Method for detecting 4-methoxybenzyl chloride impurity in cefixime

Abstract

The invention provides a method for detecting a 4-methoxybenzyl chloride impurity in cefixime, and belongs to the technical field of detection. According to the method disclosed by the invention, the content of the 4-methoxybenzyl chloride is determined by detecting the content of the 4-methoxybenzyl chloride degradation product by utilizing the characteristic that the 4-methoxybenzyl chloride degradation product is more stable, so that the accuracy of detecting the impurity of the 4-methoxybenzyl chloride in the cefixime is improved; by limiting the column temperature of the high performance liquid chromatography and the composition of the mobile phase, the detection peak area of the 4-methoxybenzyl chloride impurity in the cefixime is large, the tailing factor is low, the number of theoretical plates is high, and the detection sensitivity and accuracy are improved. According to the detection method provided by the invention, the detection limit of the 4-methoxybenzyl chloride impurity in the cefixime is 0.020 mu g / mL, the quantitation limit is 0.060 mu g / mL, and the sensitivity is high; the recovery rate of the 4-methoxybenzyl chloride is 101.15 percent to 103.94 percent; the detection time can be as low as 12.92 min.

Description

Technical Field

[0001] The invention relates to the field of detection technology, and in particular to a method for detecting 4-methoxybenzyl chloride impurities in cefixime. Background Art

[0002] Cefixime is the abbreviation of (6R,7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-2-[(carboxymethoxy)imino]acetyl]amino]-3-vinyl-8-oxy-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid trihydrate. It belongs to the third generation of cephalosporin antibiotics, has a broad antibacterial spectrum in vitro, and has strong antibacterial activity against Gram-negative and Gram-positive bacteria.

[0003] At present, the preparation method of cefixime generally comprises the following steps: using 7-phenylacetamido-3-chloromethyl-cephalosporanic acid p-methoxybenzyl ester (GCLE) as a raw material, introducing a vinyl group through a Wittig reaction, and obtaining a first intermediate 7-phenylacetamido-3-vinyl-cephalosporanic acid p-methoxybenzyl ester (GVNE); using an imino chloride method, removing the 7-position protecting group through chlorination, etherification and hydrolysis reactions, and obtaining 7-amino-3-vinyl-cephalosporanic acid p-methoxybenzyl ester (AVNE); then removing the 4-position ester group with m-cresol, and obtaining the cefixime mother nucleus 7-amino-3-vinyl-cephalosporanic acid (AVNA); using a cefixime side chain ester (MBT) and AVNA for acylation reaction, and obtaining cefixime tert-butyl ester (TBO); using methanesulfonic acid to hydrolyze TBO, and obtaining cefixime methylsulfonate, and finally further treating to obtain cefixime.

[0004] Among them, 4-methoxybenzyl chloride is introduced in the first step of the synthesis, and the structural formula is as follows:

[0005]

[0006] 4-Methoxybenzyl chloride is an impurity with a warning structure. According to the "Acceptable intake of individual impurities in Table 2" given in "7.3 Acceptable intake associated with LTL exposure" in ICH M7 "Evaluation and Control of DNA Reactive (Mutagenic) Impurities in Drugs to Limit Potential Carcinogenic Risk", the acceptable intake dose of 120 μg / day for a treatment period of <1 month is used as the PDE value of 4-methoxybenzyl chloride. The limit is calculated based on the PDE: That is, when the residual limit of 4-methoxybenzyl chloride in cefixime is greater than 300 ppm, cefixime will have a potential carcinogenic risk. Therefore, it is necessary to control the residual amount of 4-methoxybenzyl chloride in the cefixime finished product. Summary of the invention

[0007] The object of the present invention is to provide a method for detecting 4-methoxybenzyl chloride impurity in cefixime. The method for detecting 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has high accuracy, good sensitivity, high recovery rate and fast detection speed.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for detecting 4-methoxybenzyl chloride impurity in cefixime, comprising the following steps:

[0010] The cefixime to be detected is mixed with methanol and acetonitrile to obtain a cefixime solution as a test solution;

[0011] The test solution is subjected to high performance liquid chromatography detection, and the quantitative and / or qualitative results of the test solution are obtained according to the liquid chromatogram of the test solution obtained, and the conditions of the high performance liquid chromatography detection include:

[0012] Detection wavelength: 233nm;

[0013] Chromatographic column: C18 column;

[0014] Column temperature: 20-40°C;

[0015] Mobile phase: water and acetonitrile, wherein the volume ratio of water to acetonitrile is (45-60): (40-55).

