Method for detecting halogenated ester in cephalosporin-type medicine

Abstract

The invention relates to the field of drug analysis and detection, and discloses a method for detecting halogenated ester in a cephalosporin-type drug, which comprises the following steps: 1) dissolving a cephalosporin-type drug sample in a solvent as a test sample stock solution, adding n-hexane for extraction, and taking supernate to obtain a test sample solution; 2) dissolving a halogenated ester reference substance in a solvent to serve as a reference substance stock solution, adding n-hexane for extraction, and taking supernate to obtain a reference substance solution; and (3) detecting the test solution and the reference solution by adopting a gas chromatography-mass spectrometry method, and calculating by using an external standard method to obtain the content of halogenated ester in the cephalosporin type medicine. The halogenated ester is separated from the cephalosporin compound through normal hexane extraction, chromatographic condition screening and the like; the gas chromatography and mass spectrometry conditions are optimized, so that the detection limit reaches 0.055 mu g / ml or below, the quantitation limit reaches 0.2 mu g / ml or below, and the quantitation limit concentration is verified by accuracy, so that the industrial detection is more convenient, effective and accurate.

Description

Technical Field

[0001] The invention relates to the field of drug analysis and detection, and in particular to a method for detecting halogenated esters in cefuroxime-type drugs. Background Art

[0002] There are different synthetic routes for cefuroxime-type drugs, and iodoesters are essential raw materials. Iodinated esters include 1-iodoethyl isopropyl carbonate and iodomethyl pivalate. According to Sarah Dereck's software expert library and structure prediction, this type of halogenated ester is a genotoxic impurity. Halogenated esters have the properties of electrophilic reagents. Under physiological conditions, they undergo substitution reactions with nucleophilic centers in nucleic acids, proteins or other important components in the body, causing irreversible damage to these components, manifested as toxicity, mutagenicity or carcinogenicity. In order to ensure the safety of drugs, they should be strictly controlled. According to the M7 guidelines issued by the International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and the calculation of the maximum daily dose of drugs, the residues of this type of halogenated esters in cephalosporins should be controlled below 25ppm.

[0003] "Guangdong Chemical Industry" 2019, Vol. 46, No. 405, 169: A quantitative detection method for the residue of 1-iodoethyl isopropyl carbonate in cefpodoxime axetil using gas chromatography is disclosed. The FID is used as the detector and acetonitrile is used as the solvent to prepare the test sample into a 100 mg / ml solution. After preparation, the sample is directly injected for detection. The quantitative limit concentration is 30.69 μg / ml, which is equivalent to 300 ppm of the sample solution concentration. However, due to the poor stability of cephalosporin compounds, when the direct injection method is used, the amide bond of the cephalosporin compound will break at the high temperature of the injection port, decompose into small molecules and continuously enter the chromatographic column, which interferes with the determination of the impurities being measured, resulting in low detection sensitivity and inaccurate detection results.

[0004] When halogenated esters are used as genotoxic impurities, the halogenated ester content in cefuroxime drugs needs to be strictly controlled, the impurity limit is low, and the detection method requires high sensitivity. Therefore, a detection method for low-content halogenated esters in cefuroxime drugs with high sensitivity, accurate results, and simple operation is urgently needed. Summary of the invention

[0005] In order to solve the above-mentioned technical problems of low detection sensitivity and inaccurate detection results, the present invention provides a detection method for halogenated esters in cefuroxime-type drugs, which achieves concentration and enrichment of halogenated esters by extracting the test sample to be tested and eliminates the interference of cephalosporin compounds. The detection method is simple to operate, highly sensitive and produces accurate detection results.

[0006] The specific technical scheme of the present invention is: a method for detecting halogenated esters in cefuroxime type drugs, comprising the following steps: 1) dissolving a cefuroxime type drug sample in a solvent as a test sample stock solution, adding n-hexane for extraction, taking the supernatant, and obtaining a test sample solution; 2) Dissolve the halogenated ester reference substance in a solvent as a reference substance stock solution, add n-hexane for extraction, and take the supernatant to obtain a reference substance solution; 3) The test solution and the reference solution were detected by gas chromatography-mass spectrometry, and the content of the halogenated ester in the cefuroxime-type drug was calculated using the external standard method.

[0007] The pretreatment method of liquid-liquid extraction is used to achieve the concentration and enrichment of halogenated esters and eliminate most matrix interferences. By adjusting the gas phase parameters, halogenated esters are separated from other interferences, and the mass spectrometry detection maintains high sensitivity, which can complete the detection of low-concentration halogenated esters in the test sample and ensure the accuracy of the results.

