Method for detecting purity of erythromycin oxime

Abstract

The invention provides a method for detecting the purity of erythromycin oxime, and belongs to the technical field of chemical detection and analysis. The method for detecting the purity of erythromycin oxime comprises the following steps: 1) respectively detecting a to-be-detected erythromycin oxime solution and an erythromycin oxime standard substance solution by adopting a high performance liquid chromatograph, taking a mixed eluent as a mobile phase and adopting an isocratic elution method to obtain a chromatogram of the to-be-detected erythromycin oxime solution and a chromatogram of a standard substance, recording the peak area; the mixed eluent comprises acetonitrile and a monopotassium phosphate solution; and 2) calculating the purity of the erythromycin oxime to be detected by adopting an external standard method. The detection method provided by the invention is simple to operate, high in sensitivity, low in detection limit and good in stability.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical detection and analysis, and in particular relates to a method for detecting the purity of erythromycin oxime. Background Art

[0002] Erythromycin oxime, as an important chemical intermediate, is widely used in the pharmaceutical field, especially in the synthesis of macrolide antibiotics. Erythromycin oxime exists in the form of white or off-white crystalline powder and has good solubility. The preparation of erythromycin oxime usually involves the reaction of erythromycin or its economical precursor erythromycin thiocyanate with hydroxylamine, and the oximation transformation is completed under suitable temperature conditions. The importance of erythromycin oxime is not only reflected in its role as an intermediate, but also in its impact on the quality of the final drug. Its purity and yield directly affect the efficacy and safety of subsequent antibiotic products. Therefore, it is extremely important to effectively realize the quantitative detection and content analysis of erythromycin oxime.

[0003] Patent CN102617675A provides a liquid phase detection method for erythromycin oxime, which uses 85% phosphoric acid to adjust the solution pH to 6.8-7.0 and then inject the sample for testing. This method is cumbersome and has low sensitivity, and cannot accurately control the erythromycin oxime content. Summary of the invention

[0004] The invention provides a method for detecting the purity of erythromycin oxime. The detection method provided by the invention has the advantages of simple operation, high sensitivity, low detection limit and good stability, and can effectively characterize the content of erythromycin oxime.

[0005] In order to achieve the above object, the present invention provides a method for detecting the purity of erythromycin oxime, comprising the following steps:

[0006] 1) respectively subjecting the erythromycin oxime solution to be detected and the erythromycin oxime standard solution to be detected by a high performance liquid chromatograph, using a mixed eluent as a mobile phase and an isocratic elution method to obtain a chromatogram of the erythromycin oxime solution to be detected and a chromatogram of the standard, and recording the peak areas;

[0007] The mixed eluent comprises acetonitrile and potassium dihydrogen phosphate solution;

[0008] 2) The purity of the erythromycin oxime to be tested was calculated using the external standard method.

[0009] Preferably, the concentration of the potassium dihydrogen phosphate solution in step 1) is 0.025 mol / L; the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 55:45.

[0010] Preferably, the detection conditions when using high performance liquid chromatography for detection in step 1) are:

[0011] Detection wavelength: 215nm;

[0012] Column temperature 30-40°C;

[0013] Flow rate 0.5~2.0mL / min;

[0014] Injection volume: 5~20μL.

[0015] Preferably, in step 1), a reverse phase chromatography column using octadecylsilane bonded silica gel as a filler is used for high performance liquid chromatography detection.

[0016] Preferably, the column length of the chromatographic column is 4.6 mm*250 mm.

[0017] Preferably, when high performance liquid chromatography detection is performed in step 1), the detector is a diode array detector.

[0018] Preferably, a mixed solvent of a potassium dihydrogen phosphate solution with a concentration of 0.025 mol / L and acetonitrile is used to prepare the erythromycin oxime solution to be detected and the erythromycin oxime standard solution; the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 55:45.

[0019] Preferably, the concentration of the erythromycin oxime solution to be detected is 2 μg / ml to 4 mg / ml; the concentration of the erythromycin oxime standard solution is 1 μg / ml to 5 mg / ml.