[0016] If the liquid chromatogram of the test solution shows a peak at the same position as the liquid chromatogram of the reference solution or within the retention time range of ±1%, it means that the test solution contains 4-methoxybenzyl chloride impurity;

[0017] The quantitative results are obtained according to Formula I: x =A x / A R ×C R ;

[0018] Among them C x Indicates the concentration of 4-methoxybenzyl chloride impurity in the test solution; A x Represents the peak area of ​​the reference solution; A R Represents the peak area of ​​the test solution; C R It indicates the concentration of 4-methoxybenzyl chloride in the reference solution;

[0019] The difference between the reference solution and the test solution is that 4-methoxybenzyl chloride replaces the cefuroxime to be detected.

[0020] Preferably, the column temperature is 35° C.; and the volume ratio of water to acetonitrile in the mobile phase is 60:40.

[0021] Preferably, the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

[0022] Preferably, the concentration of the cefixime solution is 5-10 mg / mL.

[0023] Preferably, the volume ratio of methanol to acetonitrile in the cefixime solution is 1:(1.5-4).

[0024] Preferably, when preparing the test solution, the volume ratio of the mass of cefixime to methanol is 25-50 mg / mL.

[0025] Preferably, the preparation method of the reference solution comprises: mixing 4-methoxybenzyl chloride with methanol and acetonitrile for degradation to obtain a 4-methoxybenzyl chloride degradation product solution as the reference solution.

[0026] Preferably, when preparing the reference solution, the mass ratio of the 4-methoxybenzyl chloride to the total volume of methanol and acetonitrile is 0.06 to 3.6 μg / mL.

[0027] Preferably, the injection volume of the HPLC detection is 10 to 30 μL.

[0028] Preferably, the elution mode of the mobile phase is isocratic elution, and the flow rate of the mobile phase is 0.8 to 1.2 mL / min.

[0029] The invention provides a method for detecting 4-methoxybenzyl chloride impurity in cefixime, comprising the following steps: mixing cefixime to be detected with methanol and acetonitrile to obtain a cefixime solution as a test solution; performing high performance liquid chromatography detection on the test solution, and obtaining a quantitative and / or qualitative result of the test solution according to a liquid chromatogram of the test solution obtained, wherein the conditions of the high performance liquid chromatography detection include: detection wavelength: 233nm; chromatographic column: C18 chromatographic column; column temperature: 20-40°C; mobile phase: water and acetonitrile, wherein the volume ratio of water to acetonitrile is (45-60): (40-55); if a peak with the same peak position as that of a liquid chromatogram of a reference solution or a peak within the retention time range of ±1% appears in the liquid chromatogram of the test solution, it means that the 4-methoxybenzyl chloride impurity is contained; and the quantitative result is obtained according to formula I: C x =A x / A R ×C R ; where C x Indicates the concentration of 4-methoxybenzyl chloride impurity in the test solution; A x Represents the peak area of ​​the reference solution; A R Represents the peak area of ​​the test solution; C RIt represents the concentration of 4-methoxybenzyl chloride in the reference solution; the difference between the reference solution and the test solution is that 4-methoxybenzyl chloride replaces the cefixime to be detected. The present invention uses methanol to degrade 4-methoxybenzyl chloride, utilizes the more stable characteristics of the degradation product of 4-methoxybenzyl chloride, and determines the content of 4-methoxybenzyl chloride by detecting the content of the degradation product of 4-methoxybenzyl chloride, thereby improving the accuracy of the detection of 4-methoxybenzyl chloride impurities in cefixime, and avoiding the problem of inaccurate detection results caused by directly measuring the easily degradable 4-methoxybenzyl chloride; by limiting the column temperature of high performance liquid chromatography and the composition of the mobile phase, the detection peak area of ​​4-methoxybenzyl chloride impurities in cefixime is high, the tailing factor is low, and the theoretical plate number is high, thereby improving the sensitivity and accuracy of the detection. The results of the embodiment show that the detection method provided by the present invention has a detection limit of 0.020 μg / mL and a quantitative limit of 0.060 μg / mL for 4-methoxybenzyl chloride impurities in cefixime, and has high sensitivity; the recovery rate of 4-methoxybenzyl chloride is 101.15% to 103.94%; and the detection time can be as low as 12.92 min. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is a spectrum scanning curve diagram of the degradation product of 4-methoxybenzyl chloride in Example 1 of the present invention;

[0031] Figure 2 The chromatographic elution curve of the mixed solution of cefixime solution-4-methoxybenzyl chloride degradation products in Example 2 of the present invention is shown. DETAILED DESCRIPTION

[0032] The present invention provides a method for detecting 4-methoxybenzyl chloride impurity in cefixime, comprising the following steps:

[0033] The cefixime to be detected is mixed with methanol and acetonitrile to obtain a cefixime solution as a test solution;