[0008] Furthermore, the halogenated ester is one or more of 1-chloroethyl isopropyl carbonate, 1-iodoethyl isopropyl carbonate, chloromethyl pivalate, and iodomethyl pivalate.

[0009] Furthermore, the cefuroxime-type drug is cefpodoxime proxetil or cefditoren pivoxil.

[0010] Further, in step 3), the gas chromatography conditions in the gas chromatography-mass spectrometry are: Program temperature rise: the initial column temperature is 50-70°C, maintained for 3-8 min, then raised to 290-310°C at a rate of 10-20°C / min, maintained for 2-5 min; Inlet temperature: 150~200℃.

[0011] Although extraction can eliminate most matrix interference, when the injection port temperature is too high, a small amount of unseparated amide bonds of cefpodoxime proxetil will break and produce interference peaks. Therefore, the injection port temperature should not be too high. When the injection port temperature is in the range of 150-200℃, it can not only ensure the vaporization of the target halogenated ester, but also minimize the appearance of interference peaks. At the same time, high column temperature can make the interference peaks entering the chromatographic column flow out, so that the analytical instrument maintains a high sensitivity state.

[0012] Further, in step 3), the gas chromatography conditions in the gas chromatography-mass spectrometry are: Chromatographic column: 5% phenyl methyl polysiloxane filler column; Carrier gas: high purity helium; Injection volume: 1-2 μl; Flow rate: 1.0~2.0ml / min; Split ratio: (4-6):1.

[0013] Preferably, in the gas chromatography conditions, the chromatographic column is Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; the flow rate is 1.0ml / min; and the injection port temperature is 170°C.

[0014] Further, in step 3), the mass spectrometry analysis conditions in gas chromatography-mass spectrometry are: Ion source: EI source; Ion source temperature: 230~250℃; Quadrupole temperature: 150~170℃; Interface temperature: 300~320℃; Dwell time: 100~150ms.

[0015] Further, in step 3), the monitoring mode and quantitative ion of each test compound in the mass spectrometry of gas chromatography-mass spectrometry are: 1-Chloroethyl isopropyl carbonate, SIM, 45; 1-iodoethyl isopropyl carbonate, SIM, 45; Chloromethyl pivalate, MRM, 85>57; Iodomethyl pivalate, MRM, 85>57.

[0016] Furthermore, in step 1) and step 2), the solvent is acetonitrile or methanol.

[0017] The solubility of cefuroxime drugs in acetonitrile or methanol is much higher than that in n-hexane. According to the principle of like dissolves like, halogenated esters have a higher solubility in n-hexane and are extracted into n-hexane, while cephalosporin compounds are soluble in acetonitrile or methanol. While reducing the actual concentration of the test sample, it also enriches the impurities to be tested, so the measured halogenated ester content is accurate and reliable.

[0018] Furthermore, in step 1) and step 2), the extraction time is both 0.5 to 2 minutes.

[0019] Furthermore, in step 1), the mass volume ratio of the cefuroxime-type drug sample to the solvent is (2.4-54 mg):1 ml.

[0020] Preferably, in step 1), the mass volume ratio of the cefuroxime-type drug sample to the solvent is (12-36 mg):1 ml.

[0021] Furthermore, in step 2), the mass volume ratio of the halogenated ester reference substance to the solvent is (0.06-2.0 μg): 1 ml.

[0022] Further, in step 1), the cefuroxime-type drug sample is dissolved in a solvent to form a test sample stock solution, and the volume ratio of n-hexane to the test sample stock solution is 1:(2 to 2.5); in step 2), the halogenated ester reference substance is dissolved in a solvent to form a reference substance stock solution, and the volume ratio of n-hexane to the reference substance stock solution is 1:(2 to 2.5).

[0023] Furthermore, in step 1), 2 to 6 ml of the test sample stock solution is taken for extraction.

[0024] Furthermore, in step 2), 2 to 6 ml of the reference substance stock solution is taken for extraction.

[0025] Compared with the prior art, the present invention has the following advantages: The present invention provides a detection method capable of accurately determining halogenated esters in cefuroxime type drugs, by pre-extraction treatment of test samples and reference samples, most matrix interferences are eliminated, and the concentration and enrichment of halogenated esters are achieved. By optimizing chromatographic parameters, halogenated esters are separated from other interfering substances, the high sensitivity of mass spectrometry detection is maintained, and the accuracy of the results is guaranteed; the mass spectrometry conditions are optimized to make the detection limit reach below 0.055 μg / ml, the quantitative limit reach below 0.2 μg / ml, and after accuracy verification, extremely low content of halogenated esters can be measured. The specificity, sensitivity, accuracy, linearity, and repeatability of this method all meet the verification requirements, and are of great significance to the quality control of cefuroxime type drugs and the safety of clinical medication. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the blank solution spectrum in Example 1.

