CN110794046B - Method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediate - Google Patents
Method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediate Download PDFInfo
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Abstract
The invention belongs to the technical field of pharmaceutical analysis, and relates to a method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in a moxifloxacin intermediate, which uses convenient and quick LCMS/MS to detect the 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the moxifloxacin intermediate and can be used for monitoring the quality of moxifloxacin.
Description
Technical Field
The invention relates to a method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in a moxifloxacin intermediate, belonging to the technical field of pharmaceutical analysis.
Background
The ICHM7 is the evaluation and control of DNA activity (mutagenic) impurities in drugs to limit potential carcinogenic risk, and its purpose is to provide a framework for use in identifying, classifying, characterizing and controlling these mutagenic impurities to control potential carcinogenic risk. Genotoxic impurities refer to compounds that themselves directly or indirectly damage cellular DNA, cause genetic mutations or in vivo mutagenesis, and have carcinogenic potential or propensity.
The concept of acceptable risk intake, i.e. the toxic substance limitation (TTC), was introduced in EMA "guidelines for genotoxic impurity limits". A limit value TTC (1.5. mu.g/day) is set, which corresponds to a daily intake of 1.5. mu.g of genotoxic impurities, and is considered to be an acceptable risk for most drugs. In accordance with this threshold, acceptable impurity levels in the active agent can be calculated based on the expected daily intake.
The structural formula of the 2,4, 5-trifluoro-3-methoxybenzoyl chloride (hereinafter referred to as SM1) is as follows:
is one of starting materials of a moxifloxacin intermediate, the moxifloxacin intermediate has the chemical name: 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester, CAS: 112811-71-9, hereinafter abbreviated as moxifloxacin S1, wherein the moxifloxacin S1 may contain SM1, SM1 has a genotoxic warning structure, and the detection of SM1 in moxifloxacin S1 is very important for the quality control of moxifloxacin medicines.
I independently developed a method for testing 2,4, 5-trifluoro-3-methoxybenzoyl chloride in moxifloxacin intermediate, wherein SM1 in ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate is detected by SM1 and excessive 4- [2- (dimethylamino) ethoxy ] ethoxy]Benzylamine (structural formula:
) 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivatives are obtained by derivation according to the proportion of 1:1 (the structural formula is as follows:
) (hereinafter referred to as SM1 derivative), and then detecting the SM1 derivative by LCMS/MS.
The method accords with the guiding principle of the verification of the Chinese pharmacopoeia method in the aspects of system applicability, repeatability, stability, precision and accuracy.
Disclosure of Invention
The invention aims to provide a method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester, which is convenient, efficient and accurate, completely accords with the guide principle of Chinese pharmacopoeia method verification in the aspects of system applicability, repeatability, specificity and accuracy, and can be used for quality control of 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester.
In order to achieve the purpose, the invention provides the following technical scheme:
the detection method of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester comprises the following steps:
(1) preparing a solution, and respectively preparing a blank solution, a test solution and a reference solution, wherein the reference solution comprises a stock solution of a 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative (hereinafter referred to as SM1 derivative).
(2) The determination method comprises the following steps: determining the content of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate by adopting LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry), and after the system is stabilized, respectively adding a blank solution, a reference solution and a test solution, and recording a spectrogram;
i: the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, the flow rate is 0.5mL/min +/-0.1 mL/min, the column temperature is as follows: 30 +/-5 ℃, sample injection amount: 0.1-0.3. mu.l, mobile phase with formic acid: the water system is a mobile phase A, methanol is used as a mobile phase B, and gradient elution is adopted;
II: MS conditions: polarity: positive electrode, dry gas temperature: 350 ℃, dry air flow: 8L/min, atomizer pressure: 45psi, capillary voltage: 3000V, sheath gas temperature: 350 ℃, sheath gas flow rate: 11L/min, scanning mode: MRM, pre. ion (3.0min-5.5 min): 383.2, Pro.ion (m/z): 189.1(collision Energy 36V) (quantitation ion); 133.1(collision Energy 36V) (qualitative ion), fragment: 132V.