[0034] The test solution is subjected to high performance liquid chromatography detection, and the quantitative and / or qualitative results of the test solution are obtained according to the liquid chromatogram of the test solution obtained, and the conditions of the high performance liquid chromatography detection include:

[0035] Detection wavelength: 233nm;

[0036] Chromatographic column: C18 column;

[0037] Column temperature: 20-40°C;

[0038] Mobile phase: water and acetonitrile, the volume ratio of water to acetonitrile is (45-60): (40-55);

[0039] If the liquid chromatogram of the test solution has a peak at the same position as the liquid chromatogram of the reference solution or a peak within the retention time range of ±1%, it means that the test solution contains 4-methoxybenzyl chloride impurity;

[0040] The quantitative results are obtained according to Formula I: x =A x / A R ×C R ;

[0041] Among them C x Indicates the concentration of 4-methoxybenzyl chloride impurity in the test solution; A x Represents the peak area of ​​the reference solution; A R Represents the peak area of ​​the test solution; C R It indicates the concentration of 4-methoxybenzyl chloride in the reference solution;

[0042] The difference between the reference solution and the test solution is that 4-methoxybenzyl chloride replaces the cefuroxime to be detected.

[0043] The invention mixes the cefixime to be detected with methanol and acetonitrile to obtain a cefixime solution as a test solution.

[0044] In the present invention, the mass ratio of cefixime to methanol is preferably 25 to 50 mg / mL. In an embodiment of the present invention, the mass ratio of cefixime to methanol can be specifically 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL or 50 mg / mL. The present invention utilizes methanol to degrade 4-methoxybenzyl chloride in cefixime, and limits the mass ratio of cefixime to methanol to ensure that 4-methoxybenzyl chloride in cefixime is more fully degraded to obtain 4-methoxybenzyl chloride degradation products.

[0045] In the present invention, the concentration of the cefixime solution is preferably 5 to 10 mg / mL. In an embodiment of the present invention, the concentration of the cefixime solution can be specifically 5 mg / mL, 8 mg / mL or 10 mg / mL. The present invention limits the concentration of the cefixime solution to ensure that more symmetrical chromatographic peaks and higher resolution are obtained during high performance liquid chromatography detection, thereby improving the accuracy of high performance liquid chromatography detection.

[0046] In the present invention, the mixing of cefixime with methanol and acetonitrile preferably comprises: mixing cefixime with methanol, and then adding acetonitrile to the obtained mixture.

[0047] In the present invention, the time for mixing the cefixime to be detected with methanol is preferably 24 to 48 hours. In an embodiment of the present invention, the time for mixing the cefixime to be detected with methanol can be specifically 24 hours, 36 hours or 48 hours. In the present invention, 4-methoxybenzyl chloride is degraded during the mixing of the cefixime to be detected with methanol, and the mixing time is limited to ensure that the 4-methoxybenzyl chloride in the cefixime to be detected is more fully degraded to obtain 4-methoxybenzyl chloride degradation products. The present invention judges whether the 4-methoxybenzyl chloride peak is completely degraded by the change in the peak area of ​​the 4-methoxybenzyl chloride peak and the degradation product peak. The 4-methoxybenzyl chloride chromatographic peak is degraded to undetectable, and the peak area of ​​the degradation product peak no longer increases, which is considered to be completely degraded.

[0048] As an embodiment of the present invention, the mixing of the cefixime to be detected with methanol and acetonitrile is carried out in a volumetric flask. In the present invention, the cefixime to be detected is preferably placed in a volumetric flask, methanol is first added and mixed, and then acetonitrile is added to the volume to the scale line of the volumetric flask and shaken.

[0049] In the present invention, cefixime to be detected is placed in a volumetric flask, methanol is first added and mixed, and 4-methoxybenzyl chloride in cefixime is degraded to obtain a degradation product of 4-methoxybenzyl chloride.

[0050] In the present invention, the conditions for the HPLC detection include:

[0051] Detection wavelength: 233nm;

[0052] Chromatographic column: C18 column;

[0053] Column temperature: 20-40°C;

[0054] Mobile phase: water and acetonitrile, wherein the volume ratio of water to acetonitrile is (45-60): (40-55).

[0055] In the present invention, the detection wavelength of the high performance liquid chromatography is 233 nm. The present invention limits the detection wavelength to facilitate more accurate determination of the concentration of 4-methoxybenzyl chloride impurity in cefixime.

[0056] In the present invention, the method for determining the detection wavelength preferably includes:

[0057] 1) mixing 4-methoxybenzyl chloride with methanol to obtain a degraded 4-methoxybenzyl chloride solution sample;

[0058] 2) Using a double-beam UV-visible spectrophotometer, the degraded 4-methoxybenzyl chloride solution sample obtained in step 1) is spectrally scanned to obtain an absorption curve.

