CN115792047A - Method for detecting tedizolid phosphate intermediate related substances - Google Patents
Method for detecting tedizolid phosphate intermediate related substances Download PDFInfo
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- CN115792047A CN115792047A CN202310098290.7A CN202310098290A CN115792047A CN 115792047 A CN115792047 A CN 115792047A CN 202310098290 A CN202310098290 A CN 202310098290A CN 115792047 A CN115792047 A CN 115792047A
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- 229960003947 tedizolid phosphate Drugs 0.000 title claims abstract description 85
- QCGUSIANLFXSGE-GFCCVEGCSA-N tedizolid phosphate Chemical compound CN1N=NC(C=2N=CC(=CC=2)C=2C(=CC(=CC=2)N2C(O[C@@H](COP(O)(O)=O)C2)=O)F)=N1 QCGUSIANLFXSGE-GFCCVEGCSA-N 0.000 title claims abstract description 85
- 239000000126 substance Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 35
- JANKGNBDRWYWSN-UHFFFAOYSA-N 5-bromo-2-(2-methyltetrazol-5-yl)pyridine Chemical group CN1N=NC(C=2N=CC(Br)=CC=2)=N1 JANKGNBDRWYWSN-UHFFFAOYSA-N 0.000 claims abstract description 18
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 63
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- 238000012937 correction Methods 0.000 claims description 14
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- 238000002360 preparation method Methods 0.000 claims description 13
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- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 claims description 10
- 239000007864 aqueous solution Substances 0.000 claims description 9
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 6
- VOEFRGFXMMXFGK-UHFFFAOYSA-N 2-(2-methyltetrazol-5-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridine Chemical group CN1N=NC(C=2N=CC(=CC=2)B2OC(C)(C)C(C)(C)O2)=N1 VOEFRGFXMMXFGK-UHFFFAOYSA-N 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 4
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- 150000007522 mineralic acids Chemical class 0.000 claims description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 2
- 239000000377 silicon dioxide Substances 0.000 claims description 2
- 238000000691 measurement method Methods 0.000 claims 1
- 239000012535 impurity Substances 0.000 abstract description 77
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 abstract description 16
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- -1 2-methyl-2H-tetrazole-5-yl Chemical group 0.000 abstract description 8
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 abstract description 8
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- UAXDUQWXEJLTSR-UTONKHPSSA-N (5r)-3-[3-fluoro-4-[6-(2-methyltetrazol-5-yl)pyridin-3-yl]phenyl]-5-(hydroxymethyl)-1,3-oxazolidin-2-one;phosphoric acid Chemical group OP(O)(O)=O.CN1N=NC(C=2N=CC(=CC=2)C=2C(=CC(=CC=2)N2C(O[C@@H](CO)C2)=O)F)=N1 UAXDUQWXEJLTSR-UTONKHPSSA-N 0.000 description 1
- IZXIZTKNFFYFOF-UHFFFAOYSA-N 2-Oxazolidone Chemical compound O=C1NCCO1 IZXIZTKNFFYFOF-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention belongs to the field of pharmaceutical analysis, and particularly relates to a method for detecting a tedizolid phosphate intermediate related substance, wherein the tedizolid phosphate intermediate is a 2- (2-methyl-2H-tetrazole-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine composition, and the related substance is 5-bromo-2- (2-methyl-2H-tetrazole-5-yl) pyridine. The invention can effectively separate the tedizolid phosphate intermediate from related substances of 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine through specific solvents and chromatographic conditions, is used for detecting the impurities in the tedizolid phosphate intermediate and accurately measuring the content of the process impurity of 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine in the tedizolid phosphate intermediate drug, and has stable and reliable determination result and strong specificity.
Description
Technical Field
The invention belongs to the field of drug analysis, and particularly relates to a method for detecting substances related to a tedizolid phosphate intermediate.
