CN117191966A - Method for detecting 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride - Google Patents
Method for detecting 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride Download PDFInfo
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- IDIIJJHBXUESQI-DFIJPDEKSA-N moxifloxacin hydrochloride Chemical compound Cl.COC1=C(N2C[C@H]3NCCC[C@H]3C2)C(F)=CC(C(C(C(O)=O)=C2)=O)=C1N2C1CC1 IDIIJJHBXUESQI-DFIJPDEKSA-N 0.000 title claims abstract description 44
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- 238000000034 method Methods 0.000 title claims abstract description 19
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Abstract
The invention relates to the technical field of analysis and detection, in particular to a detection method of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride. The invention adopts a high performance liquid chromatography-tandem mass spectrometry (LC-MC/MC) method to realize rapid qualitative and quantitative analysis of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride, and has the advantages of simplicity, convenience, rapidness and high sensitivity. The detection method provided by the invention can effectively control the quality of moxifloxacin hydrochloride products by detecting whether 2,4, 5-trifluoro-3-methoxybenzoic acid and the residual quantity thereof remain in moxifloxacin hydrochloride, improves the medication safety of moxifloxacin hydrochloride medicines, makes up the blank of the existing detection method for 2,4, 5-trifluoro-3-methoxybenzoic acid substances in moxifloxacin hydrochloride, and is worthy of popularization and application.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a detection method of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride.
Background
Moxifloxacin is a fourth-generation broad-spectrum 8-methoxy fluoroquinolone antibacterial drug, belongs to a novel variety in the synthesis of antibacterial drugs, and achieves the antibacterial purpose by inhibiting bacterial DNA topoisomerase and repairing, copying, recombining and transcribing bacterial DNA. Because methoxy is introduced into the 8 th carbon atom of moxifloxacin, the combination capability and the cell membrane penetrating power of the medicine and bacteria are enhanced, and the drug resistance, phototoxicity and cytotoxicity of gram-positive bacteria are reduced, so that the medicine has the pharmacokinetic characteristics of wide antibacterial spectrum, low drug resistance, good oral absorption, high absolute bioavailability and the like. Moxifloxacin can effectively inhibit skin inflammation, has very good anti-inflammatory and anti-infective effects, has good antibacterial effects on gram-negative bacteria, gram-positive bacteria, mycoplasma, chlamydia and the like, and is clinically used for treating community-acquired pneumonia, acute bacterial sinusitis, genitourinary system infection and the like. The moxifloxacin venous preparation and the oral preparation have almost the same pharmacokinetic parameters in human bodies, and are particularly suitable for sequential treatment of antibacterial drugs. The medicine is taken for 1 time every day, has good compliance and is helpful for psychological rehabilitation of patients. In clinical application, the moxifloxacin has the characteristics of less adverse reaction, low drug resistance, reduced corresponding hospitalization time, reduced economic burden and the like, and is widely applied.
2,4, 5-trifluoro-3-methoxybenzoyl chloride is a byproduct generated in the synthesis process of moxifloxacin hydrochloride and is a potential genotoxic impurity. Because the acyl chloride compound is unstable and is easy to hydrolyze into an acid compound with stable structure, the research on the 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride directly relates to the medication safety of moxifloxacin hydrochloride. However, no report on the detection method of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride is known at present. Therefore, it is important to establish a detection method of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride.
Disclosure of Invention
The invention aims to provide a method for detecting 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride, which can rapidly and accurately realize qualitative and quantitative detection of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride.
In order to achieve the aim of the invention, the invention provides a method for detecting 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride, which comprises the following steps:
dissolving moxifloxacin hydrochloride in methanol to obtain a sample liquid to be tested;
detecting the sample liquid to be detected by adopting high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of 2,4, 5-trifluoro-3-methoxybenzoic acid; the detection result comprises a qualitative detection result and/or a quantitative detection result.
Preferably, the high performance liquid chromatography detection conditions of the high performance liquid chromatography tandem mass spectrometry include: the chromatographic column is Agilent Zorbax SB C and 18; the column temperature is 25-35 ℃; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is methanol; the flow rate of the mobile phase system is 0.4-0.6 mL/min; the sample injection amount is 5 mu L;
the elution mode is gradient elution, and the gradient elution comprises the following steps:
0.00 to 2.00min: the volume fraction of the mobile phase A is 35%;
2.00-4.50 min: the volume fraction of the mobile phase A is reduced from 35% to 0% at a constant speed;
4.50 to 6.50 minutes: the volume fraction of the mobile phase A is 0%;
6.50 to 6.51min: the volume fraction of the mobile phase A is increased from 0% to 35% at a constant speed;
6.51 to 8.50min: the volume fraction of mobile phase a was 35%.