Preferably, the blank solution is prepared by the following steps: taking a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine in a volumetric flask, adding a diluent to dilute to a scale, and shaking up;
stock solutions of the SM1 derivatives: placing a proper amount of SM1 reference substance into a volumetric flask, adding a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine, performing vortex oscillation, placing, adding a diluent to dissolve and dilute to a scale, and shaking up;
reference solution: precisely measuring a proper amount of SM1 derivative stock solution, placing the stock solution into a volumetric flask, adding diluent to dilute the stock solution to a scale, and shaking up;
test solutions: placing a proper amount of a test sample in a volumetric flask, adding a diluent to dissolve and dilute the test sample to a scale, and shaking up; precisely measuring a proper amount of the solution, placing the solution in a volumetric flask, adding a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine, performing vortex oscillation, placing the solution, adding a diluent to dilute the solution to a scale, and shaking the solution uniformly;
the diluent is acetonitrile; the mobile phase is as follows: mobile phase A: formic acid: water-1: 1000 (V/V); mobile phase B: methanol;
the formic acid is HPLC grade; the methanol is HPLC grade; the acetonitrile is of HPLC grade; the water is HPLC grade; the chromatographic column was Agilent Eclipse Plus C18RRHD 3.0X 150mm,1.8 μm.
More preferably, the measurement method of the present invention comprises the steps of:
(1) preparing a solution, namely respectively preparing a blank solution, a test solution and a reference solution, wherein the reference solution comprises a stock solution of a 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative (hereinafter referred to as SM1 derivative);
diluting liquid: acetonitrile;
blank solution: taking a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, adding a diluent to dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 10ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up;
stock solutions of SM1 derivatives: precisely weighing about 20mg of SM1 reference substance, placing in a 20ml measuring flask, adding 2ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex and shaking for about 2min, standing for about 5min, adding diluent to dissolve and dilute to scale, and shaking uniformly; precisely measuring 1.0ml of the solution, placing the solution in a 100ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 100ml measuring flask, adding the diluent to dilute to the scale, and shaking up. (SM1 derivative concentration: 100ng/ml)
Reference solution: precisely measuring 600 μ l of SM1 derivative stock solution, placing in a 10ml measuring flask, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 6ng/ml)
Test solutions: taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution, putting the solution into a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, carrying out vortex oscillation for about 2min, standing for about 5min, adding a diluent to dilute the solution to a scale, and shaking up. (Moxifloxacin S1 concentration: 1.6mg/ml)
(2) The determination method comprises the following steps: after the system is stabilized, feeding a blank solution 1 needle, a reference solution 5 needle and a test solution 1 needle, and recording a spectrogram, wherein the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, the flow rate is 0.5ml/min, and the column temperature is as follows: 30 ℃, sample introduction: 0.2. mu.l, mobile phase A: formic acid: water 1:1000(V/V), mobile phase B: methanol; gradient elution:
| T(min) | mobile phase A (%) | Mobile phase B (%) |
| 0 | 60 | 40 |
| 2 | 60 | 40 |
| 10 | 5 | 95 |
| 13 | 5 | 95 |
Post-run time: and 5 min.
The chromatographic column is Agilent Eclipse Plus C18RRHD 3.0 × 150mm,1.8 μm;
MS conditions: polarity: positive electrode, dry gas temperature: 350 ℃, dry air flow: 8L/min, atomizer pressure: 45psi, capillary voltage: 3000V, sheath gas temperature: 350 ℃, sheath gas flow rate: 11L/min, scanning mode: MRM, pre. ion (3.0min-5.5 min): 383.2, Pro.ion (m/z): 189.1(collision Energy 36V) (quantitation ion); 133.1(collision Energy 36V) (qualitative ion), fragment: 132V.
The method for detecting the 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester further comprises a method verification before detection, wherein the method verification is that according to the chromatographic conditions of formal detection, the determination result is as follows:
TABLE 1 summary of analytical results validation methods
Advantageous effects
According to the technical scheme, the detection method provided by the invention has the advantages that the accuracy of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate is high, the system applicability is high, and the detection method meets the standards in the aspects of precision and detection limit. The method utilizes convenient and quick LCMS/MS detection to detect the 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester, and can be used for monitoring the quality of the 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester. The method for detecting the 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester has the advantages of strong specificity, good system applicability, good precision, good accuracy, good linear relation and good durability, is suitable for detecting the 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester to effectively control the quality of products, and is simple to operate, the time consumption is short.