[0020] Preferably, in step 2), the purity of the erythromycin oxime to be detected is calculated by an external standard method, which specifically comprises the following steps:

[0021] a. Take a certain amount of standard in a volumetric flask and dilute it into five solutions of different concentrations. The five solutions of different concentrations obtained by dilution are respectively tested by liquid chromatography injection, and the peak area of ​​the chromatographic peak is used as the ordinate and the corresponding solution concentration is used as the abscissa to draw a graph;

[0022] b. Calculation results. The specific calculation formula is:

[0023] X = c × v × 100

[0024] 1000M

[0025] Where:

[0026] X is the mass concentration of erythromycin oxime in the sample, %;

[0027] c is the concentration of erythromycin oxime obtained from the standard curve, (mg / mL);

[0028] v is the total volume of the sample after dilution, (mL);

[0029] M is the sample mass, (g).

[0030] Compared with the prior art, the advantages and positive effects of the present invention are:

[0031] The method for detecting the purity of erythromycin oxime provided by the present invention adopts a high performance liquid chromatograph, uses a mixed eluent as a mobile phase, and adopts an isocratic elution method to detect the components in the erythromycin oxime sample solution. The present invention can effectively detect the purity of erythromycin oxime by setting a suitable eluent and elution mode, and realizes quantitative detection and quality control of erythromycin oxime.

[0032] The detection method provided by the invention has the advantages of simple operation, high sensitivity, low detection limit, good stability and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the HPLC chromatogram of the test solution in Example 1;

[0034] Figure 2 is the HPLC chromatogram of the reference substance solution in Example 1;

[0035] Figure 3 It is a comparison diagram of the HPLC chromatograms of multiple injections of the same sample in Example 2;

[0036] Figure 4 It is a comparison diagram of the HPLC chromatogram of the same batch of homemade erythromycin oxime in Example 3;

[0037] Figure 5 It is a comparison diagram of the HPLC chromatograms of multiple batches of homemade erythromycin oxime in Example 4;

[0038] Figure 6 This is the HPLC chromatogram obtained by gradient elution in Comparative Example 1;

[0039] Figure 7 This is a high performance liquid chromatogram obtained in Comparative Example 2 using methanol and potassium dihydrogen phosphate solution as mobile phases;

[0040] Figure 8 The HPLC chromatograms of erythromycin oxime under different mobile phase ratios in Comparative Example 3 are shown;

[0041] Fig. 9 The HPLC chromatograms of erythromycin oxime at different wavelengths in Comparative Example 4 are shown. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only 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.

[0043] The present invention provides a method for detecting the purity of erythromycin oxime, comprising the following steps:

[0044] 1) respectively subjecting the erythromycin oxime solution to be detected and the erythromycin oxime standard solution to be detected by a high performance liquid chromatograph, using a mixed eluent as a mobile phase and an isocratic elution method to obtain a chromatogram of the erythromycin oxime solution to be detected and a chromatogram of the standard, and recording the peak areas;

[0045] The mixed eluent comprises acetonitrile and potassium dihydrogen phosphate solution;

[0046] 2) The purity of the erythromycin oxime to be tested was calculated using the external standard method.

[0047] The present invention uses a high performance liquid chromatograph to detect the erythromycin oxime solution to be detected and the erythromycin oxime standard solution, respectively, with a mixed eluent as the mobile phase, and uses an isocratic elution method to detect, to obtain a chromatogram of the erythromycin oxime solution to be detected and a chromatogram of the standard, and record the peak area. In the present invention, it is preferred to use a mixed solvent of a potassium dihydrogen phosphate solution and acetonitrile with a concentration of 0.025mol / L to prepare the erythromycin oxime solution to be detected and the erythromycin oxime standard solution; the volume ratio of the potassium dihydrogen phosphate solution and acetonitrile is preferably 55:45. In the present invention, the concentration of the erythromycin oxime solution to be detected is preferably 2μg / ml~4mg / ml; the concentration of the erythromycin oxime standard solution is preferably 1μg / ml~5mg / ml. In the present invention, the mixed eluent includes acetonitrile and potassium dihydrogen phosphate solution; the concentration of the potassium dihydrogen phosphate solution is preferably 0.025mol / L; the volume ratio of the potassium dihydrogen phosphate solution and acetonitrile is preferably 55:45. In the present invention, acetonitrile and potassium dihydrogen phosphate solution are used as mixed eluents to effectively improve separation efficiency, and the peak shape is improved by adjusting the solvent ratio. In the present invention, isocratic elution is used for elution, which is not only simple to operate and easy to control, but also has a more stable baseline, which is conducive to quantitative analysis.