Preferably, the aqueous formic acid solution has a volume concentration of 0.1%.
Preferably, the mass spectrum detection conditions of the high performance liquid chromatography tandem mass spectrum include: the ion source is an electrospray ion source; the scanning mode is negative ion scanning; the detection mode is a multi-reaction ion monitoring mode; the temperature of the ion source is 550 ℃; the ion spray voltage is-4.5 kV; the atomizing gas flow rate is 50psi; the air curtain air flow rate was 40psi.
Preferably, the mass spectrum parameters of the 2,4, 5-trifluoro-3-methoxybenzoic acid are as follows:
preferably, the concentration of moxifloxacin hydrochloride in the sample liquid to be detected is 90 mug/mL.
Preferably, the quantitative detection result is obtained by calculating an external standard method, wherein the external standard adopted by the external standard method is a 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance.
The invention adopts a high performance liquid chromatography-tandem mass spectrometry (LC-MC/MC) method to realize rapid qualitative and quantitative analysis of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride, and has the advantages of simplicity, convenience, rapidness and high sensitivity. The detection method provided by the invention can effectively control the quality of moxifloxacin hydrochloride products by detecting whether 2,4, 5-trifluoro-3-methoxybenzoic acid and the residual quantity thereof remain in moxifloxacin hydrochloride, improves the medication safety of moxifloxacin hydrochloride medicines, makes up the blank of the existing detection method for 2,4, 5-trifluoro-3-methoxybenzoic acid substances in moxifloxacin hydrochloride, and is worthy of popularization and application.
Drawings
FIG. 1 is a typical HPLC chromatogram of a sample addition solution;
FIG. 2 is a typical HPLC chromatogram of a blank solution;
FIG. 3 is a typical HPLC chromatogram of a 2,4, 5-trifluoro-3-methoxybenzoic acid control solution;
FIG. 4 is a typical HPLC chromatogram of a test solution;
FIG. 5 is a typical plot of linearity versus range for 2,4, 5-trifluoro-3-methoxybenzoic acid.
Detailed Description
The invention provides a detection method of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride, which comprises the following steps:
dissolving moxifloxacin hydrochloride in methanol to obtain a sample liquid to be tested;
detecting the sample liquid to be detected by adopting high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of 2,4, 5-trifluoro-3-methoxybenzoic acid; the detection result comprises a qualitative detection result and/or a quantitative detection result.
In the present invention, unless otherwise specified, the reagents used are commercially available products well known to those skilled in the art.
According to the invention, moxifloxacin hydrochloride is dissolved in methanol to obtain a sample liquid to be detected. In the invention, the concentration of moxifloxacin hydrochloride in the sample liquid to be detected is preferably 90 mug/mL.
After obtaining a sample liquid to be detected, the invention adopts high performance liquid chromatography-tandem mass spectrometry to detect the sample liquid to be detected, and a detection result of 2,4, 5-trifluoro-3-methoxybenzoic acid is obtained; the detection result comprises a qualitative detection result and/or a quantitative detection result.
In the invention, the high performance liquid chromatography tandem mass spectrometry detection comprises high performance liquid chromatography detection conditions and mass spectrometry detection conditions.
In the present invention, the conditions for detecting the high performance liquid chromatography include: the chromatographic column is preferably Agilent Zorbax SB C; the column temperature is preferably 25 to 35 ℃, more preferably 30 ℃; the mobile phase system is preferably a mobile phase A and a mobile phase B, the mobile phase A is preferably an aqueous formic acid solution, and the volume concentration of the aqueous formic acid solution is preferably 0.1%; the mobile phase B is preferably methanol; the flow rate of the mobile phase system is preferably 0.4-0.6 mL/min, more preferably 0.5mL/min; the sample injection amount is preferably 5 mu L; the elution mode is preferably gradient elution, and the procedure of the gradient elution is preferably as shown in table 1.