Background
Tedizolid phosphate is a novel oxazolidinone antibiotic developed by Cubist pharmaceutical company, approved by FDA in 2014 for marketing, and is clinically used for treating acute bacterial skin and skin structure infections. Tedizolid phosphate is a prodrug which can be rapidly converted into biologically active tedizolid by phosphatase in vivo, thereby inhibiting protein synthesis. The action mechanism of the antibiotic is different from other antibiotics, and the antibiotic does not have cross drug resistance with other antibiotics. The traditional Chinese medicine composition is mainly used for treating acute bacterial skin and skin structure infection caused by gram-positive bacteria such as staphylococcus aureus (including methicillin-resistant strains and methicillin-sensitive strains) and various streptococcus and enterococcus faecalis.
The chemical name of the tedizolid phosphate is (R) -3- (4- (2- (2-methyltetrazol-5-yl) pyridine-5-yl) -3-fluorophenyl) -5-hydroxymethyl oxazolidin-2-one phosphate, and the molecular formula is C 17 H 16 FN 6 O 6 P, the structural formula is as follows:
the substance with the chemical name of 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine is a key intermediate for synthesizing tedizolid phosphate bulk drug, and in order to ensure the quality of the tedizolid phosphate API, the content of impurities in the intermediate 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine needs to be controlled so as to prevent the impurities contained in the intermediate from being too large and participating in the next reaction to form side products to cause the overproof of the related substances of the tedizolid phosphate API.
The chemical structural formula of the tedizolid phosphate intermediate 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine is as follows:
the chemical structure of the tedizolid phosphate intermediate and the synthesis process thereof are analyzed to find that: the substance with the chemical name of 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine is a main process impurity (impurity E for short) of the tedizolid phosphate intermediate, the impurity E is likely to remain in the process, an analysis and detection method for the impurity E is developed, qualitative and quantitative analysis is carried out, the quality of the tedizolid phosphate intermediate can be strictly controlled, and therefore the quality of the product tedizolid phosphate API is guaranteed to meet requirements.
Disclosure of Invention
In order to effectively control the quality of tedizolid phosphate, the invention provides a 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine composition which contains 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine with the mass ratio of not less than 99.7% and not more than 0.3% of 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine.
Further, the 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine content is not higher than 0.1%.
The invention also provides a preparation process of tedizolid phosphate, which is prepared by taking the 2- (2-methyl-2H-tetrazole-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine composition as an intermediate.
The invention also provides a detection method of the tedizolid phosphate intermediate related substances, which adopts high performance liquid chromatography for detection and comprises the following specific steps:
a. preparing a test solution: adding acetonitrile aqueous solution to dissolve a tedizolid phosphate intermediate to be detected to obtain the intermediate;
b. preparation of system suitability solution: adding acetonitrile aqueous solution into related substance reference substance to dissolve to obtain related substance solution; dissolving a tedizolid phosphate intermediate reference substance with a related substance solution, adding an acetonitrile aqueous solution, and uniformly mixing to obtain the tedizolid phosphate intermediate reference substance;
c. respectively sucking a test solution and a system applicability solution, and injecting the test solution and the system applicability solution into a chromatograph under the following chromatographic conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking 0.02 to 0.08 percent of inorganic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution procedure was:
| time/min | Mobile phase B/%) | Mobile phase A/%) |
| 0 | 10 | 90 |
| 15 | 75 | 25 |
| 20 | 75 | 25 |
| 21 | 10 | 90 |
| 33 | 10 | 90 |
Further, the mass volume ratio of the tedizolid phosphate intermediate to be detected in the step a) to the acetonitrile water solution is 0.5 to 2.5mg:1ml.
Further, the volume ratio of acetonitrile to water in the acetonitrile water solution is 60:40.
further, the system applicability solution in the step b) contains 0.01 to 0.1mg of the substances and 0.5 to 1.5mg of the tedizolid phosphate intermediate in each 1ml.
Still further, the tedizolid phosphate intermediate is 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine; the related substance is 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine.
Further, the chromatographic column is Agilent 5 TC-C18 (2), the specification is 4.6 multiplied by 250mm,5 μm; the mobile phase A is 0.05 percent of perchloric acid aqueous solution in v/v.
