CN113092644B - Method for simultaneously detecting content of moxifloxacin and intermediate thereof - Google Patents

Method for simultaneously detecting content of moxifloxacin and intermediate thereof Download PDF

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CN113092644B
CN113092644B CN202110353858.6A CN202110353858A CN113092644B CN 113092644 B CN113092644 B CN 113092644B CN 202110353858 A CN202110353858 A CN 202110353858A CN 113092644 B CN113092644 B CN 113092644B
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moxifloxacin
solution
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small ring
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王�华
杨锦明
王庆东
丁惠
孔立
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Jiangsu Baju Pharmaceutical Co ltd
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The method for simultaneously detecting the content of moxifloxacin and the intermediate thereof comprises the steps of preparing a sample solution, preparing a reference substance solution, detecting by adopting an ultra-high performance reversed phase liquid chromatography/mass spectrometry method, and determining to obtain and calculate the content of moxifloxacin and the intermediate thereof in the sample. The invention adopts the ultra-high performance reversed phase liquid chromatography method, so that the analysis time is short; the chromatographic method uses a simple isocratic elution method, has low requirements on experimental equipment and is simple to operate; the mass spectrum scanning range is set to be m/z 105-1000, so that the solvent interference is avoided, and the method specificity is high. The method directly takes the extraction ion flow diagrams of moxifloxacin and moxifloxacin small rings as quantitative chromatograms, and has high sensitivity, good specificity and accurate quantification.

Description

Method for simultaneously detecting content of moxifloxacin and intermediate thereof
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for simultaneously determining moxifloxacin and an intermediate moxifloxacin small ring thereof.
Background
Moxifloxacin is a quinolone/fluoroquinolone broad-spectrum antibiotic that may be used to treat infections caused by the following bacteria: aerobic gram-positive microorganisms: corynebacterium genus, micrococcus luteus, staphylococcus aureus, staphylococcus epidermidis, staphylococcus hemolyticus, staphylococcus hominis, staphylococcus fahrenheit, streptococcus and streptococcus pneumoniae; aerobic gram-negative microorganisms: lwiffii acinetobacter, haemophilus influenzae and haemophilus parainfluenza; other microorganisms: chlamydia trachomatis. Moxifloxacin has bactericidal effect in that its molecule is bound to bacterial DNA gyrase, thus preventing bacterial DNA replication. Since DNA gyrase is a key enzyme for DNA unwinding, and moxifloxacin has a 100-fold higher affinity for bacterial DNA gyrase than for mammals, this is an important mechanism for its action.
The inspection of the moxifloxacin product with respect to the impurity items needs to be specifically targeted. The impurity inspection items in the drug standards should include impurities and degradation products of drugs detected in quality studies and stability studies, and occurring in mass production. The moxifloxacin impurity inspection reports that 5 impurities which are explicitly required to be detected, such as USP, EP and the like, are all byproducts in the moxifloxacin synthesis process. However, for the method for simultaneously detecting moxifloxacin and its intermediate (S, S) -2, 8-diazabicyclo [4,3,0] nonane (also called moxifloxacin small ring and moxifloxacin side chain), no report is shown, and it is difficult to obtain analysis in one method simultaneously because the detection method for synthesizing the intermediate moxifloxacin small ring and the moxifloxacin detection method generally use different analysis instruments.
The detection of moxifloxacin (molecular structure shown in formula 1) is generally performed by liquid chromatography, while the detection of intermediate (S, S) -2, 8-diazabicyclo [4,3,0] nonane (moxifloxacin small ring) (molecular structure shown in formula 2) is generally performed by gas chromatography. If the two are simultaneously subjected to liquid chromatography analysis, effective detection cannot be achieved because the moxifloxacin small ring has no ultraviolet absorption. Although there are also methods of attempting chemical derivatization of moxifloxacin small rings followed by liquid chromatography, there is no simple method of detecting both compounds simultaneously. The detection method has important significance for monitoring the production process of moxifloxacin and controlling the quality of products, and comprises the steps of saving time, saving reagents, reducing the operation difficulty, avoiding chemical reaction of a derivative method, monitoring the production process in real time, controlling the feeding of the products, increasing the yield and the like.