TABLE 1 gradient elution procedure
| Time (min) | Mobile phase a volume fraction (%) | Mobile phase B volume fraction (%) |
| 0.00 | 35 | 65 |
| 2.00 | 35 | 65 |
| 4.50 | 0 | 100 |
| 6.50 | 0 | 100 |
| 6.51 | 35 | 65 |
| 8.50 | 35 | 65 |
In the present invention, the mass spectrometry detection conditions include: the ion source is preferably an electrospray ion source (ESI source); the scanning mode is preferably anion scanning; the detection mode is preferably a multi-reaction ion monitoring mode (MRM); the ion source temperature is preferably 550 ℃; the ion spray voltage is preferably-4.5 kV; the atomizing gas flow rate is preferably 50psi; the air curtain air flow rate is preferably 40psi; the atomizing gas and the air curtain gas are preferably nitrogen. In the invention, the mass spectrum parameters and the ion channel of the object to be detected of the 2,4, 5-trifluoro-3-methoxybenzoic acid are shown in table 2.
Table 2 mass spectral parameters of 2,4, 5-trifluoro-3-methoxybenzoic acid
In the invention, the instrument used for high performance liquid chromatography-tandem mass spectrometry detection is preferably a high performance liquid chromatography-mass spectrometer, and is preferably LCMS-8045 (Shimadzu Japan).
In the present invention, the detection result includes a qualitative detection result and/or a quantitative detection result. In the invention, the quantitative detection result is preferably calculated by an external standard method, and the external standard adopted by the external standard method is preferably a 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance. In the present invention, the quantitative detection method preferably comprises the steps of: detecting 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance solution by high performance liquid chromatography tandem mass spectrometry to obtain reference substance peak area; detecting the sample liquid to be detected by high performance liquid chromatography-tandem mass spectrometry to obtain the peak area of the compound to be detected; and calculating according to the peak area of the reference substance and the peak area of the compound to be detected by adopting an external standard method to obtain a quantitative detection result of the 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride. In the invention, the 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance solution is preferably obtained by dissolving 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance in methanol, and the concentration of the 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance solution is preferably 2ng/mL. In the invention, the detection conditions of the 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance solution for high performance liquid chromatography tandem mass spectrometry are the same as those of the sample solution to be detected for high performance liquid chromatography tandem mass spectrometry, and are not repeated here.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
In an embodiment of the present invention, the specifications of specific instruments, materials and samples to be tested are as follows:
the instrument is specifically: high performance liquid chromatography-mass spectrometry (LC-MC/MC): shimadzu, LCMS-8045; MSA6.6S-0CE parts per million on the day (Sartorius company, germany); model AB265-S one ten thousandth analytical balance (METTLER toldo corporation, usa);
the material and the sample to be tested: 2,4, 5-trifluoro-3-methoxybenzoic acid control (source: shenzhen Heng Feng Mo, medical science and technology Co., ltd., lot: 20200501); moxifloxacin hydrochloride (source: shandong Lu anti medicine Co., ltd., lot number: 2201001, 2201002, 2201003); methanol (source: thermo Fisher, lot number: 218273); formic acid (source: national medicine, lot number: 20200928);
the conditions for high performance liquid chromatography detection in the LC-MC/MC detection are as follows: the column was Agilent Zorbax SB C (3.0X100 mm,3.5 μm); the column temperature is 30 ℃; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is 0.1v/v% formic acid aqueous solution; the mobile phase B is methanol; the flow rate of the mobile phase system is 0.5mL/min; the sample injection amount is 5 mu L; the elution mode was gradient elution, and the procedure of the gradient elution is shown in table 1.
The mass spectrum detection conditions are as follows: the ion source is an electrospray ion source (ESI source); the scanning mode is negative ion scanning; the detection mode is a multi-reaction ion monitoring mode (MRM); the temperature of the ion source is 550 ℃; the ion spray voltage is-4.5 kV; the atomizing gas flow rates were 50psi; the air curtain air flow rate is 40psi; the atomizing gas and the air curtain gas are nitrogen; the mass spectrum parameters and the ion channel of the object to be detected of the 2,4, 5-trifluoro-3-methoxybenzoic acid are shown in table 2.
Example 1
Preparing a reference substance solution: precisely weighing 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance, adding methanol for dissolution and dilution to obtain reference substance solution with the concentration of 2ng/mL.
Sample solution preparation: precisely weighing moxifloxacin hydrochloride, adding methanol for dissolution and quantitatively diluting to obtain a sample solution with the concentration of 90 mug/mL.