Drawings
FIG. 1 is a diagram of a blank solution in a detection method of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate;
FIG. 2 is a diagram of a reference solution for the detection method of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate;
FIG. 3 is a diagram of a detection method test solution of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in ethyl 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylate;
figure 4 is a linear plot of SM1 derivatives.
Detailed Description
The invention will be further explained and illustrated by the following specific examples, which are not intended to limit the scope of the invention in any way.
Example 1
(1) Experimental materials and Instrument Condition
The instrument comprises the following steps: agilent Infinity 1290 UHPLC + Agilent 6470 ESI-QQQ-MS; a chromatographic column: agilent Eclipse Plus C18RRHD 3.0X 150mm,1.8 μm; flow rate: 0.5 ml/min; column temperature: 30 ℃; sample introduction amount: 0.2 μ l; a mobile phase A: formic acid: water 1:1000(V/V), mobile phase B: methanol; gradient elution:
| T(min) | mobile phase A (%) | Mobile phase B (%) |
| 0.0 | 60 | 40 |
| 2.0 | 60 | 40 |
| 10.0 | 5 | 95 |
| 13.0 | 5 | 95 |
Post-run time: and 5 min.
MS conditions: polarity: positive electrode, dry gas temperature: 350 ℃, dry air flow: 8L/min, atomizer pressure: 45psi, capillary voltage: 3000V, sheath gas temperature: 350 ℃, sheath gas flow rate: 11L/min, scanning mode: MRM, pre. ion (3.0min-5.5 min): 383.2, Pro.ion (m/z): 189.1(collision Energy 36V) (quantitation ion); 133.1(collision Energy 36V) (qualitative ion), fragment: 132V.
(2) Experimental procedure
Preparing a solution, namely respectively preparing a blank solution, a test solution and a reference solution, wherein the reference solution comprises a stock solution of a 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative (hereinafter referred to as SM1 derivative);
diluting liquid: acetonitrile;
blank solution: taking a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, adding a diluent to dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 10ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up;
stock solutions of SM1 derivatives: precisely weighing about 20mg of SM1 reference substance, placing in a 20ml measuring flask, adding 2ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex and shaking for about 2min, standing for about 5min, adding diluent to dissolve and dilute to scale, and shaking uniformly; precisely measuring 1.0ml of the solution, placing the solution in a 100ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 100ml measuring flask, adding the diluent to dilute to the scale, and shaking up. (SM1 derivative concentration: 100ng/ml)
Reference solution (SM1 derivative localization solution): precisely measuring 600 μ l of SM1 derivative stock solution, placing in a 10ml measuring flask, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 6ng/ml)
Test solution (moxifloxacin S1 spotting solution): taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution, putting the solution into a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, carrying out vortex oscillation for about 2min, standing for about 5min, adding a diluent to dilute the solution to a scale, and shaking up. (Moxifloxacin S1 concentration: 1.6 mg/ml).
SM1 stock solution: precisely weighing about 20mg of SM1 reference substance, placing in a 100ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up; precisely measuring 1.0ml of the solution, putting the solution into a 100ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 20ml measuring flask, adding the diluent to dilute the solution to a scale, and shaking up the solution. (SM1 concentration: 100ng/ml)
Selective solution: taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution and 600 mul of SM1 stock solution, placing into a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex shaking for about 2min, standing for about 5min, adding diluent to dilute to scale, and shaking. (Moxifloxacin S1 concentration: 1.6mg/ml, SM1 derivative concentration: 6ng/ml)
Test solutions (spiked): taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution and 600 mul of SM1 stock solution, placing into a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex shaking for about 2min, standing for about 5min, adding diluent to dilute to scale, and shaking. (concentration of moxifloxacin S1: 1.6mg/ml, concentration of SM1 derivative added: 6ng/ml) 6 parts of test solution (spiked) was prepared in the same manner.