[0048] In the present invention, the detection conditions when using high performance liquid chromatography for detection are preferably:

[0049] Detection wavelength: 215nm;

[0050] Column temperature 30-40°C;

[0051] Flow rate 0.5~2.0mL / min;

[0052] Injection volume: 5~20μL.

[0053] In the present invention, when performing high performance liquid chromatography detection, it is preferred to use a reverse phase chromatographic column with octadecylsilane bonded silica as a filler. The column length of the chromatographic column is preferably 4.6mm*250mm. When performing detection in the embodiments of the present invention and the comparative examples, the high performance liquid chromatograph is Shimadzu LC40, and the chromatographic column is a reverse phase chromatographic column with octadecylsilane bonded silica as a filler, Shimadzu Shim-packGISTC18-AQ, with a column length of 4.6mm*250mm. In the present invention, when performing high performance liquid chromatography detection, the detector is preferably a diode array detector.

[0054] In the present invention, the purity of erythromycin oxime to be detected is calculated by external standard method, which specifically comprises the following steps:

[0055] a. Take a certain amount of standard in a volumetric flask and dilute it into five solutions of different concentrations. The five solutions of different concentrations obtained by dilution are respectively tested by liquid chromatography injection, and the peak area of ​​the chromatographic peak is used as the ordinate and the corresponding solution concentration is used as the abscissa to draw a graph;

[0056] b. Calculation results. The specific calculation formula is:

[0057]

[0058] Where:

[0059] X is the mass concentration of erythromycin oxime in the sample, %;

[0060] c is the concentration of erythromycin oxime obtained from the standard curve, (mg / mL);

[0061] v is the total volume of the sample after dilution, (mL);

[0062] M is the sample mass, (g).

[0063] In the present invention, the structural formula of erythromycin oxime is as shown in Formula I:

[0064]

[0065] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0066] Example 1

[0067] (1) Preparation of test solution: Take 0.0020 g of the test sample (self-made erythromycin oxime HMSW-01) and place it in a 50 ml volumetric flask. Add solvent (potassium dihydrogen phosphate-acetonitrile solvent with a volume ratio of 55:45) and dissolve it by ultrasonic. Dilute to the scale and shake well to obtain the test solution.

[0068] (2) Preparation of reference substance solution: Take 0.0020 g of reference substance (erythromycin oxime standard substance) and place it in a 50 ml volumetric flask. Add solvent (potassium dihydrogen phosphate-acetonitrile solvent with a volume ratio of 55:45) and dissolve it by ultrasonication. Dilute to the scale and shake well to obtain the reference substance solution.

[0069] (3) The test solution obtained in (1) and the reference solution obtained in (2) are respectively injected into a liquid chromatograph for high performance liquid chromatography analysis, and an isocratic elution method is used to output a high performance liquid chromatogram of the test solution ( Figure 1 ) and the chromatogram of the reference solution ( Figure 2 ).

[0070] The above HPLC conditions are:

[0071] The detector is a diode array detector;

[0072] The detection wavelength is 215nm;

[0073] The column temperature was 40°C;

[0074] The mobile phase consisted of mobile phase A and mobile phase B, wherein mobile phase A was acetonitrile and mobile phase B was 0.025 mol / L potassium dihydrogen phosphate solution, and the volume ratio of the two was 45:55;

[0075] Flow rate: 1.0 mL / min;

[0076] Injection volume: 10 μL.

[0077] Calculate the purity of the test sample:

[0078]

[0079] Example 2

[0080] 0.0020 g of the test sample (self-made erythromycin oxime HMSW-01) was accurately weighed to prepare the test sample solution. The detection steps, detection conditions and experimental conditions were the same as those of the chromatographic detection of the test sample described in Example 1. The same sample was injected 6 times continuously, and the repeatability was evaluated using 6 measurement results. The RSD values ​​were all <2%. The precision test results are shown in Table 1 below, and the chromatogram is shown in Figure 3 shown.