[0059] In the present invention, the mass ratio of the 4-methoxybenzyl chloride to the volume ratio of methanol is preferably 0.03-0.5 mg / mL. In an embodiment of the present invention, the mass ratio of the 4-methoxybenzyl chloride to the volume ratio of methanol can be specifically 0.03 mg / ml, 0.1 mg / mL, 0.3 mg / mL or 0.5 mg / mL. The present invention ensures more complete degradation of 4-methoxybenzyl chloride by limiting the mass ratio of 4-methoxybenzyl chloride to the volume ratio of methanol.

[0060] In the present invention, the parameters of the spectral scanning are preferably:

[0061] Scanning wavelength: 190.00~400.00nm;

[0062] Scanning interval: 0.10nm;

[0063] Scanning speed: medium. The present invention further improves the accuracy and efficiency of spectrum detection by limiting the parameters of spectrum scanning.

[0064] In the present invention, the chromatographic column for high performance liquid chromatography detection is preferably a C18 chromatographic column. The present invention fully improves the separation effect, ie, accuracy, of high performance liquid chromatography detection by limiting the type of chromatographic column.

[0065] In the present invention, the specification of the chromatographic column is preferably 4.6 mm×250 mm, 5 μm. The present invention further increases the number of plates for high performance liquid chromatography detection by limiting the specification of the chromatographic column, thereby improving the sensitivity and accuracy of high performance liquid chromatography detection.

[0066] In the present invention, the column temperature of the HPLC detection is preferably 20-40° C. In an embodiment of the present invention, the column temperature of the HPLC detection can be specifically 35° C. The present invention fully prevents the loss of the stationary phase by limiting the column temperature of the HPLC detection, and more fully guarantees the accuracy of the HPLC detection.

[0067] In the present invention, the mobile phase of the high performance liquid chromatography detection is water and acetonitrile, and the volume ratio of the water to the acetonitrile is (45-60): (40-55). In an embodiment of the present invention, the volume ratio of the water to the acetonitrile can be specifically 45:50, 50:50, 50:55 or 60:40. The present invention limits the volume ratio of water to acetonitrile to ensure a more complete separation of the 4-methoxybenzyl chloride impurity in cefixime, thereby improving the accuracy of high performance liquid chromatography.

[0068] In the present invention, the elution mode of the mobile phase is preferably isocratic elution. The present invention can substantially shorten the time of HPLC detection and substantially improve the separation effect of HPLC detection by limiting the elution mode of the mobile phase.

[0069] In the present invention, the sample injection volume of the HPLC detection is preferably 10 to 30 μL. In an embodiment of the present invention, the sample injection volume of the HPLC detection can be specifically 10 μL, 15 μL, 20 μL, 25 μL or 30 μL. The present invention more fully ensures that the various components in the sample can be better separated by limiting the sample injection volume of the HPLC detection, thereby improving the accuracy and reliability of the detection.

[0070] In the present invention, the flow rate of the HPLC detection is preferably 0.8 to 1.2 mL / min. In an embodiment of the present invention, the flow rate of the HPLC detection can be specifically 1.0 mL / min. The present invention more fully ensures that the linear velocity of the mobile phase in the chromatographic column remains consistent by limiting the flow rate of the HPLC detection, more fully utilizes the separation capacity of the chromatographic column, and improves the separation effect; and more fully reduces the fluctuation of the mobile phase velocity, thereby improving the sensitivity and stability of the analysis and ensuring the accuracy of the experimental results.

[0071] The present invention performs high performance liquid chromatography on the test solution, and obtains the quantitative and / or qualitative results of the test solution according to the obtained liquid chromatogram of the test solution.

[0072] In the present invention, if the liquid chromatogram of the test solution shows a peak at the same peak position or within the error range as the liquid chromatogram of the reference solution, it means that the impurity 4-methoxybenzyl chloride is contained;

[0073] In the present invention, the quantitative results are obtained according to Formula I: x =A x / A R ×C R ;

[0074] Among them C x Indicates the concentration of 4-methoxybenzyl chloride impurity in the test solution; A x Represents the peak area of ​​the reference solution; A R Represents the peak area of ​​the test solution; C R It indicates the concentration of 4-methoxybenzyl chloride in the reference solution;

[0075] In the present invention, the reference substance solution is a 4-methoxybenzyl chloride degradation product solution.

[0076] In the present invention, the method for preparing the reference solution preferably comprises: mixing 4-methoxybenzyl chloride with methanol and acetonitrile for degradation to obtain a 4-methoxybenzyl chloride degradation product solution as the reference solution.