Precisely measuring 5 mu L of each of the reference substance solution and the test substance solution, respectively injecting into an LC-MC/MC instrument for LC-MC/MC detection, respectively recording chromatographic peak information of 2,4, 5-trifluoro-3-methoxybenzoic acid in the reference substance solution and the test substance solution, calculating the content of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride by an external standard method, and obtaining the results shown in Table 3.
TABLE 3 determination of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
| Moxifloxacin hydrochloride sample | 2,4, 5-trifluoro-3-methoxybenzoic acid content |
| 2201001 | Not detected |
| 2201002 | Not detected |
| 2201003 | Not detected |
Example 2
Methodological verification
1. Specificity verification
Control stock solution (1): precisely weighing 10mg of 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance, placing in a 10mL volumetric flask, adding methanol to dissolve and dilute to scale, shaking, and taking as reference substance stock solution.
Control stock solution (2): precisely measuring 1mL of reference substance stock solution (1) in a 100mL volumetric flask, diluting to a scale with methanol, shaking uniformly, precisely measuring 0.2mL of reference substance stock solution in the 100mL volumetric flask, diluting to the scale with methanol, shaking uniformly, and taking the reference substance stock solution as reference substance stock solution (2).
Control solution: precisely measuring 1mL of reference substance stock solution (2), placing in a 10mL volumetric flask, diluting to scale with methanol, and shaking to obtain reference substance solution.
Test solution: accurately weighing moxifloxacin hydrochloride 9mg, placing in a 10mL volumetric flask, adding methanol for dissolution and dilution to a scale, shaking uniformly, accurately weighing 1mL, placing in a 10mL volumetric flask, diluting to a scale with methanol, shaking uniformly, and taking as a sample solution.
Adding a standard solution to a test sample: accurately weighing moxifloxacin hydrochloride 9mg, placing in a 10mL volumetric flask, adding methanol for dissolution and dilution to scale, shaking uniformly, accurately weighing 1mL, placing in a 10mL volumetric flask, accurately adding 1mL reference substance stock solution (2), diluting to scale with methanol, shaking uniformly, and taking as a sample to be tested and adding a standard solution.
Precisely measuring 5 mu L of blank solution (methanol), reference substance solution, test substance solution and standard solution respectively, injecting into an LC-MC/MC instrument for LC-MC/MC detection, recording chromatograms, and obtaining results shown in Table 4 and figures 1-4, wherein figure 1 is a typical HPLC chromatogram of the test substance solution, figure 2 is a typical HPLC chromatogram of the blank solution, figure 3 is a typical HPLC chromatogram of the reference substance solution, and figure 4 is a typical HPLC chromatogram of the test substance solution.
TABLE 4 methodological-specific results of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
| Solution | Retention time (min) |
| Blank methanol | / |
| Reference substance solution | 3.09 |
| Test solution | / |
| Labeling test solution | 2.97 |
As can be seen from Table 4 and FIGS. 1 to 4, the methanol and the test solution did not interfere with the detection of 2,4, 5-trifluoro-3-methoxybenzoic acid.
2. Quantitative limit and detection limit
The detection Limit (LOD) and the quantification Limit (LOQ) are determined according to the signal-to-noise ratio method. 2,4, 5-trifluoro-3-methoxybenzoic acid control stock solution with known concentration is diluted, the concentration when S/N is approximately equal to 10 is taken as quantitative limit concentration, and the concentration when S/N is approximately equal to 3 is taken as detection limit concentration.
Precisely measuring 5 mu L of each quantitative limiting solution and 5 mu L of each detection limiting solution, respectively injecting the quantitative limiting solutions and the detection limiting solutions into an LC-MC/MC instrument for LC-MC/MC detection, and recording chromatograms, wherein the results are shown in tables 5-6.
TABLE 5 methodological-quantitative limits, detection limits for 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
TABLE 6 methodological-detection limit test results of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
As can be seen from the data in tables 5 to 6, the three-needle detection limit S/N is at least 3.1, the concentration is 0.06ng/mL, and the concentration corresponds to 3.1% of the limit concentration; the minimum S/N=10.6 in the six-needle quantitative limit is larger than 10, the concentration is 0.21ng/mL, which is equivalent to 10.3% of the limit concentration, and the RSD=1.9% of the peak area of the six-needle continuous quantitative limit is smaller than 20.0%, which indicates that the detection method provided by the invention has high sensitivity.