Further, the detection wavelength in the step c) is 254nm, the sample injection amount is 10ul, and the flow rate is 0.5-1.5 ml/min; the column temperature is 30 to 40 ℃, and preferably: the flow rate was 1.0ml/min and the column temperature was 30 ℃.
Furthermore, a chromatogram of the test solution shows a chromatographic peak corresponding to the retention time of a chromatographic peak of a related substance in the system applicability solution chromatogram, namely the test solution contains the corresponding related substance.
The invention also provides a method for measuring the content of the related substances of the tedizolid phosphate intermediate, which comprises the following specific operation steps:
1) Detecting according to the method;
2) The content of the related substances is calculated, the calculation formula is shown as figure 9, in figure 9,x ⅰ %：the content of the relevant substances in the test sample,A ⅰ : the peak area of the relevant substance in the test solution,A: the sum of all peak areas in the test solution,Fcorrection factors for the relevant substances;
further, the substance of interest is 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine with a correction factor of 0.57.
According to the method for detecting the related substances of the tedizolid phosphate intermediate, the tedizolid phosphate intermediate and the related substance 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine can be effectively separated through a specific solvent and a specific chromatographic condition, the method is used for detecting the impurity in the tedizolid phosphate intermediate and accurately measuring the content of the process impurity 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine in the tedizolid phosphate intermediate drug, and the detection result is stable and reliable and has strong specificity.
Proved by methodology verification, the invention meets the requirement of 'Chinese pharmacopoeia' 2020 edition on analysis methodology. The detection method can effectively monitor the synthesis process of the tedizolid phosphate intermediate, is used for quality detection of the tedizolid phosphate intermediate, ensures the quality of the tedizolid phosphate intermediate, further ensures the quality safety of the medicine tedizolid phosphate, and has wide application prospect.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 HNMR spectra of a tedizolid phosphate intermediate, 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine control;
FIG. 2 MS spectrum of tedizolid phosphate intermediate 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine control;
FIG. 3 HPLC purity profile of the tedizolid phosphate intermediate 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine control;
FIG. 4 is a HPLC plot of a suitable solution of a tedizolid phosphate intermediate system;
FIG. 5 HPLC chart of the sample solution of example 1;
FIG. 6 HPLC chart of the sample solution of example 2;
FIG. 7 HPLC chart of blank solution;
FIG. 8 HPLC plot of a localized solution of impurity E;
FIG. 9 is a graph of a calculation formula for the content of a substance;
FIG. 10 is a graph of the results of the evaluation of the system suitability;
FIG. 11 is a graph of the results of a specificity experiment;
FIG. 12 is a graph of quantitation limit and detection limit test results for impurity E;
FIG. 13 is a plot of the linearity versus range test results for impurity E;
FIG. 14 is a graph of the results of the linearity and range test of the tedizolid phosphate intermediate;
FIG. 15 is a graph of the substance correction factor;
FIG. 16 is a graph of repeatability test results;
FIG. 17 is a graph of intermediate precision;
FIG. 18 is a graph showing the recovery rate of impurity E.
Detailed Description
(1) Principal material
Tedizolid phosphate intermediate samples: homemade, lot numbers 220401, 220402;
tedizolid phosphate intermediate control: homemade, lot No. 210101 (tedizolid intermediate 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl) pyridine control HNMR spectrum is shown in figure 1, MS spectrum is shown in figure 2, HPLC purity spectrum is shown in figure 3).
Impurity E control: heilongjiang Hao Yun Yao pharmaceutical Co., ltd, lot number 210501. Impurity E control was 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine.
(2) Main instrument
High performance liquid chromatograph: shimadzu LC-20AT;
a chromatographic column: agilent 5 TC-C18 (2), specification 4.6X 250mm,5 μm.
Example 1 determination of impurity E in tedizolid phosphate intermediate sample
(1) Preparation of a test solution: a tedizolid phosphate intermediate sample (batch number 220401) is precisely weighed and placed in a volumetric flask, and the volume ratio of the sample to the intermediate sample is 60:40 of acetonitrile and water, and preparing a 1.0mg/ml tedizolid phosphate intermediate test solution.