[0077] In the present invention, the mass ratio of the 4-methoxybenzyl chloride to the volume ratio of methanol is preferably 0.03-0.5 mg / mL. In an embodiment of the present invention, the mass ratio of the 4-methoxybenzyl chloride to the volume ratio of methanol can be specifically 0.03 mg / ml, 0.1 mg / mL, 0.3 mg / mL or 0.5 mg / mL. The present invention limits the mass ratio of 4-methoxybenzyl chloride to the volume ratio of methanol to ensure that 4-methoxybenzyl chloride is more fully degraded to obtain 4-methoxybenzyl chloride degradation products.

[0078] In the present invention, the ratio of the mass of 4-methoxybenzyl chloride to the total volume of methanol and acetonitrile is preferably used to represent the concentration of the 4-methoxybenzyl chloride solution. In the present invention, the mass of 4-methoxybenzyl chloride to the total volume of methanol and acetonitrile is preferably 0.06 to 3.6 μg / mL. In an embodiment of the present invention, the mass of 4-methoxybenzyl chloride to the total volume of methanol and acetonitrile can be specifically 0.06 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL or 3.6 μg / mL.

[0079] In the present invention, the mixing of 4-methoxybenzyl chloride with methanol and acetonitrile preferably comprises: firstly mixing 4-methoxybenzyl chloride with methanol, and then adding acetonitrile to the obtained mixed solution.

[0080] In the present invention, the mixing time of 4-methoxybenzyl chloride and methanol is preferably 24 to 48 hours. In an embodiment of the present invention, the mixing time of 4-methoxybenzyl chloride and methanol can be specifically 24 hours, 36 hours or 48 hours. In the present invention, 4-methoxybenzyl chloride is degraded during the mixing process of 4-methoxybenzyl chloride and methanol, and the mixing time is limited to ensure that 4-methoxybenzyl chloride is more fully degraded to obtain 4-methoxybenzyl chloride degradation products.

[0081] As an embodiment of the present invention, the mixing of 4-methoxybenzyl chloride with methanol and acetonitrile is carried out in a volumetric flask. In the present invention, preferably, the 4-methoxybenzyl chloride to be detected is placed in a volumetric flask, methanol is added to the scale line of the volumetric flask to obtain a first mixed solution; then a portion of the first mixed solution is taken in a volumetric flask, methanol is added to the scale line of the volumetric flask to obtain a second mixed solution; finally, a portion of the second mixed solution is taken in a volumetric flask, acetonitrile is added to the scale line of the volumetric flask to obtain a 4-methoxybenzyl chloride degradation product solution.

[0082] The invention performs high performance liquid chromatography detection on the reference substance solution and obtains a liquid chromatogram of the reference substance solution.

[0083] The invention utilizes the more stable characteristic of 4-methoxybenzyl chloride degradation products, determines the content of 4-methoxybenzyl chloride by detecting the content of the 4-methoxybenzyl chloride degradation products, and improves the accuracy of detecting 4-methoxybenzyl chloride impurities in cefixime; and by limiting the column temperature of high performance liquid chromatography and the composition of the mobile phase, the detection sensitivity of the 4-methoxybenzyl chloride impurities in cefixime is high, the recovery rate is high and the detection time is short.

[0084] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0085] Example 1

[0086] Method for determining the detection wavelength:

[0087] 1) mixing 10 mg of 4-methoxybenzyl chloride with 10 mL of methanol to obtain a degraded 4-methoxybenzyl chloride solution sample; the mass ratio of the 4-methoxybenzyl chloride to the volume of methanol is preferably 1 mg:1 mL;

[0088] 2) Using a double-beam UV-visible spectrophotometer to perform spectral scanning on the degraded 4-methoxybenzyl chloride solution sample obtained in step 1) to obtain an absorption curve; the parameters of the spectral scanning are:

[0089] Scanning wavelength: 190.00~400.00nm;

[0090] Scanning interval: 0.10nm;

[0091] Scan speed:Medium.

[0092] The 4-methoxybenzyl chloride solution sample after degradation in step 1) of Example 1 was subjected to spectral scanning using a double-beam UV-visible spectrophotometer. The parameters of the spectral scanning were: scanning wavelength: 190.00-400.00 nm; scanning interval: 0.10 nm; scanning speed: medium. The spectral scanning curve was obtained as shown in FIG. Figure 1 As shown by Figure 1 The obtained wavelength and absorbance values ​​are shown in Table 1.

[0093] Table 14-Spectral data of methoxybenzyl chloride degradation products

[0094] Serial number Peak / Valley wavelength Absorbance 1 peak 233.10 4.593 2 peak 274.70 1.156

[0095] Depend on Figure 1As shown in Table 1, the degradation product of 4-methoxybenzyl chloride has a maximum absorption at 233.10 nm, so 233 nm is selected as the detection wavelength of HPLC detection.