LOQ stock solution: precisely weighing about 32mg of SM1 reference substance, placing in a 100ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution in a 100ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 200ml measuring flask, adding the diluent to dilute to the scale, and shaking up. (SM1 derivative concentration: 16ng/ml)
Test solution (a): precisely measuring 1.0ml of LOQ stock solution, placing in a 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex and shake for about 2min, standing for about 5min, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 1.6ng/ml)
LOQ solution: adjusting the dilution ratio according to the S/N value of the SM1 derivative obtained in the test solution (a) until the S/N value of the SM1 derivative is more than 10, and preparing 6 parts of the solution by the same method;
LOD solution: precisely measuring 3.0ml of LOQ solution, placing in a 10ml measuring flask, adding the diluent to dilute to the scale, and shaking up.
Linear solution-50%: precisely measuring 300 mul of linear-50% stock solution, placing into a 10ml measuring flask, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 3ng/ml)
Linear solution-80%: precisely measuring 480 mul of linear-80% stock solution, placing the linear-80% stock solution into a 10ml measuring flask, adding diluent to dilute the stock solution to a scale, and shaking up. (SM1 derivative concentration: 4.8ng/ml)
Linear solution-100%: precisely measuring 600 mul of linear-100% stock solution, placing in a 10ml measuring flask, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 6ng/ml)
Linear solution-120%: 720 mul of linear-120% stock solution is precisely measured, placed in a 10ml measuring flask, diluted to the scale by adding diluent, and shaken up. (SM1 derivative concentration: 7.2ng/ml)
Linear solution-150%: precisely measuring 900 mul of linear-150% stock solution, placing in a 10ml measuring flask, adding diluent to dilute to scale, and shaking up. (SM1 derivative concentration: 9ng/ml)
Accuracy stock solution: precisely weighing about 20mg of SM1 reference substance, placing in a 100ml measuring flask, adding diluent to dissolve and dilute to scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 100ml measuring flask, adding a diluent to dilute the solution to a scale, and shaking up; precisely measuring 1.0ml of the solution, placing the solution into a 20ml measuring flask, adding the diluent to dilute the solution to a scale, and shaking up the solution. (SM1 concentration: 100ng/ml) (SM1 stock solution under 5.2 specialization can be cited)
Accuracy LOQ solutions: taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution and a proper amount of accurate LOQ stock solution, placing the solution in a same 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, performing vortex oscillation for about 2min, standing for about 5min, adding a diluent to dilute the solution to a scale, and shaking up to enable the concentration of moxifloxacin S1 in the solution to be 1.6mg/ml and the concentration of SM1 derivatives to be LOQ. 3 portions of the mixture are prepared by the same method.
Accuracy solution-100%: taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent for dissolving, diluting to a scale, and shaking up; precisely measuring 1.0ml of the solution and 600 mul of the accurate stock solution respectively, placing the solution and the accurate stock solution into the same 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, carrying out vortex oscillation for about 2min, standing for about 5min, adding the diluent to dilute to scale, and shaking up. (concentration of moxifloxacin S1: 1.6mg/ml, concentration of SM1 derivative added: 6ng/ml) 3 parts were prepared in the same manner.
Accuracy solution-150%: taking about 160mg of a test sample, precisely weighing, placing in a 10ml measuring flask, adding a diluent to dissolve and dilute to a scale, and shaking up; precisely measuring 1.0ml of the solution and 900 μ l of the accuracy stock solution, placing into the same 10ml measuring flask, adding 1ml of 4- [2- (dimethylamino) ethoxy ] benzylamine, vortex shaking for about 2min, standing for about 5min, adding diluent to dilute to scale, and shaking. (concentration of moxifloxacin S1: 1.6mg/ml, concentration of SM1 derivative: 9ng/ml) was prepared in 3 parts by the same method.
Sample introduction procedure: after the system is stabilized, 1 pin of blank solution, 5 pins of reference solution and 1 pin of test solution are added, and the spectrogram is recorded.