Figure BDA0003002933290000021
Disclosure of Invention
The invention aims to solve the problem of providing a rapid, simple, good-precision and high-specificity analysis method which is used for simultaneously measuring the content information of moxifloxacin and an intermediate moxifloxacin small ring thereof, namely (S, S) -2, 8-diazabicyclo [4,3,0] nonane (also known as moxifloxacin small ring and moxifloxacin side chain).
In order to solve the technical problems, the invention provides the following technical scheme:
a method for simultaneously detecting the content of moxifloxacin and an intermediate thereof, wherein the intermediate is a moxifloxacin small ring, and the method comprises the following steps:
A. preparing a test solution: dissolving a moxifloxacin sample to be detected in a mobile phase to obtain a moxifloxacin sample solution;
or dissolving a moxifloxacin sample to be detected by using a cosolvent to obtain an initial solution of the sample to be detected, and diluting the initial solution of the sample to be detected by using a mobile phase to obtain a moxifloxacin sample solution;
preferably, the cosolvent is pure methanol, acetonitrile, dimethyl sulfoxide (DMSO);
further preferably, the cosolvent is pure methanol;
B. preparing a reference substance solution:
the reference substance solution is a moxifloxacin single reference substance solution and a moxifloxacin small ring single reference substance solution; or the reference substance solution is a mixed reference substance solution of moxifloxacin and moxifloxacin small ring;
C. the detection is carried out by adopting an ultra-high performance reversed phase liquid chromatography/mass spectrometry combination method, and the detection conditions are as follows:
chromatographic column: an ultra-high performance reverse phase chromatographic column bonded with nonpolar octadecyl functional groups (ODS) with high water resistance;
mobile phase: formic acid, methanol and water in a volume ratio of 0.1:50:50, and isocratic eluting;
column temperature: 15-45 ℃;
mobile phase flow rate: 0.2-0.3mL/min;
the mass spectrum detection conditions are as follows:
ionization mode ESI + The method comprises the steps of carrying out a first treatment on the surface of the Ionization voltage +5kV; the atomizing gas stream is 55psi; auxiliary gas 55psi; the air curtain gas is 35psi; the temperature is 550 ℃; mass spectrometry scanning mode primary mass spectrometry scanning; the mass scanning range m/z is 105-1000;
analysis time: less than or equal to 2min;
D. and (3) measuring: and B, precisely measuring 1 mu l of the sample solution obtained in the step A and 1 mu l of the reference substance solution obtained in the step B, respectively injecting into a liquid chromatograph, obtaining a standard curve according to the result of the ultra-high performance reversed phase liquid chromatograph/mass spectrometer combined analysis of the reference substance solution, and calculating the content of moxifloxacin and intermediates thereof in the sample solution according to the standard curve.
Further, the moxifloxacin sample to be detected in the step A is a moxifloxacin crude product, a raw material or a preparation.
Further, when the moxifloxacin sample to be detected is a moxifloxacin crude product which is mutually dissolved with the flowing water, the moxifloxacin sample to be detected is dissolved by a mobile phase to obtain a moxifloxacin sample solution; in a particular embodiment, the crude product is a product solution of a condensation reaction step of a nonane side chain (moxifloxacin small ring) and a pre-intermediate in the synthesis process of moxifloxacin
When the moxifloxacin sample to be detected is a moxifloxacin raw material or preparation, dissolving the moxifloxacin sample with a cosolvent to obtain an initial solution of the sample to be detected, and diluting the initial solution of the sample to be detected with a mobile phase to obtain a moxifloxacin sample solution;
further, in step A, the moxifloxacin sample to be tested or the initial solution of the sample to be tested is quantitatively diluted step by using a mobile phase, and after shaking and mixing uniformly, a microporous filter membrane with the thickness of 0.22 mu m is used for filtering to prepare a moxifloxacin sample solution, preferably, the step by step quantitative dilution is 10 times based on the volume of the initial solution 4 ,10 5 ,10 6 Dilution step by step.