[0081] Table 1

[0082] Serial number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Injection 1 0.237 0.588 0.674 97.569 00205 0.133 0.594 Injection 2 0.239 0.592 0.681 97.556 0.211 0.130 0.591 Injection 3 0.229 0.581 0.673 97.59 0.206 0.132 0.589 Injection 4 0.237 0.582 0.669 97.567 0.216 0.135 0.594 Injection 5 0.241 0.586 0.679 97.563 0.208 0.134 0.589 Injection 6 0.236 0.59 0.667 97.567 0.208 0.135 0.597 RSD / % 1.73 0.75 0.79 0.01 1.91 1.46 0.54

[0083] It can be seen from the data in Table 1 that when the same erythromycin oxime sample solution was injected 6 times, the relative peak area RSD was less than 2%, indicating that the detection method provided by the present invention has good precision in detecting erythromycin oxime.

[0084] Example 3

[0085] Weigh 0.0020 g of the test sample (self-made erythromycin oxime HMSW-01) accurately, weigh 6 portions in parallel, and prepare the test sample solution. The detection steps, detection conditions and experimental conditions are the same as those of the chromatographic detection of the test sample described in Example 1. Record the chromatogram, which is as follows: Figure 4 As shown, the relative peak areas of the main chromatographic peaks were calculated, and the results of the repeatability experiment are shown in Table 2 below.

[0086] Table 2

[0087] Serial number Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 Peak 7 Parallel 1 0.228 0.59 0.676 97.581 0.212 0.131 0.597 Parallel 2 0.231 0.586 0.674 97.579 0.209 0.128 0.593 Parallel 3 0.229 0.588 0.679 97.582 0.210 0.132 0.597 Parallel 4 0.234 0.579 0.676 97.588 0.212 0.133 0.596 Parallel 5 0.235 0.586 0.681 97.586 0.218 0.133 0.600 Parallel 6 0.228 0.591 0.679 97.574 0.211 0.136 0.598 RSD / % 1.33 0.73 0.38 0.01 1.49 2.00 0.39

[0088] As can be seen from Table 2, the same erythromycin oxime sample solution was injected 6 times, and the relative peak area RSD was less than 2%, indicating that the method provided by the present invention has good repeatability in detecting erythromycin oxime.

[0089] Example 4

[0090] In this example, six batches of erythromycin oxime (homemade erythromycin oxime HMSW-01, HMSW-02, HMSW-03, HMSW-04, HMSW-05, HMSW-06) were tested for test solutions. Accurately weigh the test samples, wherein the sample weights of HMSW-01, HMSW-02, HMSW-03, HMSW-04, HMSW-05, and HMSW-06 were 0.0020 g, 0.0022 g, 0.0020 g, 0.0021 g, 0.0021 g, and 0.0020 g, respectively, and prepare the test solutions. The detection steps, detection conditions, and experimental conditions are the same as those for the chromatographic detection of the test samples described in Example 1. Use isocratic elution and record the chromatogram, as shown in detail. Figure 5 The measurement results of six batches of test products are shown in Table 3 below.

[0091] Table 3

[0092]

[0093]

[0094] The data in Table 3 show that the RSDs are all less than 2%, and this method has good adaptability to erythromycin oxime samples.

[0095] Comparative Example 1

[0096] The difference from Example 1 is that high performance liquid chromatography analysis is performed, a mixed eluent is used as the mobile phase (the mobile phase consists of mobile phase A and mobile phase B, mobile phase A is acetonitrile, and mobile phase B is 0.025 mol / L potassium dihydrogen phosphate solution), and a gradient elution method is used for detection. The other operations are exactly the same as in Example 1.

[0097] The specific gradient process is shown in Table 4. The results show that the gradient elution cannot completely separate the impurity peaks. The specific chromatogram obtained by the test is as follows: Figure 6 shown.

[0098] Table 4

[0099] Time (min) Mobile phase A (%) Mobile phase B (%) 0 55 45 20 55 45 25 60 40 40 60 40 45 50 50

[0100] Comparative Example 2

[0101] The difference from Example 1 is that the mobile phase is different, and the other detection steps, detection conditions and experimental conditions are the same as the basic steps of chromatographic detection of the test sample described in Example 1. The specific mobile phase composition and detection results are shown in Table 5.