[0027] Figure 2 This is the spectrum of the reference solution in Example 1.

[0028] Figure 3 This is the spectrum of the test solution in Example 1.

[0029] Figure 4 This is the detection limit solution spectrum in Example 1.

[0030] Figure 5 This is the quantitative limit solution spectrum in Example 1.

[0031] Figure 6 This is the spectrum of the 100% spiked solution in Example 1.

[0032] Figure 7 This is the blank solution spectrum in Example 2.

[0033] Figure 8 This is the spectrum of the reference solution in Example 2.

[0034] Fig. 9This is the spectrum of the test solution in Example 2.

[0035] Fig.10 This is the blank spectrum in Comparative Example 1.

[0036] Fig.11 This is the spectrum of reference solution 1 in Comparative Example 1.

[0037] Fig.12 This is the spectrum of the test solution in Comparative Example 1.

[0038] Fig.13 This is the spectrum of reference solution 2 in comparative example 1.

[0039] Fig.14 This is the spectrum obtained for gas phase parameter 1 in test example 2.

[0040] Fig.15 This is the spectrum obtained for gas phase parameter 2 in test example 2.

[0041] Fig.16 This is the spectrum obtained for gas phase parameter 3 in test example 2. DETAILED DESCRIPTION

[0042] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0043] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0044] In the present invention, the ratios of the mobile phases mentioned are all volume ratios unless otherwise specified.

[0045] 1. Instrument Analytical balance: XPR105; Gas Spectrometer: Shimadzu GC2030-8050NX.

[0046] 2. Reagent acetonitrile: HPLC; n-Hexane: GC.

[0047] 3. Reference 1-iodoethyl isopropyl carbonate; 1-chloroethyl isopropyl carbonate; iodomethyl pivalate; chloromethyl pivalate.

[0048] 4. Test products Cefpodoxime proxetil API was provided by Zhejiang Prodebon Pharmaceutical Co., Ltd.; Cefditoren pivoxil API was provided by Zhejiang Prodebang Pharmaceutical Co., Ltd.

[0049] Example 1 This example detects 1-iodoethyl isopropyl carbonate and 1-chloroethyl isopropyl carbonate in cefpodoxime axetil.

[0050] 1. Instrument parameters The gas chromatography conditions were: Chromatographic column: Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; carrier gas: high-purity helium; injection volume: 1μl; flow rate: 1.0ml / min; split ratio: 5:1; injection port temperature: 170℃; programmed temperature: the initial column temperature is 70℃, maintained for 8min, and then increased to 300℃ at a rate of 15℃ / min and maintained for 2min.

[0051] The mass spectrometry analysis conditions were: Ion source: EI source; ion source temperature: 230°C; quadrupole temperature: 150°C; interface temperature: 300°C; detection mode: selected ion monitoring SIM mode, quantitative ion is 45; dwell time: 100ms.

[0052] 2. Experimental process 1) Solution Preparation Blank solution: Take 3.0 ml of acetonitrile into a 10 ml centrifuge tube, add 1.5 ml of n-hexane, vortex for 1 min, separate the layers, and take the supernatant.

[0053] Test solution: Weigh 108 mg of cefpodoxime axetil sample and dissolve it in 3.0 ml of acetonitrile to obtain a test stock solution. Take 3.0 ml of the test stock solution into a 10 ml centrifuge tube, add 1.5 ml of n-hexane, vortex for 1 min, and then separate the supernatant.

[0054] Impurity stock solution: Weigh 45 mg of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate into a 100 ml volumetric flask, dissolve and dilute to volume with acetonitrile, and mix well. Pipette 1.0 ml of the above solution into a 50 ml volumetric flask, dilute to scale with acetonitrile, and mix well.

[0055] Reference substance solution: accurately transfer 2.0 ml of impurity stock solution to a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain reference substance stock solution, transfer 3.0 ml of reference substance stock solution to a 10 ml centrifuge tube, add 1.5 ml of n-hexane, vortex for 1 min, and then separate the supernatant.

[0056] 2) Sampling and analysis Accurately measure 1 μl of the test solution and each reference solution, inject them into the gas chromatography-mass spectrometer, and use the external standard method to calculate the content of the halogenated ester in the test sample.

[0057] 3. Effectiveness of the detection method 1. Exclusivity Take blank solution, reference solution and test solution and inject them into GC-MS. Record the interference of blank solution, reference solution and test solution at the retention time of target peak. See the spectrum for details. Figure 1 to Figure 3 .