Further, in the step B,
the preparation method of the moxifloxacin single reference substance solution comprises the following steps: taking a moxifloxacin reference substance, dissolving the moxifloxacin reference substance in a cosolvent to obtain an initial solution of the moxifloxacin reference substance, and diluting the initial solution with a mobile phase to obtain a moxifloxacin reference substance solution;
the preparation method of the moxifloxacin small-ring single reference substance solution comprises the following steps: taking a moxifloxacin small ring reference substance, dissolving the moxifloxacin small ring reference substance in a cosolvent to obtain an initial solution of the moxifloxacin small ring reference substance, and diluting the initial solution with a mobile phase to obtain a moxifloxacin small ring reference substance solution;
the preparation method of the mixed reference substance solution of the moxifloxacin and the moxifloxacin small ring comprises the following steps: respectively taking a moxifloxacin reference substance and a moxifloxacin small ring reference substance, dissolving in a cosolvent to respectively obtain an initial solution of the moxifloxacin reference substance and an initial solution of the moxifloxacin small ring reference substance; mixing the initial solution of moxifloxacin reference substance and the initial solution of moxifloxacin small ring reference substance, diluting with mobile phase to obtain mixed reference substance solution,
preferably, moxifloxacin in the mixed reference solution: the moxifloxacin small ring concentration ratio is 10:1, a step of;
preferably, the cosolvent is pure methanol, acetonitrile, dimethyl sulfoxide (DMSO),
further preferably, the cosolvent is pure methanol.
Further, in the step A and the step B, the initial solution concentration is 10-50mg/mL.
Further, in the step B, the concentration range of moxifloxacin in the moxifloxacin control solution is 50ng/mL-500ng/mL;
preferably, the concentration range of moxifloxacin in the moxifloxacin control solution is 100ng/mL-500ng/mL;
further preferably, the concentration of moxifloxacin in the moxifloxacin control solution comprises one or more of 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL and 500 ng/mL.
Further, in the step B, the concentration range of moxifloxacin small ring in the moxifloxacin small ring reference substance solution is 5ng/mL-100ng/mL;
preferably, the concentration range of moxifloxacin small ring in the moxifloxacin small ring reference substance solution is 10ng/mL-100ng/mL;
further preferably, the concentration range of moxifloxacin small ring in the moxifloxacin small ring reference solution is 20ng/mL-100ng/mL;
still more preferably, the concentration of moxifloxacin small ring in the moxifloxacin small ring control solution comprises one, two or more of 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 80ng/mL and 100ng/mL.
Further, in step C, the specification of the chromatographic column is: inner diameter 2.1mm, length 50mm, filler C 18 Particle size 1.7 μm.
And D, calculating the content of moxifloxacin and an intermediate moxifloxacin small ring in the sample according to an external standard method and by using the peak area in a total ion flow diagram or an extraction ion flow diagram of a mass spectrum result. The method specifically comprises the following steps:
d-1: quantitatively measuring the series of reference substance solutions in the step B for liquid chromatography-mass spectrometry determination, taking the retention time of each moxifloxacin and moxifloxacin small ring in a total ion flow (TIC) chromatogram as a first specificity parameter, and taking the excimer ion peak ([ M+H) in the mass chromatogram corresponding to the retention time] + Compound proton addition peak) is a second qualitative parameter, and the chromatographic peaks of moxifloxacin and moxifloxacin small rings are determined;
d-2: excimer ion peak [ M+H ] of moxifloxacin small ring] + The peak area in an ion flow chromatogram (EIC) is a quantitative parameter, and a standard curve of moxifloxacin and a standard curve of a moxifloxacin small ring are calculated;
d-3: after standard curves of moxifloxacin and moxifloxacin small rings are obtained, measuring the sample solution in the step A, and measuring under the same conditions; and calculating the content of moxifloxacin and/or moxifloxacin small rings in the test sample according to an external standard method and peak area.
In the invention, all prepared sample solutions and control solutions are filtered by using a microporous filter membrane with the diameter of 0.22 mu m.
Compared with the prior art, the technical scheme of the invention has the following beneficial effects:
the adoption of the ultra-high performance reversed phase liquid chromatography method ensures that the analysis time is short; the chromatographic method uses a simple isocratic elution method, has low requirements on experimental equipment and is simple to operate; the mass spectrum scanning range is set to be m/z 105-1000, so that the solvent interference is avoided, and the method specificity is high. The method directly takes the extraction ion flow diagrams of moxifloxacin and moxifloxacin small rings as quantitative chromatograms, and has high sensitivity, good specificity and accurate quantification.