The method comprises the following steps: the RSD of the 5 reference solutions should not be more than 10.0% based on the peak area of the SM1 derivative.
And (3) calculating:
wherein: rU: testing the peak area of SM1 derivative in the solution map;
RS: average peak area of SM1 derivative in 5 reference solution spectra;
CS: concentration of SM1 derivative in the reference solution (ng/ml);
CU: the concentration of moxifloxacin S1 (mg/ml) in the test solution;
remarking: the result of measurement of the SM1 derivative was SM 1.
TABLE 2 limits
| Name (R) | RT(min) | Limit (ppm) | LOQ(ppm) | LOD(ppm) |
| SM1 derivative (SM1) | ≈4.25min | ≤3.75 | 1.024 | 0.307 |
Remarking: SM1 has a genotoxic warning structure, reference ICHM7, which has a TTC value of 1.5 μ g/day:
TTC/maximum daily dose
The maximum daily dose of moxifloxacin hydrochloride was 400mg, the calculated limit of SM1 in finished moxifloxacin hydrochloride was 3.75ppm, and in order to strictly control the starting material S1, the limit of SM1 in the starting material S1 was determined to be 3.75 ppm.
Example 2 detection method of the invention System suitability test
The applicability of the system is realized by RSD of the peak area of the SM1 derivative in 5 reference solutions, the RSD of the peak area of the SM1 derivative of the 5 reference solutions is required to be not more than 10.0%, after the system is stabilized, a blank solution 1 needle is added, the reference solution 5 needles are added, a spectrogram is recorded, and the RSD of the peak area of the SM1 derivative of the 5 reference solutions is not more than 10.0%.
TABLE 3 results of the System suitability measurement
The specificity is that the blank solution is determined to have no interference on the detection of the SM1 derivative; separation and recovery of the SM1 derivative in the selective solution before and after loading. Blank solutions should not interfere with the detection of SM1 derivatives; the degree of separation of the SM1 derivative from the adjacent peaks in the selective solution should be no less than 1.5; before and after loading, the recovery rate of the SM1 derivative in the selective solution is determined
70.0-130.0%, after the system is stabilized, feeding a blank solution 1 needle, an SM1 derivative positioning solution (reference solution) 3 needles, a moxifloxacin S1 positioning solution (test solution) 1 needle, a selective solution 3 needles, recording a spectrogram, wherein the blank solution has no interference on the detection of the SM1 derivative; the degree of separation of the SM1 derivative from the adjacent peaks in the selective solution should be no less than 1.5; before and after loading, the recovery rate of the SM1 derivative in the selective solution is 70.0-130.0%.
TABLE 4 results of specificity determination
TABLE 5 Selective solution assay results
Remarking: recovery (%) — peak area of SM1 derivative in selective solution ÷ average peak area of three-pin reference solution × concentration of SM1 derivative in reference solution ÷ concentration of SM1 added in selective solution.
Example 4 precision of the assay of the invention
The precision is realized by the RSD of the measurement result of the SM1 derivative in 6 parts of test solution (added marks), the RSD of the measurement result of the SM1 derivative in 6 parts of test solution (added marks) is required to meet the acceptable standard, after the system is stabilized, 1 needle of blank solution, 5 needles of reference solution and 1 needle of 6 parts of moxifloxacin S1 test solution (added marks) are respectively used, and the RSD of the measurement result of the SM1 derivative in 6 parts of test solution (added marks) is not more than 10.0 percent.
TABLE 6 results of precision measurement
Example 5 detection and quantitation limits of the detection methods of the invention
The detection limit is determined by detecting that the ratio of the response signal to the noise is not less than 3: 1, the limit of quantitation is determined by the signal-to-noise ratio of not less than 10: 1, and (b). At the concentration level, 6 parts of quantitative limit test solution are repeatedly inspected, and in a spectrogram obtained by 6 times, the RSD of the SM1 derivative unit concentration peak area accords with the regulation to confirm that the quantitative limit determination result has certain precision, after a system is stabilized, 1 needle of blank solution, 1 needle of test solution (a), 1 needle of 6 parts of LOQ solution and 1 needle of LOD solution are respectively added, the spectrogram is recorded, the LOQ is not more than 1.125ppm, the S/N value is not less than 10, and the RSD of the SM1 derivative unit concentration peak area of 6 parts of LOQ solution is not more than 10.0%; LOD should be less than LOQ and S/N should be no less than 3.