[0058] Exclusive results: from Figure 2 It can be seen that the retention time of 1-chloroethyl isopropyl carbonate in the reference solution is 10.00 min, and the retention time of 1-iodoethyl isopropyl carbonate is 12.36 min; Figure 1 and Figure 2 , the blank solution has no interference at the retention time of the target peak; Figure 3 and Figure 2 , the test solution has no interference at the target peak retention time, and the test solution has a flat baseline. This proves that the method described in this example has good specificity.

[0059] 2. Sensitivity Take the reference solution, dilute it step by step with the diluent, and extract it in the same way as the reference solution before entering the instrument for detection and recording the chromatogram. The detection limit (LOD) is calculated as S / N ≥ 3, and the quantification limit (LOQ) is calculated as S / N ≥ 10. The detection limit and quantification limit of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate are determined by injection respectively. The experimental results are shown in Table 1. The LOD and LOQ spectra are shown in Figure 4 and Figure 5 .

[0060] Table 1 Detection limit and quantitation limit test results of Example 1 From the data in Table 1, it can be seen that the detection limit of 1-chloroethyl isopropyl carbonate is 0.054 μg / ml, and the signal-to-noise ratio is 10, which is equivalent to 1.5ppm of the test solution concentration; the quantitative limit is 0.18 μg / ml, and the signal-to-noise ratio is 27, which is equivalent to 5ppm of the test solution concentration. The detection limit of 1-iodoethyl isopropyl carbonate is 0.042 μg / ml, and the signal-to-noise ratio is 13, which is equivalent to 1.2ppm of the test solution concentration; the quantitative limit is 0.14 μg / ml, and the signal-to-noise ratio is 38, which is equivalent to 4ppm of the test solution concentration. This proves that the method described in this embodiment has high sensitivity.

[0061] 3. Linear Take an appropriate amount of impurity stock solution, dilute the impurity stock solution with acetonitrile to prepare the standard series mixed solution shown in the table below, extract with n-hexane and enter into gas chromatography-mass spectrometry for detection, and perform linear regression with concentration as the horizontal axis and peak area as the vertical axis. The results are shown in Table 2.

[0062] Table 2 Linear results of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate From the data in Table 2, it can be seen that the linear regression equation of 1-chloroethyl isopropyl carbonate is y=54025.6291x-401.2263, and the regression coefficient r is 0.9999, indicating that it has a good linear relationship in the range of 0.1789μg / ml to 1.7894μg / ml; the linear regression equation of 1-iodoethyl isopropyl carbonate is y=48428.5750x-728.0321, and the regression coefficient r is 0.9999, indicating that it has a good linear relationship in the range of 0.1975μg / ml to 1.9746μg / ml.

[0063] 4. Accuracy Quantitation limit spiked stock solution: Accurately pipette 0.4 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain the quantitation limit spiked stock solution.

[0064] 100% spiked stock solution: Accurately pipette 2.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain the 100% spiked stock solution.

[0065] 150% spiked stock solution: Accurately pipette 3.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain a 150% spiked stock solution.

[0066] Quantitation limit spike solution: weigh 108 mg of cefpodoxime axetil sample into a 10 ml centrifuge tube, add 3.0 ml of quantitation limit spike stock solution to completely dissolve it, add 1.5 ml of n-hexane, vortex and separate the supernatant to obtain quantitation limit spike solution. Prepare 3 portions in the same way.

[0067] 100% spiked solution: weigh 108 mg of cefpodoxime axetil sample into a 10 ml centrifuge tube, add 3.0 ml of 100% spiked stock solution to completely dissolve it, add 1.5 ml of n-hexane, vortex and separate the supernatant to obtain 100% spiked solution. Prepare 3 portions in the same way.

[0068] 150% spiked solution: weigh 108 mg of cefpodoxime axetil sample into a 10 ml centrifuge tube, add 3.0 ml of 150% spiked stock solution to completely dissolve it, add 1.5 ml of n-hexane, vortex and separate the supernatant to obtain 150% spiked solution. Prepare 3 portions in the same way.

[0069] Take the above solution and test it according to the gas chromatography-mass spectrometry conditions, record the spectrum, and the spectrum of 100% spiked solution is shown in Figure 6 The results are shown in Tables 3 and 4.

[0070] Table 3 1-Chloroethyl isopropyl carbonate accuracy test results Table 4 Accuracy determination results of 1-iodoethyl isopropyl carbonate The known acceptable standard is that the recovery rate is between 75% and 120%, and the RSD value is less than 10.0%. Tables 3 and 4 show that at three spike levels, the recovery rate of the halogenated ester in cefpodoxime axetil detected by the method described in this embodiment is 89.22% to 104.10%, and the maximum RSD is 2.0%, which meets the standard. This proves that the recovery rate of the method described in this embodiment is high.