Drawings
FIG. 1 Total ion flow diagram (TIC) of moxifloxacin small ring and moxifloxacin mixed sample
FIG. 2 is a schematic diagram of an exemplary moxifloxacin small ring proton adduct ion extraction ion flow diagram
FIG. 3 representative Mass Spectrometry for Moxifloxacin small Ring
FIG. 4 is a schematic diagram of an exemplary moxifloxacin proton adduct ion extraction ion flow diagram
FIG. 5 representative Mass Spectrometry for moxifloxacin
FIG. 6 Standard regression curve of Moxifloxacin
FIG. 7 Standard regression curve of Moxifloxacin small Ring
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and all other embodiments obtained by those skilled in the art without making creative efforts based on the embodiments of the present invention are included in the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below.
The moxifloxacin control and moxifloxacin small ring control used in the examples can be obtained by commercial or self-made products, the purity is required to be higher than 98.0%, and the content of other single impurities is not required to be higher than 0.5%.
Example 1
1. Instrument and reagent
High performance liquid chromatograph (Agilent 1290 equipped with DAD detector, usa); analytical balance (sartorius bsa224S, certolisco instruments (beijing); ultrasonic cleaner (KQ 100DE type, kunshan ultrasonic instruments Co., ltd.). Moxifloxacin hydrochloride (CAS: 186826-86-8) control (> 99.5%, nanjing sea-tangle pharmaceutical limited); (s, s) -2, 8-diazabicyclo (4, 3, 0) nonane (moxifloxacin small ring, CAS: 151213-42-2) control (99.9%, hemsl, japan sea Biochemical technology Co., ltd.); methanol is a chromatographic grade; pure water was prepared by a pure water meter (Milli-QDirect 8, millipore, france) and the other reagents were analytically pure reagents.
2. Content detection of moxifloxacin and moxifloxacin small ring in sample
A. Preparing test article
According to the method of patent (CN 101941969A), in acetonitrile solvent, in the presence of organic alkali, precisely measuring nonane side chain (moxifloxacin small ring) and a pre-intermediate 1-cyclopropyl-6, 7-difluoro-8-methoxy-4-oxo-1, 4-dihydro-3-quinoline carboxylic acid in the moxifloxacin synthesis process, refluxing at 80 ℃ for 5 hours, taking a product solution of the reaction step as a sample to be detected, and using a mobile phase formic acid/methanol/water (0.1:50:50; v/v/v) solution according to 10 of the volume of the sample to be detected 4 ,10 5 ,10 6 Quantitatively diluting step by step, shaking and uniformly mixing, and filtering with a 0.22 mu m microporous filter membrane to prepare a sample to be tested.
B. Preparing standard solution
The preparation method of the reference substance solution B-1 is that the reference substance solution is a moxifloxacin single reference substance solution and a moxifloxacin small ring single reference substance solution, and the preparation method is as follows: commercial "moxifloxacin" controls were all provided as "moxifloxacin hydrochloride", which was dissolved in solution to dissociate Cheng Mo moxifloxacin and hydrochloric acid. Accurately weighing 21.8mg of moxifloxacin hydrochloride reference substance, which is equivalent to 20.0mg of moxifloxacin, and dissolving in 2mL of pure methanol in an ultrasonic-assisted manner to prepare an initial solution; the initial solution was diluted stepwise with a mobile phase formic acid/methanol/water (0.1:50:50; v/v/v) solution to give a series of solutions at concentrations of 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL, and filtered through a 0.22 μm microporous filter membrane to be tested.
Accurately weighing 20mg of moxifloxacin small-ring reference substance, diluting with pure methanol to 2mL, and preparing into an initial solution; the initial solution was diluted stepwise with a mobile phase formic acid/methanol/water (0.1:50:50; v/v/v) solution to give a series of solutions at 5ng/mL, 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL, 100ng/mL, and filtered through a 0.22 μm microporous filter membrane to be tested.