[0102] Table 5 Experimental results of different mobile phase tests

[0103]

[0104]

[0105] A series of test results show that when acetonitrile and potassium dihydrogen phosphate are selected as mobile phases, the peak separation effect is good.

[0106] Comparative Example 3

[0107] The difference from Example 1 is that the mobile phase ratio is different, and the other detection steps, detection conditions and experimental conditions are the same as the basic steps of chromatographic detection of the test sample described in Example 1. The specific mobile phase ratio is shown in Table 6, and the specific chromatogram obtained by the detection is shown in Figure 8 shown.

[0108] Table 6 Mobile phase ratio conditions

[0109] Acetonitrile / % 0.025 mol / L potassium dihydrogen phosphate solution / % 65 35 55 45 45 55

[0110] Depend on Figure 8 It can be seen that the best effect is achieved when acetonitrile: 0.025 mol / L potassium dihydrogen phosphate solution = 45:55 is used as the mobile phase.

[0111] Comparative Example 4

[0112] The difference from Example 1 is that the detection wavelength is different, and the other detection steps, detection conditions and experimental conditions are the same as the basic steps of chromatographic detection of the test sample described in Example 1. The specific chromatogram obtained by the detection is as follows Fig. 9 As shown. Fig. 9 It can be seen that 215 nm wavelength is the best detection wavelength for erythromycin oxime HPLC.

[0113] 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 the purity of erythromycin oxime, characterized in that: The steps include: 1) respectively subjecting the erythromycin oxime solution to be detected and the erythromycin oxime standard solution to be detected by a high performance liquid chromatograph, using a mixed eluent as a mobile phase and an isocratic elution method to obtain a chromatogram of the erythromycin oxime solution to be detected and a chromatogram of the standard, and recording the peak areas; The mixed eluent comprises acetonitrile and potassium dihydrogen phosphate solution; 2) The purity of the erythromycin oxime to be tested was calculated using the external standard method.

2. The detection method according to claim 1, characterized in that: The concentration of the potassium dihydrogen phosphate solution in step 1) is 0.025 mol / L; the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile is 55:

45.

3. The detection method according to claim 1, characterized in that: The detection conditions when using high performance liquid chromatography for detection in step 1) are: Detection wavelength: 215nm; Column temperature 30-40°C; Flow rate 0.5~2.0mL / min; Injection volume: 5~20μL.

4. The detection method according to claim 1, characterized in that: In step 1), a reverse phase chromatography column using octadecylsilane bonded silica gel as a filler is used for high performance liquid chromatography detection.

5. The detection method according to claim 4, characterized in that: The column length of the chromatographic column is 4.6 mm*250 mm.

6. The detection method according to claim 1, characterized in that: When high performance liquid chromatography detection is performed in step 1), the detector is a diode array detector.

7. The detection method according to claim 1, characterized in that: The erythromycin oxime solution to be detected and the erythromycin oxime standard solution were prepared by using a mixed solvent of a potassium dihydrogen phosphate solution with a concentration of 0.025 mol / L and acetonitrile; the volume ratio of the potassium dihydrogen phosphate solution to acetonitrile was 55:

45.

8. The detection method according to claim 7, characterized in that: The concentration of the erythromycin oxime solution to be detected is 2 μg / ml to 4 mg / ml; the concentration of the erythromycin oxime standard solution is 1 μg / ml to 5 mg / ml.

9. The detection method according to claim 1, characterized in that: In step 2), the purity of erythromycin oxime to be detected is calculated by external standard method, which specifically comprises the following steps: a. Take a certain amount of standard in a volumetric flask and dilute it into five solutions of different concentrations. The five solutions of different concentrations obtained by dilution are respectively tested by liquid chromatography injection, and the peak area of ​​the chromatographic peak is used as the ordinate and the corresponding solution concentration is used as the abscissa to draw a graph; b. Calculation results. The specific calculation formula is: Where: X is the mass concentration of erythromycin oxime in the sample, %; c is the concentration of erythromycin oxime obtained from the standard curve, (mg / mL); v is the total volume of the sample after dilution, (mL); M is the sample mass, (g).

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