Determination of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry

Abstract

The invention discloses a method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry, wherein isopropanol is used as both a solvent and a derivatization reagent when the method is determined by gas chromatography-mass spectrometry. The test results of the applicant show that the method of the present invention has good linearity within a concentration range of 2.4 to 19.3 ng / mL, and the average relative standard deviation is low. It can be seen that the method of the present invention is qualitatively and quantitatively accurate and can meet the detection requirements of MEM in roxithromycin.

Description

Technical Field

[0001] The invention relates to the determination of the content of residual components in medicines, and in particular to a method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry. Background Art

[0002] Roxithromycin is a new generation of macrolide antibiotics, which mainly acts on Gram-positive bacteria, anaerobic bacteria, Chlamydia and Mycoplasma, etc., and is usually used to treat respiratory tract, urinary tract and soft tissue infections. The production method of Roxithromycin is to use erythromycin as a raw material, react with hydroxylamine hydrochloride in triethylamine and methanol to generate oxime, and then react with 2-methoxyethoxymethyl chloride (MEM) in acetone to obtain it.

[0003] MEM is an intermediate in the synthesis of roxithromycin. Its chemical structure contains a methyl chloride structure, which has potential genotoxicity. Therefore, it is necessary to establish a method for detecting MEM in roxithromycin. After searching, there is no report on the use of gas chromatography-mass spectrometry to detect the content of MEM in roxithromycin. Summary of the invention

[0004] The technical problem to be solved by the invention is to provide a method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry with simple and rapid operation, high sensitivity and accuracy.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] The method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry is characterized in that isopropanol is used as both solvent and derivatization reagent during the gas chromatography-mass spectrometry determination.

[0007] According to the regulatory guidelines for genotoxic impurities in active drugs issued by the International Council for Harmonization (ICH) and the European Medicines Agency, the threshold value of toxicological concern of 1.5 μg / day is considered to be an acceptable risk for potential genotoxic impurities (DNA-reactive mutagenic impurities) in most drugs and APIs. Based on the dosage of 0.3 g / day of roxithromycin, the limit of MEM is 5 ppm. The applicant found in the experiment that when other methods (such as GC-FID (gas chromatography-flame ionization detector method)) are used for detection, the sensitivity cannot meet the detection requirements; only when gas chromatography-mass spectrometry (GC-MS) is used for detection can the detection requirements be met. In addition, the applicant also found through experiments that when different alcohols are used as derivatization agents to react with MEM, there are significant differences in the stability of the products obtained, and the stability of the products obtained by the reaction directly affects the accuracy of the test results. The test results of the applicant show that when ethanol is used as both a solvent and a derivatization agent, the product after its reaction with MEM is only stable within 3 hours; and when isopropanol is used as both a solvent and a derivatization agent, the product after its reaction with MEM is stable within 8 hours, which brings great convenience to the detection operation. Therefore, the present application selects isopropanol as both a solvent and a derivatization agent. In the present application, using isopropanol as both a solvent and a derivatization agent means using isopropanol as a solvent for dissolving roxithromycin, and also using isopropanol as a derivatization agent to convert MEM into the corresponding ether, and then using a gas chromatography-mass spectrometry device for analysis.

[0008] In the method of the present invention, when the gas chromatography-mass spectrometry is used for determination, the chromatographic column of the gas chromatography is preferably an HP-5MS chromatographic column, an HP-5MS UI chromatographic column, a DB-5MS chromatographic column or a DB-5MS UI chromatographic column.

[0009] In the method of the present invention, during the gas chromatography-mass spectrometry determination, the mass spectrometer used is a quadrupole mass spectrometer, specifically a triple quadrupole tandem mass spectrometer or a single quadrupole tandem mass spectrometer.

[0010] In the method of the present invention, when measuring by gas chromatography-mass spectrometry, the device used is preferably Agilent 7890B-7000C.

[0011] Compared with the prior art, the present invention first proposes a method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry with isopropanol as both solvent and derivatization reagent. The method is simple and fast to operate, and has high sensitivity and accuracy. The test results of the applicant show that the method of the present invention has good linearity within the concentration range of 2.4-19.3 ng / mL, r=0.9995, LOQ is 0.9ppm, the average recovery is 82.3% (n=9), and the average relative standard deviation is 9.3%. It can be seen that the method of the present invention is qualitatively and quantitatively accurate and can meet the detection requirements of MEM in roxithromycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a chromatogram of the blank solution in Example 1 of the present invention, wherein 1-1 is a total ion current graph, 1-2 is a chromatogram with an extracted ion mass-to-charge ratio of 73, and 1-3 is a chromatogram with an extracted ion mass-to-charge ratio of 89.

[0013] Figure 2 2-1 is a total ion current diagram, 2-2 is a chromatogram with an extracted ion mass-to-charge ratio of 73, and 2-3 is a chromatogram with an extracted ion mass-to-charge ratio of 89.