B-2, the preparation method of the reference substance solution which is a mixed reference substance solution of moxifloxacin and moxifloxacin small ring is as follows: taking initial concentrated solutions of two standard substances, and mixing moxifloxacin solutions in a volume ratio: moxifloxacin small ring solution = 10:1, and then gradually diluting with a mobile phase formic acid/methanol/water (0.1:50:50; v/v/v) solution to obtain 5 concentration levels of mixed standard solution. In each grade of mixed standard solution, the concentration of the moxifloxacin is sequentially 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL and 500ng/mL, and the concentration of the moxifloxacin small ring is sequentially 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL and 50ng/mL. The mixed standard solution is filtered by a microporous filter membrane with the diameter of 0.22 mu m, and is to be measured.
C. Sample analysis
ACQUITY UPLC BEH C is selected for chromatographic column 18 (2.1X105 mm,1.7 μm) (Waters, USA);
the mobile phase was formic acid/methanol/water (0.1:50:50; v/v/v) solution, isocratic elution.
The elution speed of the mobile phase is 0.2mL/min, the column temperature is 35 ℃, and the sample injection amount is 1.0 mu L.
The chromatographic time was 2.0min.
Mass spectra were TripleTOF 5600+ (AB Sciex, usa);
mass spectrometry conditions: ionization mode ESI + The method comprises the steps of carrying out a first treatment on the surface of the Ionization voltage +5kV; the atomizing gas stream is 55psi; auxiliary gas 55psi; the air curtain gas is 35psi; the temperature is 550 ℃; mass spectrometry scanning mode primary mass spectrometry scanning; the mass scanning range m/z is 105-1000.
D. The method is used for investigation and content measurement:
(1) The mixed sample (the mixed sample is the fourth grade mixed standard of the mixed reference substance solution in the step 2B-2, which comprises 400ng/mL of moxifloxacin and 40ng/mL of moxifloxacin small ring) is analyzed by the liquid chromatography-mass spectrometry method, and the chromatographic separation process is completed within 2 min. In the obtained total ion flow (TIC) chromatogram, the separation of moxifloxacin small ring and moxifloxacin above the base line is realized, the retention time of the moxifloxacin small ring is about 0.67min, and the peak shape is symmetrical; the retention time of the moxifloxacin is about 0.92min, and the peak shape is symmetrical; as shown in fig. 1.
(2) Extraction of M/z 127.08 ion Signal from Total ion flow graph (excimer ion peak of moxifloxacin Small Ring [ M+H ]] + ) And obtaining a moxifloxacin small ring proton addition ion flow diagram as shown in figure 2. The moxifloxacin small ring has symmetrical peak shape, high signal to noise ratio (S/N), good specificity, and the specificity can be confirmed by a mass spectrogram (figure 3) at the moment of 0.690 min.
(3) Extraction of M/z 402.06 ion Signal from Total ion flow graph (excimer ion peak of moxifloxacin [ M+H ]] + ) And obtaining a moxifloxacin proton addition ion flow diagram as shown in fig. 4. The moxifloxacin has symmetrical peak shape, high signal-to-noise ratio (S/N), good specificity, and the specificity can be confirmed by a mass spectrum (figure 5) at the time of 0.977 min.
The separation degree of the two compounds is high, the peak shape is good, and the signal to noise ratio is high, which indicates that the method has high specificity and satisfactory test result.
(4) Linear range investigation
Serial single control solutions with moxifloxacin concentration of 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500ng/mL were taken and analyzed sequentially. 1 μl of each standard solution was injected into the chromatograph, and the LC-MS analysis was repeated 3 times. Determining, recording and calculating the peak area in an extracted ion flow graph according to the method; and drawing a standard curve chart of moxifloxacin by taking the concentration C (ng/mL) as an abscissa and the corresponding 3 times peak area average value as an ordinate (figure 6). Through standard curve data calculation, a linear regression equation y=138.44x+1032.8 of moxifloxacin and a linear correlation coefficient R 2 =0.9997, indicating that the method has good linearity for moxifloxacin in the range of 100-500 ng/mL.