TABLE 7 determination of quantitation Limit and detection Limit
TABLE 8 LOQ precision results
Example 6 the detection method of the invention linearity and range
6 points were uniformly set in the range of the LOQ concentration to the 150% limit concentration, and the curve was drawn with the concentration as the abscissa and the peak area of the SM1 derivative as the ordinate. The peak area of the SM1 derivative is required to be linear in the range of LOQ concentration to 150% limit concentration, the square of the linear correlation coefficient R (R2) meets the acceptable standard, after the system is stabilized, 3 needles of linear solution at each concentration are added, the spectrogram is recorded, the SM1 derivative is required to be linear in the range of LOQ concentration to 150% limit concentration, and the square of the linear correlation coefficient R (R2) is required to be not less than 0.99.
TABLE 9-SM1 derivatives Linear assay results
Example 7 accuracy (recovery) of the detection method of the invention
The accuracy is the degree that the actual concentration is close to the theoretical concentration measured by the method, and is realized by measuring the recovery rate of standard solutions with different concentrations added into a test solution, the recovery rate of the SM1 derivative in the accuracy solution with the LOQ concentration of 150% limit concentration is required to meet an acceptable standard, after a system is stabilized, 1 needle of blank solution (added with an internal standard) is added, 1 needle of the test solution, 1 needle of a reference solution, 3 parts of accuracy LOQ solution-100%, and 3 parts of accuracy solution-150% are added, 1 needle of each, a spectrogram is recorded, and the recovery rate of the SM1 derivative in the accuracy solution with the LOQ concentration of 150% limit concentration is 70.0-130.0%.
TABLE 10 accuracy measurement results
Remarking: measured concentration ═ reference solution concentration ÷ reference solution average peak area × accuracy solution calculated peak area.
EXAMPLE 8 durability of the detection method of the invention
Observing the rule that the detection results change along with time after the test solution, the reference solution and the selective solution are placed at room temperature for a period of time, providing reference for the placement time of the test solution and the reference solution during detection, and after the system is stable, feeding 1 needle of blank solution and 1 needle of each of the test solution, the reference solution and the selective solution at different times; recording a spectrogram, and if the components of the test solution are not detected within 0 day and are still not detected within a period of time after being placed at room temperature, stabilizing the test solution during the investigation period; if the components of the test solution are detected in 0 day and are placed at room temperature for a period of time, the change value of the component measurement result is within 20% of the limit compared with the change value in 0 day, and no obvious change trend exists, the test solution is stable in the investigation period;
compared with 0 day, the recovery rate of the SM1 derivative is 70.0-130.0% when the reference solution is placed at room temperature for a period of time, and no obvious change trend exists, so that the reference solution is stable during the room temperature investigation;
the selective solution is placed at room temperature for a period of time, the recovery rate of the SM1 derivative is 70.0-130.0%, and no obvious change trend exists, and then the selective solution is examined at room temperature.
TABLE 11 solution stability-System suitability assay results
TABLE 12 solution stability test solution assay results
Remarking: the test solution (day 0), the test solution (day 1), the test solution (day 3) and the test solution (day 4) are respectively test solution-1 injected after being placed for 0 day, test solution-2 injected after being placed for 1 day, test solution-3 injected after being placed for 3 days and test solution-4 injected after being placed for 4 days.