[0014] Figure 3 3-1 is a total ion current graph, 3-2 is a chromatogram with an extracted ion mass-to-charge ratio of 73, and 3-3 is a chromatogram with an extracted ion mass-to-charge ratio of 89.

[0015] Figure 4 4-1 is a total ion current graph, 4-2 is a chromatogram with an extracted ion mass-to-charge ratio of 73, and 4-3 is a chromatogram with an extracted ion mass-to-charge ratio of 89.

[0016] Figure 5 It is the chromatogram of the blank solution in Comparative Example 2 of the present invention.

[0017] Figure 6 It is the chromatogram of the reference substance solution in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0018] In order to better explain the technical solution of the present invention, the present invention is further described in detail below in conjunction with embodiments, but the embodiments of the present invention are not limited thereto.

[0019] Example 1

[0020] 1. Instruments and reagents

[0021] 1.1 Instruments Gas chromatography tandem mass spectrometer (Agilent 7890B-7000C) (Agilent); XS205DU electronic balance (Mettler-Toledo).

[0022] 1.2 Reagent: 2-methoxyethoxymethyl chloride (batch number LB90T28, content 97.58%); isopropanol and acetonitrile are chromatographically pure reagents.

[0023] The three batches of roxithromycin were all provided by a certain manufacturer (batch numbers R190627003, R191028017, and R191028018).

[0024] 2 Methods and Results

[0025] 2.1 Chromatographic conditions

[0026] Gas chromatography conditions: Agilent HP-5MS UI (30m×0.25mm×0.25μm) (serial number USN387665H) column; column temperature: programmed temperature rise, starting temperature at 40°C, maintained for 3 minutes, then raised to 110°C at a rate of 15°C per minute, and then raised to 220°C at a rate of 40°C per minute, maintained for 5 minutes; injection port temperature was 110°C, carrier gas: helium, flow rate was 1.0mL / min; split ratio: splitless. Injection volume: 1μL.

[0027] The mass spectrometry conditions were detected by a triple quadrupole tandem mass spectrometer, the ion source was an electron impact source (EI), the voltage was 70 eV, the ion source temperature was 230°C, the mass spectrometry transmission interface temperature was 220°C, the gain factor was 10, and the solvent delay was 6 minutes; the detection mode was selected ion detection mode (SIM), the quantitative ion m / z was 73, and the qualitative ion m / z was 89.

[0028] 2.2 Preparation of test solution Take about 40 mg of roxithromycin, accurately weigh it, put it in a 20 mL volumetric flask, add isopropanol to dissolve it and dilute it to the scale, shake it well, and use it as the test solution.

[0029] 2.3 Preparation of reference solution Weigh an appropriate amount of MEM reference substance, place it in a 25mL volumetric flask, dilute to the mark with acetonitrile, shake well, and obtain a 1mg / mL MEM standard stock solution. Accurately measure 0.1mL of MEM standard stock solution, place it in a 100mL volumetric flask, dilute to the mark with acetonitrile, shake well, and obtain a 1μg / mL MEM standard working solution. Accurately measure 0.2mL of MEM standard working solution, place it in a 20mL volumetric flask, dilute to the mark with isopropanol, shake well, and obtain the reference solution.

[0030] 2.4 Preparation of blank solution Take 200 μL of acetonitrile and place it in a 20 mL volumetric flask, dilute to the mark with isopropanol, and shake well to prepare the blank solution.

[0031] 2.5 Specificity and system suitability tests

[0032] The blank solution, reference solution, test solution and spiked solution (100% limit level) were injected and analyzed to investigate the specificity of the method. The results showed that the blank solution had no interference at the peak position to be measured, and the method had good specificity. The chromatogram is shown in Figures 1 to 4 shown.

[0033] 2.6 Linearity and Range

[0034] The MEM standard working solution was accurately measured and prepared into a series of reference solutions of different concentrations. The solutions were injected for measurement and linear regression was performed with the peak area (Y) and the corresponding concentration (X). The results are shown in Table 1 below.

[0035] Table 1 Linear range, linear equation, and correlation coefficient (r)

[0036]

[0037] 2.7 Detection limit

[0038] Weigh 40 mg of roxithromycin, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 25 μL of the standard working solution, dissolve it with isopropanol and dilute it to the mark, shake it well, and use it as the detection limit solution. The concentration when the signal-to-noise ratio (S / N) is about 3 is the lower limit of detection (LOD) of the analyte, and its detection limit is calculated to be 0.6 ppm.

[0039] 2.8 Limit of quantitation

[0040] Weigh 40 mg of roxithromycin, accurately weigh it, place it in a 20 mL volumetric flask, accurately add 50 μL of the standard working solution, dissolve it with isopropanol and dilute it to the mark, shake it well, and use it as the quantitative limit solution. The concentration when the signal-to-noise ratio (S / N) is about 10 is the lower limit of quantification (LOQ) of the analyte, and its quantitative limit is calculated to be 0.9 ppm.