[0071] 5. Repeatability Three portions of the test solution and six portions of 100% spiked solution were prepared and tested under the above-mentioned gas chromatography-mass spectrometry conditions. The results are shown in Tables 5 and 6.

[0072] Table 5 Repeatability test results of 1-chloroethyl isopropyl carbonate Table 6 Repeatability test results of 1-iodoethyl isopropyl carbonate It is known that the acceptable standard RSD value is less than 8%, and the maximum RSD of the test result of 100% spiked solution is 2.8%, which proves that the method described in this example has good repeatability.

[0073] Example 2 This example detects chloromethyl pivalate and iodomethyl pivalate in cefditoren pivoxil.

[0074] 1. Instrument parameters The gas chromatography conditions were: Chromatographic column: Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; carrier gas: high-purity helium; injection volume: 1μl; flow rate: 1.5ml / min; split ratio: 5:1; injection port temperature: 200℃; programmed temperature: the initial column temperature is 50℃, maintained for 3min, and then increased to 300℃ at a rate of 10℃ / min and maintained for 2min.

[0075] The mass spectrometry analysis conditions were: Ion source: EI source; ion source temperature: 230°C; quadrupole temperature: 150°C; interface temperature: 300°C; detection mode: multiple reaction monitoring MRM mode, parent ion is 85, daughter ion is 57; dwell time: 100ms.

[0076] 2. Experimental process 1) Solution Preparation Blank solution: Take 5.0 ml of acetonitrile into a 15 ml centrifuge tube, add 2.0 ml of n-hexane, vortex for 1.5 min, and then separate the supernatant.

[0077] Test solution: Accurately weigh 60 mg of cefditoren pivoxil sample and dissolve it in 5.0 ml of acetonitrile to obtain the test stock solution. Take 5.0 ml of the test stock solution into a 15 ml centrifuge tube, add 2.0 ml of n-hexane, vortex for 1.5 min, and then separate the supernatant.

[0078] Reference solution: Weigh 30 mg of each of chloromethyl pivalate and iodomethyl pivalate into a 100 ml volumetric flask, dissolve and dilute to scale with acetonitrile. Pipette 1.0 ml of the above solution into a 100 ml volumetric flask, dilute to scale with acetonitrile, and shake well to obtain an impurity stock solution. Accurately pipette 2.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute to scale with acetonitrile, and shake well to obtain a reference stock solution. Take 5.0 ml of the reference stock solution into a 15 ml centrifuge tube, add 2.0 ml of n-hexane, vortex for 1.5 min, and then separate and take the supernatant.

[0079] 2) Sampling and analysis Accurately measure 1 μl of the test solution and each reference solution, inject them into the gas chromatography-mass spectrometer, and use the external standard method to calculate the content of the halogenated ester in the test sample.

[0080] 3. Effectiveness of the detection method 1. Exclusivity Take blank solution, reference solution and test solution and inject them into GC-MS. Record the interference of blank solution, reference solution and test solution at the retention time of target peak. See the spectrum for details. Figure 7 to Figure 9 .

[0081] Exclusive results: from Figure 8It can be seen that the retention time of chloromethyl pivalate in the reference solution is 8.82 min, and the retention time of iodomethyl pivalate is 11.58 min; Figure 7 and Figure 8 , the blank solution has no interference at the retention time of the target peak; Fig. 9 and Figure 8 , the test solution has no interference at the target peak retention time, and the test solution has a flat baseline. This proves that the method described in this example has good specificity.

[0082] 2. Sensitivity Take the reference solution, dilute it step by step with the diluent, and extract it in the same way as the reference solution, then enter the gas chromatography-mass spectrometer for detection, and record the chromatogram. The detection limit (LOD) is calculated as S / N ≥ 3, and the quantification limit (LOQ) is calculated as S / N ≥ 10. The detection limit and quantification limit of chloromethyl pivalate and iodomethyl pivalate are determined by injection, respectively. The experimental results are shown in Table 7.

[0083] Table 7 Detection limit and quantitative limit test results of Example 2 As shown in Table 7, the detection limit of chloromethyl pivalate is 0.019 μg / ml, and the signal-to-noise ratio is 10.4, which is equivalent to 1.6ppm of the test solution concentration; the quantitative limit is 0.062 μg / ml, and the signal-to-noise ratio is 24.8, which is equivalent to 5.2ppm of the test solution concentration. The detection limit of iodomethyl pivalate is 0.019 μg / ml, and the signal-to-noise ratio is 5.8, which is equivalent to 1.6ppm of the test solution concentration; the quantitative limit is 0.064 μg / ml, and the signal-to-noise ratio is 11.9, which is equivalent to 5.3ppm of the test solution concentration. This proves that the method described in this embodiment has high sensitivity.