3. Linearity and range
The concentration of the test solution is 90 mug/mL, the limit of 2,4, 5-trifluoro-3-methoxybenzoic acid is 22ppm, and the limit concentration is 2ng/mL; the linear investigation range was set to a quantitative limit of 500% of the limit concentration.
Control stock solution (1): precisely weighing 10mg of 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance, placing in a 10mL volumetric flask, adding methanol to dissolve and dilute to scale, shaking, and taking as reference substance stock solution (1).
Linear stock solution: 1mL of the reference substance stock solution (1) is precisely measured and placed in a 100mL volumetric flask, methanol is added for dissolution and dilution to a scale, shaking is carried out, then 0.2mL of the reference substance stock solution is precisely measured and placed in the 100mL volumetric flask, methanol is used for dilution to the scale, shaking is carried out, and the reference substance stock solution is used as the linear stock solution.
Preparation of a linear solution: the control stock solution (2) was precisely measured according to Table 7 and placed in a volumetric flask, diluted to the scale with methanol, and shaken well as a linear solution.
TABLE 7 preparation of linear solutions of 2,4, 5-trifluoro-3-methoxybenzoic acid
Precisely measuring 5 mu L of each linear solution, respectively injecting the linear solutions into an LC-MC/MC instrument for LC-MC/MC detection, recording a chromatogram, plotting a linear relation according to a function of the measured peak area to the concentration, taking the concentration as an abscissa (x), taking the peak area as an ordinate (y), and carrying out linear regression, wherein the linear test results of the 2,4, 5-trifluoro-3-methoxybenzoic acid are shown in fig. 5 and table 8.
TABLE 8 Linear test results of 2,4, 5-trifluoro-3-methoxybenzoic acid
As is clear from Table 8, 2,4, 5-trifluoro-3-methoxybenzoic acid has good linear relationship in the concentration range of 0.2066-10.33 ng/mL, and the linear equation is y= 3.901 ×10 4 x+5.332×10 3 The correlation coefficient r= 0.9991 shows that the detection method provided by the invention has wide linear range and good linear relation.
4. Accuracy of
Control stock solution: precisely weighing 10mg of 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance, placing in a 10mL volumetric flask, adding methanol for dissolution and dilution to scale, shaking uniformly, precisely weighing 1mL, placing in a 100mL volumetric flask, diluting to scale with methanol, shaking uniformly, precisely weighing 0.2mL, placing in a 100mL volumetric flask, diluting to scale with methanol, shaking uniformly, and taking as reference substance stock solution.
Control solution: precisely measuring 1mL of reference substance stock solution, placing in a 10mL volumetric flask, diluting to scale with methanol, and shaking to obtain reference substance solution.
50% strength solution: accurately weighing 9mg of moxifloxacin raw material medicine, placing in a 10mL volumetric flask, adding methanol for dissolving and diluting to scale, shaking uniformly, accurately weighing 1mL, placing in a 10mL volumetric flask, accurately adding 0.5mL of reference substance stock solution, dissolving with methanol and diluting to scale, and shaking uniformly. 3 parts of a 50% strength solution was prepared in the same manner.
100% strength solution: accurately weighing 9mg of moxifloxacin raw material medicine, placing in a 10mL volumetric flask, adding methanol for dissolving and diluting to scale, shaking uniformly, accurately weighing 1mL, placing in a 10mL volumetric flask, accurately adding 1.0mL of reference substance stock solution, dissolving with methanol and diluting to scale, and shaking uniformly. 3 parts of a 100% strength solution was prepared by the same method.
150% strength solution: accurately weighing 9mg of moxifloxacin raw material medicine, placing in a 10mL volumetric flask, adding methanol for dissolving and diluting to scale, shaking uniformly, accurately weighing 1mL, placing in a 10mL volumetric flask, accurately adding 1.5mL of reference substance stock solution, dissolving with methanol and diluting to scale, and shaking uniformly. 3 parts of 150% strength solution were prepared in the same manner.
The control solution and the recovery solution were measured precisely at 5. Mu.L each, and the samples were injected into an LC-MC/MC instrument for LC-MC/MC detection, and the chromatograms were recorded, and the results are shown in Table 9.