TABLE 13 solution stability-reference solution and Selective solution assay results
Remarking: (1) recovery (%) — calculated peak area ÷ 0 peak area of day-ginseng-to-solution × 0 concentration of day-ginseng-to-solution ÷ input concentration × 100%; (2) the reference solution (day 0) refers to a first needle of reference solution with system applicability under the system applicability solution stability, and the test solution (day 0) and the selective solution (day 0) are respectively a test solution-1 and a selective solution-1 which are placed at room temperature for 0 day for sample injection; the test solution (1 day), the reference solution (1 day) and the selective solution (1 day) are respectively a test solution-2, a reference solution-2 and a selective solution-2 which are injected after being placed for 1 day at room temperature; the test solution (3 days), the reference solution (3 days) and the selective solution (3 days) are respectively a test solution-3, a reference solution-3 and a selective solution-3 which are injected after being placed at room temperature for 3 days; the test solution (4 days), the reference solution (4 days) and the selective solution (4 days) are respectively a test solution-4, a reference solution-4 and a selective solution-4 which are injected after being placed at room temperature for 4 days.
The above examples are only preferred embodiments of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (2)
1. A method for detecting 2,4, 5-trifluoro-3-methoxybenzoyl chloride in a moxifloxacin intermediate, wherein the moxifloxacin intermediate is 1-cyclopropyl-6, 7-difluoro-1, 4-dihydro-8-methoxy-4-oxoquinoline-3-carboxylic acid ethyl ester, and the method comprises the following steps:
(1) preparing a solution, and respectively preparing a blank solution, a test solution and a reference solution, wherein the reference solution comprises a stock solution of a 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative, the 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative is abbreviated as SM1 derivative, and the 2,4, 5-trifluoro-3-methoxybenzoyl chloride derivative is a derivative generated by the reaction of 2,4, 5-trifluoro-3-methoxybenzoyl chloride and 4- [2- (dimethylamino) ethoxy ] benzylamine;
(2) the determination method comprises the following steps: determining the content of 2,4, 5-trifluoro-3-methoxybenzoyl chloride in the moxifloxacin intermediate by adopting LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry), after the system is stabilized, respectively adding a blank solution, a reference solution and a test solution, and recording a spectrogram;
i: the chromatographic conditions are as follows: octadecylsilane chemically bonded silica is used as a filling agent, the flow rate is 0.5mL/min +/-0.1 mL/min, the column temperature is as follows: 30 +/-5 ℃, sample injection amount: 0.1-0.3. mu.l, mobile phase with formic acid: the water system is a mobile phase A, methanol is used as a mobile phase B, and gradient elution is adopted, wherein the mobile phase gradient process is as follows:
Post-run time: 5 min;
II: MS conditions: polarity: positive electrode, dry gas temperature: 350 ℃, dry air flow: 8L/min, atomizer pressure: 45psi, capillary voltage: 3000V, sheath gas temperature: 350 ℃, sheath gas flow rate: 11L/min, scanning mode: MRM, pre.ion, 3.0min-5.5 min: 383.2, Pro.ion (m/z): 189.1, collision Energy 36V, quantitative ion, 133.1, collision Energy 36V, qualitative ion, fragment: 132V.
2. The method of claim 1, wherein:
the preparation steps of the blank solution are as follows: taking a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine in a volumetric flask, adding a diluent to dilute to a scale, and shaking up;
stock solutions of the SM1 derivatives: placing a proper amount of SM1 reference substance into a volumetric flask, adding a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine, performing vortex oscillation, placing, adding a diluent to dissolve and dilute to a scale, and shaking up;
reference solution: precisely measuring an appropriate amount of SM1 derivative stock solution, placing the stock solution into a volumetric flask, adding diluent to dilute the stock solution to a scale, and shaking up;
test solutions: placing a proper amount of a sample in a volumetric flask, adding a diluent to dissolve and dilute the sample to a scale, and shaking up; precisely measuring a proper amount of solution, placing the solution in a volumetric flask, adding a proper amount of 4- [2- (dimethylamino) ethoxy ] benzylamine, performing vortex oscillation, placing the solution, adding diluent to dilute the solution to a scale, and shaking the solution uniformly;
the diluent is acetonitrile;
the mobile phase is as follows:
mobile phase A: formic acid: water volume ratio =1: 1000;
mobile phase B: methanol;
the formic acid is HPLC grade;
the methanol is HPLC grade;
the acetonitrile is HPLC grade;
the water is HPLC grade;
the chromatographic column is Agilent Eclipse Plus C18RRHD 3.0 × 150mm,1.8 μm.
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