(2) Preparation of system suitability solution: precisely weighing an impurity E reference substance, adding the reference substance in a volume ratio of 60:40 of acetonitrile and water was dissolved to prepare a solution having a concentration of 1.0mg/ml as an impurity E stock solution. Precisely weighing 25mg of the tedizolid phosphate intermediate reference substance into a 25ml volumetric flask, precisely adding 0.25ml of impurity E stock solution, and mixing the reference substance and the impurity E stock solution according to a volume ratio of 60: and (4) fixing the volume of the mixed solution of the acetonitrile and the water to obtain the system applicability solution.
(3) Chromatographic conditions are as follows:
a chromatographic column: agilent 5 TC-C18 (2), specification 4.6X 250mm,5 μm;
mobile phase: a mobile phase A:0.05% aqueous perchloric acid; mobile phase B: acetonitrile; detection wavelength: 254nm;
column oven: 30 ℃;
flow rate of the chromatographic column: 1ml/min, the sample volume is 10 mul;
gradient elution conditions:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0 | 10 | 90 |
| 15 | 75 | 25 |
| 20 | 75 | 25 |
| 21 | 10 | 90 |
| 33 | 10 | 90 |
(4) Respectively and precisely absorbing 10 mu l of the test solution and the system applicability solution, injecting the test solution and the system applicability solution into a chromatograph, recording a chromatogram, and calculating the content of the impurity E in the test solution according to a formula in figure 9 by an area normalization method with correction factors;A ⅰ : the peak area value of the impurity E in the test solution,A: the sum of all peak areas in the test solution,F: the correction factor for impurity E was 0.57.
x ⅰ % =0.0%, i.e. 0.0% of impurity E in tedizolid phosphate intermediate sample lot No. 220401. The HPLC chromatogram of this sample solution is shown in FIG. 5.
Example 2 determination of impurity E in tedizolid phosphate intermediate sample
(1) Preparation of a test solution: precisely weighing a tedizolid phosphate intermediate sample (batch number 220402) and placing the tedizolid phosphate intermediate sample in a volumetric flask, adding a volume ratio of 60:40 of acetonitrile and water to prepare a 1.0mg/ml tedizolid phosphate intermediate test solution.
(2) Preparation of system suitability solution: precisely weighing an impurity E reference substance, adding the reference substance in a volume ratio of 60:40 of acetonitrile and water was dissolved to prepare a solution having a concentration of 1.0mg/ml as an impurity E stock solution. Precisely weighing 25mg of tedizolid phosphate intermediate reference substance into a 25ml volumetric flask, precisely adding 0.25ml of impurity E stock solution, and mixing the reference substance and the impurity E stock solution according to a volume ratio of 60: and (4) fixing the volume of the mixed solution of acetonitrile and water to 40 to obtain the system applicability solution.
(3) Chromatographic conditions are as follows:
a chromatographic column: agilent 5 TC-C18 (2), specification 4.6X 250mm,5 μm;
mobile phase: mobile phase A:0.05% aqueous perchloric acid; mobile phase B: acetonitrile; detection wavelength: 254nm;
column oven: 30 ℃;
flow rate of the chromatographic column: 1ml/min, and the sample injection amount is 10 mu l;
gradient elution conditions:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0 | 10 | 90 |
| 15 | 75 | 25 |
| 20 | 75 | 25 |
| 21 | 10 | 90 |
| 33 | 10 | 90 |
(4) Respectively and precisely absorbing 10 mu l of the test solution and the system applicability solution, injecting the test solution and the system applicability solution into a chromatograph, recording a chromatogram, and calculating the content of the impurity E in the test solution according to a formula in figure 9 by an area normalization method with a correction factor;A ⅰ : the peak area value of the impurity E in the test solution,A: sum of all peak areas in the test solution, F: the correction factor for impurity E was 0.57.
x ⅰ % =0.05%, i.e. 0.05% of impurity E in tedizolid phosphate intermediate sample lot No. 220402. The HPLC chart of this test solution is shown in FIG. 6.