[0041] 2.9 Recovery Test About 40 mg of the same batch of roxithromycin samples were accurately weighed for a total of 9 portions, and a certain amount of standard working solution was added to each portion. After treatment according to the method under the aforementioned "2.2", the sample was injected for determination. The recovery rate and RSD of the component to be measured were calculated, and the results are shown in the following Table 2. The results show that the recovery rate of the method described in the present invention is good.

[0042] Table 2 Recovery test results

[0043]

[0044] 2.10 Repeatability test

[0045] Weigh 6 samples of batch number R191028018, each about 40 mg, accurately weigh, put into 20 mL volumetric flask, accurately add 200 μL of MEM standard working solution, dissolve and dilute to scale with isopropanol, shake well, and use as repeatability solution, and measure in the same way. The RSD of the 6 results is 5.9%, indicating that the repeatability of the method described in the present invention is good.

[0046] 2.11 Stability test

[0047] The recovery solution was taken and injected at 0, 5, 11, 16 and 19 hours after preparation, and the chromatogram was recorded. The RSD (n=5) of the peak area of ​​MEM was calculated to be 4.1%, indicating that the analyte was stable within 19 hours.

[0048] 2.12 Intermediate precision test

[0049] Another analyst prepared 6 100% limit spiked sample solutions at different times, injected and analyzed them, and the RSD of the recoveries of 12 100% limit spiked solutions was calculated to be 4.1%, which met the methodological requirements.

[0050] 2.13 Durability test

[0051] By changing the carrier gas flow rate ±0.1mL / min, the heating rate ±1℃, and replacing the chromatographic column with the same type of filler, the blank had no interference and the separation degree with the adjacent peaks was ≥1.5, which could meet the detection requirements, indicating that the method has good durability.

[0052] 2.14 Sample determination

[0053] Take 3 batches of samples and prepare test solutions according to the method under "2.2". No MEM was detected in the samples.

[0054] 3 Conclusion

[0055] The above experiments show that the method of the present invention is simple, rapid, sensitive and accurate, and can be used for the determination of MEM in roxithromycin.

[0056] Comparative Example 1

[0057] Example 1 was repeated, except that ethanol was used instead of the isopropanol in Example 1.

[0058] In the stability test, the reference solution prepared with ethanol was sampled at 0, 3.5, 6, 9, 12, and 14 hours after preparation, and the chromatogram was recorded. The RSD (n=6) of the peak area of ​​MEM was calculated to be 28.2%. The specific data are shown in Table 3 below.

[0059] Table 3 Stability results of reference solution (solvent ethanol)

[0060]

[0061] Comparative Example 2

[0062] GC-FID was used to detect MEM in roxithromycin, where:

[0063] The instrument and chromatographic conditions of the GC-FID method are as follows: Instrument Gas Chromatograph (GC-2010plus) (Shimadzu Corporation)

[0064] Gas chromatography conditions: Agilent HP-5 column (30m×0.32mm×0.25μm); column temperature: programmed temperature rise, starting temperature at 35°C, maintained for 5 minutes, then raised to 245°C at a rate of 10°C per minute, maintained for 2 minutes; injection port temperature was 150°C, detector temperature was 250°C, carrier gas: nitrogen, flow rate was 3.0mL / min; split ratio: 10:1. Injection volume: 2μL.

[0065] Weigh an appropriate amount of MEM reference substance, dissolve it in N,N-dimethylformamide and dilute it to prepare a reference substance solution with a concentration of 200 μg / mL.

[0066] Take N,N-dimethylformamide as the blank solution.

[0067] Take blank solution and reference solution for analysis, the chromatogram is as follows Figures 5 and 6 shown.

[0068] For the reference solution with a concentration of 200 μg / mL, the signal-to-noise ratio of MEM is 30.8. Experiments have shown that 1 g of roxithromycin can be dissolved in 2 ml of N,N-dimethylformamide. When the signal-to-noise ratio (S / N) is 3, the detection limit is 38.96 ppm, and when the signal-to-noise ratio (S / N) is 10, the quantification limit is 129.87 ppm.

Claims

1. A method for determining the content of 2-methoxyethoxymethyl chloride in roxithromycin by gas chromatography-mass spectrometry, characterized in that: In the gas chromatography-mass spectrometry (GC-MS) determination, isopropanol was used as both solvent and derivatization reagent.

2. The method according to claim 1, characterized in that: In the gas chromatography-mass spectrometry determination, the gas chromatography column is an HP-5MS column, an HP-5MS UI column, a DB-5MS column or a DB-5MS UI column.

3. The method according to claim 1, characterized in that: In the gas chromatography-mass spectrometry (GC-MS) measurement, the mass spectrometer used is a quadrupole mass spectrometer.

4. The method according to claim 1, characterized in that: In the gas chromatography-mass spectrometry (GC-MS) analysis, the instrument used was Agilent 7890B-7000C.

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