TABLE 9 methodological-accuracy test results of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
As shown in Table 9, the recovery rate of 9 parts of recovery solution is 99.6-109.3%, the recovery rates are all in the range of 90-110%, the RSD values are 1.0-3.8%, and the RSD values are less than 10%, which shows that the detection method provided by the invention has high accuracy and meets the verification requirement.
5. Repeatability of
Control stock solution: precisely weighing about 10mg of 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance, placing in a 10mL volumetric flask, adding methanol for dissolution and dilution to scale, shaking uniformly, precisely weighing 1mL, placing in a 100mL volumetric flask, diluting to scale with methanol, shaking uniformly, precisely weighing 0.2mL, placing in a 100mL volumetric flask, diluting to scale with methanol, shaking uniformly, and taking as reference substance stock solution.
Control solution: precisely measuring 1mL of reference substance stock solution, placing in a 10mL volumetric flask, diluting to scale with methanol, and shaking to obtain reference substance solution.
Adding a standard solution to a test sample: accurately weighing 9mg of moxifloxacin raw material medicine, placing in a 10mL volumetric flask, adding methanol for dissolving and diluting to scale, shaking uniformly, accurately weighing 1mL, placing in the 10mL volumetric flask, accurately adding 1mL of reference substance stock solution, dissolving and diluting to scale with methanol, and shaking uniformly. 6 parts of sample adding and marking solution are prepared by adopting the same method.
Respectively precisely measuring 5 mu L of each of the reference substance solution and the sample adding standard solution, respectively injecting into an LC-MC/MC instrument for LC-MC/MC detection, and recording the chromatograms, wherein the results are shown in Table 10.
TABLE 10 methodological-repeatability test results of 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride
As shown in Table 10, the RSD of the measurement result of the 6-part labeled sample solution is 1.0% and less than 10%, which indicates that the detection method provided by the invention has good repeatability and high precision and meets the verification requirement.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. The method for detecting 2,4, 5-trifluoro-3-methoxybenzoic acid in moxifloxacin hydrochloride comprises the following steps:
dissolving moxifloxacin hydrochloride in methanol to obtain a sample liquid to be tested;
detecting the sample liquid to be detected by adopting high performance liquid chromatography-tandem mass spectrometry to obtain a detection result of 2,4, 5-trifluoro-3-methoxybenzoic acid; the detection result comprises a qualitative detection result and/or a quantitative detection result.
2. The method according to claim 1, wherein the hplc detection conditions of the hplc tandem mass spectrometry include: the chromatographic column is AgilentZorbax SB C and 18; the column temperature is 25-35 ℃; the mobile phase system comprises a mobile phase A and a mobile phase B, wherein the mobile phase A is a formic acid aqueous solution, and the mobile phase B is methanol; the flow rate of the mobile phase system is 0.4-0.6 mL/min; the sample injection amount is 5 mu L;
the elution mode is gradient elution, and the gradient elution comprises the following steps:
0.00 to 2.00min: the volume fraction of the mobile phase A is 35%;
2.00-4.50 min: the volume fraction of the mobile phase A is reduced from 35% to 0% at a constant speed;
4.50 to 6.50 minutes: the volume fraction of the mobile phase A is 0%;
6.50 to 6.51min: the volume fraction of the mobile phase A is increased from 0% to 35% at a constant speed;
6.51 to 8.50min: the volume fraction of mobile phase a was 35%.
3. The method according to claim 2, wherein the aqueous formic acid solution has a volume concentration of 0.1%.
4. The method according to claim 1, wherein the mass spectrometry detection conditions of the high performance liquid chromatography tandem mass spectrometry include: the ion source is an electrospray ion source; the scanning mode is negative ion scanning; the detection mode is a multi-reaction ion monitoring mode; the temperature of the ion source is 550 ℃; the ion spray voltage is-4.5 kV; the atomizing gas flow rate is 50psi; the air curtain air flow rate was 40psi.
5. The method according to claim 1 or 4, wherein the mass spectrum parameters of the 2,4, 5-trifluoro-3-methoxybenzoic acid are as follows:
6. the detection method according to claim 1, wherein the concentration of moxifloxacin hydrochloride in the sample liquid to be detected is 90 μg/mL.
7. The method according to claim 1, wherein the quantitative detection result is calculated by an external standard method, and the external standard adopted by the external standard method is a 2,4, 5-trifluoro-3-methoxybenzoic acid reference substance.
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