[0096] Example 2

[0097] A method for detecting 4-methoxybenzyl chloride impurity in cefixime, comprising the following steps:

[0098] 100 mg of cefixime to be tested was placed in a 10 mL volumetric flask, 2 mL of methanol was added and mixed, and then placed at room temperature for 24 h, and then acetonitrile was added to the volume to the scale line of the volumetric flask and shaken to obtain a cefixime solution with a concentration of 10 mg / mL as the test solution; the mass ratio of cefixime to methanol was 100 mg: 2 mL;

[0099] Conducting high performance liquid chromatography on the test solution;

[0100] The conditions for the HPLC detection are:

[0101] Detection wavelength: 233nm;

[0102] Chromatographic column: GL Sciences InertSustain C18 column;

[0103] Column temperature: 35 °C;

[0104] Mobile phase: water and acetonitrile, the volume ratio of water to acetonitrile is 60:40;

[0105] The elution mode of the mobile phase was isocratic elution;

[0106] The injection volume was 30 μL;

[0107] Flow rate: 1 mL / min.

[0108] The test solution was subjected to high performance liquid chromatography to obtain a chromatographic elution curve of the test solution, and its data statistics are shown in Table 2. As shown in Table 2, the test results of the test solution were all ND, which indicates that the cefixime solution detected by the present invention does not contain 4-methoxybenzyl chloride impurities or contains 4-methoxybenzyl chloride impurities below the detection limit.

[0109] In order to further determine whether the cefixime solution in Example 2 of the present invention does not contain 4-methoxybenzyl chloride impurities or contains 4-methoxybenzyl chloride impurities below the detection limit, the present invention places 100 mg of cefixime to be detected in a 10 mL volumetric flask, adds 2 mL of methanol and 1 mL of the second mixed solution in Example 3 in sequence, mixes, then adds acetonitrile to the scale line of the volumetric flask and shakes well to obtain a cefixime-4-methoxybenzyl chloride degradation product mixed solution; the cefixime concentration in the cefixime-4-methoxybenzyl chloride degradation product mixed solution is 10 mg / mL; the 4-methoxybenzyl chloride concentration in the cefixime-4-methoxybenzyl chloride degradation product mixed solution is 0.003 mg / mL; then the cefixime-4-methoxybenzyl chloride degradation product mixed solution is subjected to high performance liquid chromatography detection under the same detection conditions as in Example 2 to obtain a chromatographic elution curve and statistical data of the cefixime-4-methoxybenzyl chloride degradation product mixed solution, as shown respectively. Figure 2 And as shown in Table 3.

[0110] Table 3 Statistical table of chromatographic elution curves of mixed solution of cefixime-4-methoxybenzyl chloride degradation products

[0111]

[0112] According to C x =A x / A R ×C R , it can be calculated that the concentration of 4-methoxybenzyl chloride when the cefixime+4-methoxybenzyl chloride degradation product solution is prepared is 12.928 / 12.94×0.003mg / mL=0.0029mg / mL; and the cefixime-4-methoxybenzyl chloride degradation product mixed solution itself contains a 4-methoxybenzyl chloride solution with a concentration of 0.003mg / mL. It can be seen that the cefixime to be detected in the present invention does not contain 4-methoxybenzyl chloride impurities. On this basis, through the separation degree of 19.78 in Table 3, it can be seen that other impurities in cefixime have no effect on the detection of 4-methoxybenzyl chloride impurities, and the detection method of 4-methoxybenzyl chloride impurities in cefixime provided by the present invention has high detection accuracy.

[0113] Example 3

[0114] 50 mg of 4-methoxybenzyl chloride was placed in a 100 mL volumetric flask, methanol was added to make up to the scale line of the volumetric flask, and the mixture was shaken and placed at room temperature for 24 hours to obtain a first mixed solution; then 6 mL of the first mixed solution was placed in a 100 mL volumetric flask, methanol was added to make up to the scale line of the volumetric flask, and the mixture was shaken to obtain a second mixed solution; finally, 1 mL of the second mixed solution was placed in a 10 mL volumetric flask, acetonitrile was added to make up to the scale line of the volumetric flask, and the mixture was shaken to obtain a 4-methoxybenzyl chloride degradation product solution as a reference solution; the mass ratio of the 4-methoxybenzyl chloride to the volume of methanol was 0.03 mg:1 mL; the mass ratio of the 4-methoxybenzyl chloride to the volume of acetonitrile was 0.03 mg:9 mL; the concentration of the 4-methoxybenzyl chloride solution was 0.003 mg / mL;

[0115] Performing high performance liquid chromatography on the reference solution;

[0116] The conditions for the HPLC detection are:

[0117] Detection wavelength: 233nm;

[0118] Chromatographic column: GL Sciences InertSustain C18 column;

[0119] Column temperature: 35 °C;

[0120] Mobile phase: water and acetonitrile, the volume ratio of water to acetonitrile is 60:40;

[0121] The elution mode of the mobile phase was isocratic elution;

[0122] The injection volume was 30 μL;

[0123] Flow rate: 1 mL / min.