The beneficial effects of the invention are further illustrated by the following test examples:
test example 1 evaluation of System applicability
Instrument and chromatographic conditions: shimadzu LC-20AT; a chromatographic column: agilent 5 TC-C18 (2), specification 4.6X 250mm,5 μm; flow rate: 1.0ml/min; temperature of the column: 30 ℃; the sample amount is 10 mul; detector wavelength: 254nm; mobile phase A:0.05% aqueous perchloric acid, mobile phase B: acetonitrile; gradient elution conditions:
| time (min) | Mobile phase B (%) | Mobile phase A (%) |
| 0 | 10 | 90 |
| 15 | 75 | 25 |
| 20 | 75 | 25 |
| 21 | 10 | 90 |
| 33 | 10 | 90 |
Solution preparation:
(1) The blank solution (the following dilutions) was: mixing acetonitrile and water according to a volume ratio of 60;
(2) Impurity E stock solution: precisely weighing 25mg of the impurity E reference substance into a 25ml volumetric flask, adding the reference substance in a volume ratio of 60: dissolving 40 parts of mixed solution of acetonitrile and water, diluting to a scale, and shaking up to obtain an impurity E stock solution, wherein the concentration of an impurity E reference substance is 1.0mg/ml; the reference substance of the impurity E is a reference substance of 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine;
(3) System applicability solution: precisely weighing 25mg of the tedizolid phosphate intermediate reference substance into a 25ml volumetric flask, precisely adding 0.25ml of impurity E stock solution, and then adding a diluent to dilute to scale to obtain a system applicability solution (the concentration of the tedizolid phosphate intermediate reference substance in the system applicability solution is 1.0mg/ml, and the concentration of the impurity E reference substance is 10 mu g/ml);
(4) Impurity E localization solution: precisely transferring 1.0ml of impurity stock solution into a 10ml volumetric flask, adding a diluent to dissolve and dilute the impurity stock solution to a scale to obtain an impurity E positioning solution, wherein the concentration of an impurity E reference substance is 100 mu g/ml;
(5) Test solution: precisely weighing 25mg of tedizolid phosphate intermediate sample (batch number 220401) in a 25ml volumetric flask, adding a diluent to dissolve and diluting to a scale mark to obtain a test solution.
And (3) detection: respectively injecting blank solution, impurity E positioning solution, system applicability solution and sample solution according to the above chromatographic conditions, and recording the chromatographic process, wherein the HPLC chart of the system applicability solution is shown in FIG. 4, and FIG. 7 is the HPLC chart of the blank solution; FIG. 8 is an HPLC plot of a localized solution of impurity E; the sample solution was the same as that in example 1, and its chromatogram was as shown in FIG. 5, and the results of degrees of separation were shown in FIG. 10.
The experimental results show that: the blank is free of interference, the separation degree of the solution with the system applicability is 10.91, the separation degree is far more than 2.0, and the requirements of China pharmacopoeia 2020 edition are met. The method can well separate the tedizolid phosphate intermediate and the impurity E thereof.
Test example 2, specificity test
The apparatus, the chromatographic conditions and the solution preparation method were the same as those in test example 1.
And (3) detection: the method specifically inspects peak identification and selectivity, samples blank solution and impurity E positioning solution respectively according to the chromatographic conditions, records chromatogram, and the result is shown in figure 11.
The experimental results show that: the detection method has no interference of other impurities, and can be used for identifying the peak of the impurity E5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine.
Test example 3, detection Limit and quantitation Limit test
The apparatus, chromatographic conditions and solution preparation method were the same as in test example 1.
And (3) detection: the limit of detection (LOD) and limit of quantitation (LOQ) are determined from the signal-to-noise ratio. Diluting the stock solution of the impurity E with known concentration to low concentration for sample injection, determining that the detection signal-to-noise ratio S/N is more than or equal to 10 as a quantitative limit, and determining that S/N = 2-4 as a detection limit. The test results are shown in FIG. 12.
The experimental results show that: the quantitative limit and the detection limit of the impurity E are far less than 0.1 percent, meet the requirements of universal regulation of international pharmacopoeia (more than 0.1 percent of the impurity should be quantified), and can be used for qualitative and quantitative analysis of the impurity in the tedizolid phosphate intermediate.