[0084] 3. Accuracy Quantitation limit spiked stock solution: Accurately pipette 0.4 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain the quantitation limit spiked stock solution.

[0085] 100% spiked stock solution: Accurately pipette 2.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain the 100% spiked stock solution.

[0086] 150% spiked stock solution: Accurately pipette 3.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and make up to the mark, shake well to obtain a 150% spiked stock solution.

[0087] Quantitation limit spike solution: weigh 60 mg of cefditoren pivoxil sample into a 15 ml centrifuge tube, add 5.0 ml of quantitation limit spike stock solution to completely dissolve it, add 2.0 ml of n-hexane, vortex and separate the supernatant to obtain quantitation limit spike solution. Prepare 3 portions in the same way.

[0088] 100% spiked solution: weigh 60 mg of cefditoren pivoxil sample into a 15 ml centrifuge tube, add 5.0 ml of 100% spiked stock solution to completely dissolve it, add 2.0 ml of n-hexane, vortex and separate the supernatant to obtain 100% spiked solution. Prepare 3 portions in the same way.

[0089] 150% spiked solution: weigh 60 mg of cefditoren pivoxil sample into a 15 ml centrifuge tube, add 5.0 ml of 150% spiked stock solution to completely dissolve it, add 2.0 ml of n-hexane, vortex and separate the supernatant to obtain 150% spiked solution. Prepare 3 portions in the same way.

[0090] The above solutions were tested under gas chromatography-mass spectrometry conditions. The results are shown in Tables 8 and 9.

[0091] Table 8 Chloromethyl pivalate accuracy determination results Table 9 Iodomethyl pivalate accuracy determination results The known acceptable standard is that the recovery rate is between 75-120% and the RSD value is less than 10%. Tables 8 and 9 show that at three spike levels, the recovery rate of halogenated esters in cefditoren pivoxil detected by the method described in this embodiment is 88.15% to 99.90%, and the maximum RSD is 4.2%, which meets the standard. This proves that the recovery rate of the method described in this embodiment is high.

[0092] Comparative Example 1 In this comparative example, the direct injection method was used to determine 1-chloroethyl isopropyl carbonate in cefpodoxime axetil.

[0093] 1. Instrument parameters The gas chromatography conditions were: Chromatographic column: Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; carrier gas: high-purity helium; injection volume: 1μl; flow rate: 1.0ml / min; split ratio: 5:1; injection port temperature: 170℃; programmed temperature: the initial column temperature is 70℃, maintained for 8min, and then increased to 300℃ at a rate of 15℃ / min and maintained for 2min.

[0094] The mass spectrometry analysis conditions were: Ion source: EI source; ion source temperature: 230°C; quadrupole temperature: 150°C; interface temperature: 300°C; detection mode: selected ion monitoring SIM mode, quantitative ion is 45; dwell time: 100ms.

[0095] 2. Experimental process 1) Solution preparation Test solution: Weigh 108 mg of cefpodoxime axetil sample, dissolve in 3 ml of acetonitrile and mix well.

[0096] Reference solution: Weigh 45 mg of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate into a 100 ml volumetric flask, dissolve and dilute to volume with acetonitrile, and mix well. Pipette 1.0 ml of the above solution into a 50 ml volumetric flask, dilute to scale with acetonitrile, and mix well to obtain an impurity stock solution. Accurately pipette 2.0 ml of the impurity stock solution into a 20 ml volumetric flask, dilute with acetonitrile and dilute to scale, and shake well to obtain a reference solution.

[0097] 2) Sampling and analysis Accurately measure 1 μl of the test solution and each reference solution, inject them into the gas chromatography-mass spectrometer, and use the external standard method to calculate the content of the halogenated ester in the test sample.

[0098] 3. Experimental Results Take the diluent acetonitrile, the reference solution and the test solution and inject them into the gas chromatography-mass spectrometer. Record the interference of the blank solution, the reference solution and the test solution at the retention time of the target peak. For the spectrum, see Figure 10 to Figure 13 .

[0099] Exclusive results: from Fig.11 It can be seen that the retention time of 1-chloroethyl isopropyl carbonate in the reference solution is 10.85min; Fig.10 and Fig.11 , the blank solution has no interference at the retention time of the target peak and the baseline is flat; Fig.12 and Fig.11 , the test solution showed more unknown peaks at the retention time of the target peak, and the baseline had large fluctuations; after completing the analysis of the test solution, the reference solution was re-injected for analysis, and the results were Fig.13 , compared to Fig.11 , unknown peaks appeared and the baseline was not flat. The reason was that the test sample was decomposed by heat at the injection port, which contaminated the injection port, and the decomposition products continued to enter the chromatographic column, resulting in an uneven baseline in the spectrum and low sensitivity. If the experiment is continued, the contamination of the instrument may be aggravated. Therefore, this method is not suitable for the detection of halogenated esters in cefpodoxime axetil.

