4 mg of SM1-B reference, 2.4 mg of SM1-C reference, 2.4 mg of SM1-D reference, 2.4 mg of SM1-E reference and 2.4 mg of SM1-G reference, accurately weigh, place in a 20 ml volumetric flask, add about 12 ml of 2% acetic acid (v / v) solution-acetonitrile (50:50) to dissolve, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 20 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well. Accurately measure 1 ml, place in a 50 ml volumetric flask, dilute to scale with 2% acetic acid (v / v) solution-acetonitrile (50:50), and shake well.

[0346] (2) Brand and model of HPLC: Agilent 1260;

[0347] (3) Brand and model of triple quadrupole mass spectrometer: AB SCIEX API4000;

[0348] (4) Chromatographic column: Pheromon Titank F5, specification: 4.6 mm × 150 mm, 3 μm;

[0349] (5) Mobile phase A: acetic acid-water (2:100), mobile phase B: acetonitrile; isocratic elution: 50% mobile phase B by volume, maintaining this mobile phase ratio for 30 min.

[0350] (6) Flow rate is 0.5 ml per minute;

[0351] (7) Column temperature is 30°C;

[0352] (8) The sample tray temperature is 25°C;

[0353] (9) Injection volume: 5 μl;

[0354] (10) Detection was performed using a triple quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550 °C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

[0355]

[0356] (11) Take a sample of the reference solution and inject it.

[0357] (12) Fig.26 The XIC graph of impurity SM1-C in the reference solution of Example 19. By comparing Example 1, Example 18 and Example 19, it is shown that the chromatographic column (ACE Excel C18-PFP, 4.6×150 mm, 3 μm) is the best.

Claims

1. A method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology, characterized in that: The detection method is as follows: using ESI-LC-MSMS technology, adopting a reverse phase chromatographic column with octadecylsilane bonded silica gel as filler, using an aqueous phase as mobile phase A, an organic phase as mobile phase B, setting liquid phase conditions and mass spectrometry conditions, the detection method can simultaneously and quantitatively detect six benzoic acid genotoxic impurities in chloroprocaine hydrochloride, especially can effectively separate and accurately determine the isomeric impurities SM1 (4-nitro-2-chlorobenzoic acid) and SM1-B (4-nitro-3-chlorobenzoic acid).

2. A method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 1, characterized in that: The reverse phase chromatography column is selected from ACE ExcelC18-PFP, with specifications of 4.6mm×150mm and 3.0µm.

3. The method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 1, characterized in that: The mobile phase A contains an acid, and the acid is selected from formic acid or acetic acid.

4. The method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 3, characterized in that: The acid is selected from acetic acid, and the volume ratio of acetic acid to water is selected from 2:

100.

5. The method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 1, characterized in that: The organic phase in the mobile phase B is acetonitrile or methanol.

6. The method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 5, characterized in that: The mobile phase B is selected from acetonitrile.

7. The method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 1, characterized in that: The liquid phase conditions in the ESI-LC-MSMS technology are as follows: the flow rate is selected from 0.45~0.55ml / min, the column temperature is selected from 28~32℃, the injection plate temperature is selected from 20~30℃, the injection volume is selected from 5μL, and the isocratic elution is: by volume ratio, with 50% mobile phase B, and the mobile phase ratio is maintained for 10min.

8. A method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claim 7, characterized in that: The liquid phase conditions in the ESI-LC-MSMS technology are as follows: the flow rate is selected from 0.5 ml / min, the column temperature is selected from 30° C., the injection plate temperature is selected from 25° C., and the injection volume is selected from 5 μL.

9. A method for simultaneously determining six benzoic acid genotoxic impurities in chloroprocaine hydrochloride or its preparations using ESI-LC-MSMS technology according to claims 1 to 8, characterized in that: In the mass spectrometry conditions of the ESI-LC-MSMS technique, detection was performed using the multiple reaction monitoring (MRM) mode of a triple quadrupole mass spectrometer, electrospray ionization, and negative ion mode scanning; the curtain gas was 35 psi, the nebulizer 1 was 40 psi, the nebulizer 2 was 45 psi, the spray needle voltage was -4500 V, and the drying temperature was 550°C; the quantitative ion pairs, declustering voltage (DP), and collision voltage (CE) of each impurity are shown in the following table:

Images