(5) Serial standard solutions with moxifloxacin small ring concentration of 20ng/mL, 40ng/mL, 60ng/mL, 80ng/mL and 100ng/mL are taken and analyzed sequentially. 1 μl of each standard solution was injected into the chromatograph, and the LC-MS analysis was performed, and the test was repeated 3 times. Determining, recording and calculating the peak area in an extracted ion flow graph according to the method; and (3) drawing a standard curve chart of the moxifloxacin small ring by taking the concentration C (ng/mL) as an abscissa and the corresponding 3 times of peak area mean value as an ordinate. Through standard curve data calculation, a linear regression equation y=7459.4x+39265 of the moxifloxacin small ring is shown in fig. 7, and a linear correlation coefficient R2= 0.9983 shows that the linear relation of the moxifloxacin small ring in the range of 20-100ng/mL is good.
(6) Detection of moxifloxacin small ring and moxifloxacin content in test sample
Respectively taking the dilution 10 in the step 2.A 4 ,10 5 ,10 6 Multiple 3 samples, each 1 μl, were injected into the liquid chromatograph and developed according to the method of the present invention.
Substituting the peak area in the extract ion flow (m/z 127.08) chromatogram of the moxifloxacin small ring into a linear regression equation of the moxifloxacin small ring, and calculating the content of the moxifloxacin small ring in the sample to be tested. Chromatographic results showed dilution 10 5 In the multiplied test sample, the moxifloxacin small ring content is within the linear range (20-100 ng/mL), 3 times of sample injection analysis are repeated, and the peak area average value is substituted into a linear regression equation, so that the moxifloxacin small ring concentration in the test sample is 32.62ng/mL. Diluting 10 with the sample to be tested as sampling sample 5 Calculated by doubling, the moxifloxacin small ring content in the sampled sample was 0.326%.
Substituting the peak area in the moxifloxacin extraction ion flow (m/z 402.26) chromatogram into a linear regression equation of moxifloxacin to obtain the content of moxifloxacin in the sample. Chromatographic results showed dilution 10 6 In the multiplied sample, the content of moxifloxacin is within the linear range (100-500 ng/mL). Repeating the sample injection analysis for 3 times, and calculating the content of moxifloxacin in the sampled sample to be 80.5%.
(7) Precision investigation
Taking a second grade mixed standard (moxifloxacin 200ng/mL and moxifloxacin small ring 20 ng/mL) of the mixed reference substance solution in the step 2B-2, injecting 1 mu L into a liquid chromatograph, continuously injecting the sample for 9 times, taking the peak areas of the 2 compounds, and calculating the average value and the Relative Standard Deviation (RSD) result shown in the table 1.
TABLE 1 precision test results (peak area)
Figure BDA0003002933290000081
Figure BDA0003002933290000091
The test result shows that under the detection method of the invention, the precision of the quantitative analysis result of the target compound is good.
(8) Inspection of recovery rate by adding marks
Taking a third-level mixed standard (300 ng/mL of moxifloxacin and 30ng/mL of moxifloxacin small ring) of the mixed reference substance solution in the step 2B-2, and simultaneously adding the single reference substance solution of moxifloxacin and moxifloxacin small ring prepared by the step B-1 to prepare a standard-added sample; the adding scalar of the moxifloxacin is 30ng/mL, and the adding scalar of the moxifloxacin small ring is 3ng/mL; according to the method of the invention, 5 determinations are performed in parallel; the measurement result is expressed by the following formula:
labeling recovery = (concentration measured by labeling sample-concentration measured by test article)/labeling quantity x 100%
Calculating the recovery rate; the recovery of the label, the average recovery of the label and the RSD value are obtained in 5 times in parallel, and the results are shown in Table 2.
TABLE 2 recovery test results
Figure BDA0003002933290000092
The test result shows that the detection method has good recovery rate and accurate and reliable detection result.
Example two
1. Instrument and reagent
As in the first embodiment.
2. Content detection of moxifloxacin and moxifloxacin small ring in sample
A. Preparing test article
Accurately weighing 20mg of moxifloxacin synthesized crude product (after drying) and dissolving in 2mL of methanol under the assistance of ultrasonic, and then gradually diluting with mobile phase formic acid/methanol/water (0.1:50:50; v/v/v) solution by 50000 times in volume multiple, and filtering with a 0.22 mu m microporous filter membrane to prepare a sample to be tested.
B. Preparing standard solution
As in the first embodiment.
C. Chromatography/mass spectrometry combined analysis steps
As in the first embodiment.