[0100] Test Example 1 This test example uses n-hexane as the extractant to study the effect of different solvents on the extraction rate of target impurities.

[0101] 1. Instrument parameters The gas chromatography conditions were: Chromatographic column: Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; carrier gas: high-purity helium; flow rate: 1.0ml / min; split ratio: 5:1; injection port temperature: 170℃; programmed temperature: the initial column temperature is 70℃, maintained for 8min, and then increased to 300℃ at a rate of 15℃ / min and maintained for 2min.

[0102] The mass spectrometry analysis conditions were: Ion source: EI source; ion source temperature: 230°C; quadrupole temperature: 150°C; interface temperature: 300°C; detection mode: selected ion monitoring SIM mode, quantitative ion is 45; dwell time: 100ms.

[0103] 2. Experimental process 1) Solution Preparation Reference solution: Weigh 45 mg each of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate into a 100 ml volumetric flask, dissolve and dilute to volume with n-hexane, mix well, transfer 1.0 ml of the solution into a 50 ml volumetric flask, dilute to scale with n-hexane, mix well, transfer another 1.0 ml of the solution into a 10 ml volumetric flask, dilute to scale with n-hexane, and mix well.

[0104] Acetonitrile solution to be extracted: Weigh 45 mg each of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate into a 100 ml volumetric flask, dissolve and dilute to volume with acetonitrile, mix well, transfer 1.0 ml of the solution to a 50 ml volumetric flask, dilute to the mark with acetonitrile, mix well, transfer 1.0 ml of the above solution to a 10 ml volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0105] Methanol solution to be extracted: Weigh 45 mg each of 1-chloroethyl isopropyl carbonate and 1-iodoethyl isopropyl carbonate into a 100 ml volumetric flask, dissolve and dilute to volume with methanol, mix well, transfer 1.0 ml of the solution to a 50 ml volumetric flask, dilute to the mark with methanol, mix well, transfer another 1.0 ml of the solution to a 10 ml volumetric flask, dilute to the mark with methanol, and mix well.

[0106] 2) Extraction: Pipette 2.0 ml of the acetonitrile solution to be extracted into a 10 ml centrifuge tube, add 2.0 ml of n-hexane, vortex for 1 min, separate the layers, take the supernatant, and obtain an acetonitrile extraction solution; Pipette 2.0 ml of the methanol solution to be extracted into a 10 ml centrifuge tube, add 2.0 ml of n-hexane, vortex for 1 min, separate the layers, take the supernatant, and obtain a methanol extraction solution.

[0107] 3. Experimental Results Precisely measure 1 μl of the blank solution and each extraction solution and inject them into the gas chromatography-mass spectrometer. The extraction rate of impurities is calculated with the peak area of ​​the reference solution as 100%. The results are shown in Table 10.

[0108] Table 10 Target impurity extraction rate of different solvents It can be seen from Table 10 that when methanol or acetonitrile is used as the solvent, the target impurities in the solvent can be extracted by n-hexane, and the extraction rate is higher than 78.5%.

[0109] Test Example 2 This test case studies the effect of gas phase parameters on the baseline.

[0110] 1. Instrument parameters The gas chromatography conditions were: (1) Gas phase parameters 1 Chromatographic column: Agilent DB-5MS, 30m×0.25mm, 1μm chromatographic column; carrier gas: high-purity helium; flow rate: 1.0ml / min; split ratio: 5:1; injection port temperature: 250℃; programmed temperature: the initial column temperature is 70℃, maintained for 8min, and then increased to 250℃ at a rate of 15℃ / min and maintained for 2min.

[0111] (2) Gas phase parameters 2 Inlet temperature: 170°C. Other conditions are the same as gas phase parameters 1.

[0112] (3) Gas phase parameters 3 Inlet temperature: 170°C; Program temperature: Initial column temperature is 70°C, maintained for 8 min, then increased to 300°C at a rate of 15°C / min, maintained for 2 min. Other conditions are the same as gas phase parameter 1.

[0113] The mass spectrometry analysis conditions were: Ion source: EI source; ion source temperature: 230°C; quadrupole temperature: 150°C; interface temperature: 300°C; detection mode: selected ion monitoring SIM mode, quantitative ion is 45; dwell time: 100ms.