[0124] The reference solution was subjected to high performance liquid chromatography to obtain a chromatographic elution curve of the reference solution, and its data statistics are shown in Table 2.

[0125] Table 2 Statistics of chromatographic elution curves of reference solution and test solution

[0126]

[0127] As can be seen from Table 2, the peak elution time of the reference solution is short, indicating that under the same HPLC conditions as in Example 2, the detection speed of 4-methoxybenzyl chloride is fast, the peak area of ​​the detection is high, the tailing factor is low, and the theoretical plate number is high, indicating that under the HPLC conditions of Example 2, the detection sensitivity of 4-methoxybenzyl chloride is high and the separation efficiency is high; while the detection of the test solution is ND, it is inferred that the cefixime solution detected by the present invention does not contain 4-methoxybenzyl chloride impurities or contains 4-methoxybenzyl chloride impurities below the detection limit.

[0128] The blank solution was subjected to HPLC detection under the same conditions as in Example 3. The specific operation was as follows: 2 mL of methanol was placed in a 10 mL volumetric flask, acetonitrile was added to the volume to the mark, and the blank solution was obtained by shaking; then, HPLC detection was performed to obtain a chromatographic elution curve of the blank solution. The statistical data are shown in Table 4.

[0129] Table 4 Statistics of chromatographic elution curves of blank solutions

[0130]

[0131] It can be seen from Table 4 that no peak was detected in the blank solution, indicating that methanol and acetonitrile solvents had no interference with the detection of 4-methoxybenzyl chloride, thereby improving the accuracy of the detection method of 4-methoxybenzyl chloride impurity in cefixime.

[0132] The present invention also detects the detection limit and the quantitative limit of the detection method of 4-methoxybenzyl chloride impurity in cefixime, and the specific operation is: taking 1 mL of the 4-methoxybenzyl chloride degradation product solution in Example 3 in a 50 mL volumetric flask, adding acetonitrile to the scale line and shaking well to obtain a quantitative limit solution with a concentration of 0.06 μg / mL; taking 3 mL of the quantitative limit solution in a 10 mL volumetric flask, adding 6 mL of acetonitrile and shaking well to obtain a detection limit solution with a concentration of 0.02 μg / mL; then using the same conditions as in Example 3, respectively, the quantitative limit solution and the detection limit solution are subjected to high performance liquid chromatography detection to obtain a chromatographic elution curve of the quantitative limit solution and a chromatographic elution curve of the detection line solution, and the statistical data are shown in Table 5.

[0133] Table 5 Statistics of chromatographic elution curves of quantitative limit solution and detection limit solution

[0134] Solution name Concentration (μg / mL) Signal-to-Noise Ratio Quantitative limit solution 0.06 11.19 Detection limit solution 0.02 4.09

[0135] It can be seen from Table 5 that the detection limit of the detection method for 4-methoxybenzyl chloride impurity in cefixime provided by the present invention can be as low as 0.02 μg / mL, and the quantitative limit can be as low as 0.06 μg / mL, indicating that the detection method for 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has high sensitivity.

[0136] The present invention also detects the linear relationship of the detection method of 4-methoxybenzyl chloride impurity in cefixime, and the specific operation is: taking 2.5mL, 4mL, 5mL, 5.5mL and 6mL of the second mixed solution in Example 3 in a 50mL volumetric flask, adding acetonitrile to the scale line and shaking well to obtain a series of 4-methoxybenzyl chloride degradation product solutions with different concentrations; then using the same conditions as Example 3, respectively, the quantitative limit solution and a series of 4-methoxybenzyl chloride degradation product solutions with different concentrations are subjected to high performance liquid chromatography detection to obtain a series of chromatographic elution curves of 4-methoxybenzyl chloride degradation product solutions with different concentrations, and calculating the linear equation data of the high performance liquid chromatography as shown in Table 6.

[0137] Table 6 High performance liquid chromatography linear equation data table

[0138]

[0139]

[0140] It can be seen from Table 6 that the detection method of 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has a good linear relationship between peak area and concentration within the range of 0.060 μg / mL to 3.629 μg / mL.