3. Detection of moxifloxacin small ring and moxifloxacin content in test sample
Taking the sample to be tested in the step 2.A, injecting 1 mu L into a liquid chromatograph, developing according to the method of the invention, and repeating the sample injection analysis for 5 times. Substituting the peak area in the moxifloxacin extraction ion flow (m/z 402.26) chromatogram into a linear regression equation of moxifloxacin (same as the embodiment I), and calculating the content of the folded moxifloxacin in the sample to be tested. 5 times of averaging, the result shows that the concentration of the folded moxifloxacin in the test sample is 197.44ng/mL (RSD=0.34%, n=5), namely the content of the moxifloxacin in the sampling sample (moxifloxacin synthetic crude product) is 98.72%.
In the extract ion flow (m/z 127.08) chromatogram of the moxifloxacin small ring, no obvious chromatographic peak is seen, so the moxifloxacin small ring in the moxifloxacin synthesized crude product is not detected.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and additions to the present invention may be made by those skilled in the art without departing from the principles of the present invention and such modifications and additions are to be considered as well as within the scope of the present invention.

Claims (19)

1. A method for simultaneously detecting the content of moxifloxacin and an intermediate thereof, which is characterized in that the intermediate is a moxifloxacin small ring, and the method comprises the following steps:
A. preparing a test solution: dissolving a moxifloxacin sample to be detected in a mobile phase to obtain a moxifloxacin sample solution;
or dissolving a moxifloxacin sample to be detected by using a cosolvent to obtain an initial solution of the sample to be detected, and diluting the initial solution of the sample to be detected by using a mobile phase to obtain a moxifloxacin sample solution;
the cosolvent is pure methanol, acetonitrile and dimethyl sulfoxide (DMSO);
B. preparing a reference substance solution: the reference substance solution is a moxifloxacin single reference substance solution and a moxifloxacin small ring single reference substance solution; or the reference substance solution is a mixed reference substance solution of moxifloxacin and moxifloxacin small ring;
C. the detection is carried out by adopting an ultra-high performance reversed phase liquid chromatography/mass spectrometry combination method, and the detection conditions are as follows:
chromatographic column: an ultra-high performance reverse phase chromatographic column with high water resistance and bonded nonpolar octadecyl functional groups;
mobile phase: formic acid, methanol and water in a volume ratio of 0.1:50:50, and isocratic eluting;
column temperature: 15-45 ℃;
mobile phase flow rate: 0.2-0.3mL/min;
chromatographic analysis time: less than or equal to 2min;
the mass spectrum detection conditions are as follows:
ionization mode ESI + The method comprises the steps of carrying out a first treatment on the surface of the Ionization voltage +5kV; the atomizing gas stream is 55psi; auxiliary gas 55psi; the air curtain gas is 35psi; the temperature is 550 ℃; mass spectrometry scanning mode primary mass spectrometry scanning; the mass scanning range m/z is 105-1000;
D. and (3) measuring: precisely measuring 1 mu l of the sample solution obtained in the step A and 1 mu l of the reference substance solution obtained in the step B, respectively injecting into a chromatograph, obtaining a standard curve according to the result of the ultra-high performance reversed phase liquid chromatography/mass spectrometry analysis of the reference substance solution, and calculating the content of moxifloxacin and intermediates thereof in the sample solution according to the standard curve.
2. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 1, wherein the cosolvent in the step a is pure methanol.
3. The method for simultaneously detecting moxifloxacin and an intermediate content thereof according to claim 1, wherein the moxifloxacin sample to be detected in the step a is a crude moxifloxacin product, a raw material or a preparation.
4. The method for simultaneously detecting moxifloxacin and an intermediate content thereof according to claim 1, wherein in the step A, the moxifloxacin sample to be detected or an initial solution of the moxifloxacin sample to be detected is quantitatively diluted step by using a mobile phase, and after shaking and mixing, a 0.22 μm microporous filter membrane is used for filtering to prepare a moxifloxacin sample solution.
5. The method for simultaneous detection of moxifloxacin and its intermediates according to claim 4, wherein in step a the stepwise quantitative dilution is 10% by volume of the initial solution 4 ,10 5 ,10 6 Dilution step by step.