[0114] 2. Experimental process 1) Solution preparation Test solution: Accurately weigh 108 mg of cefpodoxime axetil sample and dissolve it in 3.0 ml of acetonitrile to obtain a test stock solution. Take 3.0 ml of the test stock solution into a 10 ml centrifuge tube, add 1.5 ml of n-hexane, vortex for 1 min, separate the supernatant and obtain the test solution.

[0115] 2) Sampling and analysis Accurately measure 1 μl of the test solution and each reference solution and inject them into the gas chromatography-mass spectrometer.

[0116] 3. Experimental Results The sample solution was injected and analyzed under the conditions of gas phase parameter 1, gas phase parameter 2 and gas phase parameter 3, and the baselines of the spectra of the sample solution under the three conditions were compared. The analysis results are shown in Figure 14 to Figure 16 As can be seen from the figure, under the condition of gas phase parameter 1, there are more interference peaks near the peak of 1-iodoethyl isopropyl carbonate (12-13.5min), while under gas phase parameter 2, there are fewer interference peaks in this area. This is because although extraction can eliminate most of the matrix interference, a small amount of cefpodoxime proxetil is still not separated. The injection port temperature of gas phase parameter 1 is too high, and the amide bond of a small amount of extracted cefpodoxime proxetil will break to produce interference peaks. Compared with gas phase parameter 2, gas phase parameter 3 has fewer interference peaks. This is because the column temperature is raised to a higher temperature, which can make the interference peaks entering the chromatographic column flow out, so that each analysis can make the instrument in a high-sensitivity state.

[0117] The raw materials and equipment used in the present invention, unless otherwise specified, are all commonly used raw materials and equipment in the art; the methods used in the present invention, unless otherwise specified, are all conventional methods in the art.

[0118] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting halogenated esters in cefuroxime-type drugs, characterized in that: The following steps are involved: 1) dissolving a cefuroxime-type drug sample in a solvent as a test sample stock solution, adding n-hexane for extraction, and taking the supernatant to obtain a test sample solution; 2) Dissolve the halogenated ester reference substance in a solvent as a reference substance stock solution, add n-hexane for extraction, and take the supernatant to obtain a reference substance solution; 3) The test solution and the reference solution were detected by gas chromatography-mass spectrometry, and the content of the halogenated ester in the cefuroxime-type drug was calculated using the external standard method.

2. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: The halogenated ester is one or more of 1-chloroethyl isopropyl carbonate, 1-iodoethyl isopropyl carbonate, chloromethyl pivalate, and iodomethyl pivalate.

3. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: The cefuroxime-type drug is cefpodoxime proxetil or cefditoren pivoxil.

4. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: In step 3), the gas chromatography conditions in the gas chromatography-mass spectrometry are: Program temperature rise: the initial column temperature is 50-70°C, maintained for 3-8 min, then raised to 290-310°C at a rate of 10-20°C / min, maintained for 2-5 min; Inlet temperature: 150~200℃.

5. A method for detecting halogenated esters in a cefuroxime-type drug according to claim 1 or 2, characterized in that: In step 3), the mass spectrometry analysis conditions in gas chromatography-mass spectrometry are: Ion source: EI source; Ion source temperature: 230~250℃; Quadrupole temperature: 150~170℃; Interface temperature: 300~320℃; Dwell time: 100~150ms.

6. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 2, characterized in that: In step 3), the monitoring mode and quantitative ion of each compound to be tested in the mass spectrometry of gas chromatography-mass spectrometry are: 1-Chloroethyl isopropyl carbonate, SIM, 45; 1-iodoethyl isopropyl carbonate, SIM, 45; Chloromethyl pivalate, MRM, 85>57; Iodomethyl pivalate, MRM, 85>57.

7. A method for detecting halogenated esters in a cefuroxime-type drug according to claim 1 or 3, characterized in that: In step 1) and step 2), the solvent is acetonitrile or methanol.

8. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: In step 1), the mass volume ratio of the cefuroxime-type drug sample to the solvent is (2.4-54 mg): 1 ml.

9. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: In step 2), the mass volume ratio of the halogenated ester reference substance to the solvent is (0.06-2.0 μg): 1 ml.

10. The method for detecting halogenated esters in a cefuroxime-type drug according to claim 1, characterized in that: In step 1), a cefuroxime-type drug sample is dissolved in a solvent to form a test sample stock solution, and the volume ratio of n-hexane to the test sample stock solution is 1:(2-2.5); in step 2), a halogenated ester reference substance is dissolved in a solvent to form a reference substance stock solution, and the volume ratio of n-hexane to the reference substance stock solution is 1:(2-2.5).

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