[0141] The present invention also detects the spiked recovery rate of 4-methoxybenzyl chloride impurity in cefixime. The specific operation is as follows: 100 mg of cefixime in Example 3 is put into a 10 mL volumetric flask, 2 mL of methanol is added for ultrasonic dissolution, 0.8 mL, 1 mL and 1.2 mL of the second mixed solution in Example 3 are added respectively, acetonitrile is used to make the volume to the scale line and shaken well, so as to obtain 80%, 100% and 120% 4-methoxybenzyl chloride spiked sample solutions, and 3 portions are prepared for each concentration; then, a series of 4-methoxybenzyl chloride spiked sample solutions with different concentrations are respectively subjected to high performance liquid chromatography detection under the same conditions as in Example 3, a series of chromatographic elution curves of 4-methoxybenzyl chloride spiked sample solutions with different concentrations are obtained, and the spiked recovery rates are calculated as shown in Table 7.

[0142] Table 74-Methoxybenzyl chloride spiked sample solution spiked recovery data table

[0143] name Spike recovery RSD value of spike recovery 4-Methoxybenzyl chloride spiked sample solution 101.15％～103.94％ 1.11％

[0144] It can be seen from Table 7 that the spiked recovery of 4-methoxybenzyl chloride spiked sample solution is between 101.15% and 103.94%, and the RSD value of the spiked recovery is 1.11%, indicating that the detection method of 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has high accuracy.

[0145] The present invention performs high performance liquid phase detection on the 4-methoxybenzyl chloride degradation product solution in Example 3 and the mixed solution of cefixime + 4-methoxybenzyl chloride degradation product to be detected at injection times of 0h, 2h, 5h, 10h, 20h and 48h, and obtains statistical data as shown in Table 8.

[0146] Table 8 4-methoxybenzyl chloride degradation product solution and the chromatographic elution curve data statistics of the cefixime + 4-methoxybenzyl chloride degradation product solution to be detected

[0147]

[0148] It can be seen from Table 8 that the peak area of ​​the detection method for 4-methoxybenzyl chloride impurity in cefixime provided by the present invention does not change much at different injection times, indicating that the detection method for 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has good stability and high accuracy.

[0149] In summary, it can be seen that the detection method of 4-methoxybenzyl chloride impurity in cefixime provided by the present invention has high accuracy, good sensitivity, high recovery rate and fast detection speed.

[0150] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting 4-methoxybenzyl chloride impurity in cefixime, comprising the following steps: The cefixime to be detected is mixed with methanol and acetonitrile to obtain a cefixime solution as a test solution; The test solution is subjected to high performance liquid chromatography detection, and the quantitative and / or qualitative results of the test solution are obtained according to the liquid chromatogram of the test solution obtained, and the conditions of the high performance liquid chromatography detection include: Detection wavelength: 233nm; Chromatographic column: C18 column; Column temperature: 20-40°C; Mobile phase: water and acetonitrile, the volume ratio of water to acetonitrile is (45-60): (40-55); If the liquid chromatogram of the test solution has a peak at the same position as the liquid chromatogram of the reference solution or a peak within the retention time range of ±1%, it means that the test solution contains 4-methoxybenzyl chloride impurity; The quantitative results are obtained according to Formula I: x =A x / A R ×C R ; Among them C x Indicates the concentration of 4-methoxybenzyl chloride impurity in the test solution; A x Represents the peak area of ​​the reference solution; A R Represents the peak area of ​​the test solution; C R It indicates the concentration of 4-methoxybenzyl chloride in the reference solution; The difference between the reference solution and the test solution is that 4-methoxybenzyl chloride replaces the cefuroxime to be detected.

2. The detection method according to claim 1, characterized in that: The column temperature is 35°C; The volume ratio of water to acetonitrile in the mobile phase is 60:

40.

3. The detection method according to claim 1 or 2, characterized in that: The specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

4. The detection method according to claim 1 or 2, characterized in that: The concentration of the cefixime solution is 5-10 mg / mL.

5. The detection method according to claim 1 or 2, characterized in that: The volume ratio of methanol to acetonitrile in the cefixime solution is 1:(1.5-4).

6. The detection method according to claim 1 or 2, characterized in that: When preparing the test solution, the volume ratio of the mass of cefixime to methanol is 25-50 mg / mL.

7. The detection method according to claim 1 or 2, characterized in that: The preparation method of the reference solution comprises: mixing 4-methoxybenzyl chloride with methanol and acetonitrile for degradation to obtain a 4-methoxybenzyl chloride degradation product solution as the reference solution.

8. The detection method according to claim 7, characterized in that: When preparing the reference solution, the mass ratio of the 4-methoxybenzyl chloride to the total volume of methanol and acetonitrile is 0.06 to 3.6 μg / mL.

9. The detection method according to claim 1 or 2, characterized in that: The injection volume of the high performance liquid chromatography detection is 10 to 30 μL.

10. The detection method according to claim 1 or 2, characterized in that: The elution mode of the mobile phase is isocratic elution, and the flow rate of the mobile phase is 0.8-1.2 mL / min.

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