6. The method for simultaneous detection of moxifloxacin and an intermediate thereof according to claim 1, wherein in step B,
the preparation method of the moxifloxacin single reference substance solution comprises the following steps: taking a moxifloxacin reference substance, dissolving the moxifloxacin reference substance in a cosolvent to obtain an initial solution of the moxifloxacin reference substance, and diluting the initial solution with a mobile phase to obtain a moxifloxacin reference substance solution;
the preparation method of the moxifloxacin small-ring single reference substance solution comprises the following steps: taking a moxifloxacin small ring reference substance, dissolving the moxifloxacin small ring reference substance in a cosolvent to obtain an initial solution of the moxifloxacin small ring reference substance, and diluting the initial solution with a mobile phase to obtain a moxifloxacin small ring reference substance solution;
the preparation method of the mixed reference substance solution of the moxifloxacin and the moxifloxacin small ring comprises the following steps: respectively taking a moxifloxacin reference substance and a moxifloxacin small ring reference substance, dissolving in a cosolvent to respectively obtain an initial solution of the moxifloxacin reference substance and an initial solution of the moxifloxacin small ring reference substance; mixing the initial solution of moxifloxacin reference substance and the initial solution of moxifloxacin small ring reference substance, and diluting with mobile phase to obtain mixed reference substance solution.
7. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 6, wherein the moxifloxacin in the mixed reference solution: the moxifloxacin small ring concentration ratio is 10:1.
8. the method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 6, wherein the cosolvent is pure methanol, acetonitrile, dimethyl sulfoxide (DMSO).
9. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 8, wherein the cosolvent is pure methanol.
10. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 1, wherein the initial solution concentration in step a and step B is 10-50mg/mL.
11. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 1, wherein in the step B, the concentration of the moxifloxacin reference solution ranges from 50ng/mL to 500 ng/mL.
12. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 11, wherein in the step B, the concentration of the moxifloxacin reference solution ranges from 100ng/mL to 500 ng/mL.
13. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 12, wherein in the step B, the concentration of the moxifloxacin control solution comprises one, two or more of 50ng/mL, 100ng/mL, 200ng/mL, 300ng/mL, 400ng/mL, 500 ng/mL.
14. The method for simultaneously detecting moxifloxacin and an intermediate content thereof according to claim 1, wherein in the step B, the concentration of the moxifloxacin small-ring reference solution ranges from 5ng/mL to 100ng/mL.
15. The method for simultaneously detecting moxifloxacin and an intermediate content thereof according to claim 14, wherein in the step B, the concentration of the moxifloxacin small-ring reference solution ranges from 10ng/mL to 100ng/mL.
16. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 15, wherein in the step B, the concentration of the moxifloxacin small-ring reference solution ranges from 20ng/mL to 100ng/mL.
17. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 16, wherein in the step B, the concentration of the moxifloxacin small-ring control solution comprises one, two or more of 5ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 80ng/mL, 100ng/mL.
18. The method for simultaneously detecting moxifloxacin and an intermediate thereof according to claim 1, wherein in step C, the specification of the chromatographic column is: inner diameter 2.1mm, length 50mm, filler C 18 Particle size 1.7 μm.
19. The method for simultaneously detecting the content of moxifloxacin and an intermediate thereof according to claim 1, wherein the step D specifically comprises the following steps:
d-1: quantitatively measuring the series of reference substance solutions in the step B for liquid chromatography-mass spectrometry determination, wherein the retention time of each moxifloxacin and moxifloxacin small ring in the total ion flow chromatogram is taken as a first definite parameter, and the excimer ion peak [ M+H ] in the mass chromatogram corresponding to the retention time is taken as a first definite parameter] + Determining chromatographic peaks of moxifloxacin and moxifloxacin small rings as second qualitative parameters;
d-2: excimer ion peak [ M+H ] of moxifloxacin small ring] + The peak area in the ion flow chromatogram is a quantitative parameter, and a standard curve of moxifloxacin and a standard curve of a moxifloxacin small ring are calculated;
d-3: after standard curves of moxifloxacin and moxifloxacin small rings are obtained, measuring the sample solution in the step A, and measuring under the same conditions; and calculating the content of moxifloxacin and/or moxifloxacin small rings in the test sample according to an external standard method and peak area.
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