Test example 4, linearity and Range testing
The apparatus, the chromatographic conditions and the solution preparation method were the same as those in test example 1.
And (3) detection: for impurity E, 6 points are studied in the range from the LOQ concentration to the index concentration of not less than 150%, the linear relationship is plotted as a function of the measured response signal (peak area) against the concentration of the analyte (i.e., impurity E control), linear regression is performed using the least squares method, and at least the correlation coefficient is reportedR 2 To confirm a good linear relationship, the linear regression coefficients are requiredR 2 Should not be less than 0.990.
The results are shown in FIGS. 13 to 15 for the linearity and range of the main component tedizolid phosphate intermediate.
The experimental results show that: the impurity E has good linearity in the range of 0.1202 mu g/ml-2.0134 mu g/ml, the measured correction factor of the impurity E is 0.57 and is in the range of 0.2-5.0 (since the correction factor is not in the range of 0.9-1.1, the correction factor needs to be added for correction), so the content of the impurity E can be calculated by an area normalization method with the addition of the correction factor.
Test example 5 measurement of precision
The apparatus and conditions and the solution preparation method were the same as those in test example 1.
And (3) detection: and adding a standard sample solution of 0.1 percent of impurity E into the tedizolid phosphate intermediate sample as a test solution, preparing 6 parts in parallel, sequentially injecting samples according to the conditions, and obtaining a repeatability result shown in figure 16.
Intermediate precisions were performed by the same procedure, by different personnel on different instruments, and the results are shown in FIG. 17.
The experimental results show that: the repeatability of the impurity E and the RSD value of the intermediate precision are less than 2.0 percent, and the detection requirements of the international pharmacopoeia and the Chinese pharmacopoeia are met.
Test example 6 test of accuracy
The apparatus, conditions and solution preparation method were the same as those in test example 1.
And (3) detection: the accuracy is obtained by adding 50 percent, 100 percent and 150 percent of three different concentrations of impurities E with the limit of 0.1 percent of the impurities E into a tedizolid phosphate intermediate sample and measuring the recovery rate of the impurities E. The accuracy of the known impurity E is determined by adding a known amount of the impurity E and measuring the ratio (recovery) between the content of the known impurity E in the sample to the theoretical value, expressed in% by weight. The results are shown in FIG. 18.
The experimental results show that: the recovery rate of the impurity E is 100.77% -106.55%, and the average recovery rate is as follows: 103.92%, has good accuracy, and meets the requirements of international pharmacopoeia and Chinese pharmacopoeia.
The research result of the test example 1~6 shows that the method for determining the process impurity 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine of the tedizolid phosphate intermediate has good specificity, good precision, high accuracy and high sensitivity, meets the requirement of 'Chinese pharmacopoeia' 2020 edition on analytical methodology, and is suitable for quality control of the process impurity 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine in the synthesis of the tedizolid phosphate intermediate. The method summarizes the quality analysis of the tedizolid phosphate intermediate, and in order to ensure the quality of the tedizolid phosphate API prepared by using the tedizolid phosphate intermediate as a raw material, the content of the impurity E in the tedizolid phosphate intermediate is controlled to be 0.1%.
In conclusion, the method for detecting the tedizolid phosphate intermediate impurity E can effectively separate the tedizolid phosphate intermediate from the impurity E (namely 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine) of the tedizolid phosphate intermediate, can be used for detecting the impurity in the tedizolid phosphate intermediate, accurately measures the content of the process impurity 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine in the tedizolid phosphate intermediate medicament, and has stable and reliable determination result and strong specificity. The result of methodology verification shows that the invention meets the requirement of 'Chinese pharmacopoeia' 2020 edition on analysis methodology. The detection method can effectively monitor the synthesis process of the tedizolid phosphate intermediate, is used for quality detection of the tedizolid phosphate intermediate, and ensures the quality of the tedizolid phosphate intermediate.
Claims (10)
1. A detection method of tedizolid phosphate intermediate related substances is characterized by comprising the following steps: the detection is carried out by adopting a high performance liquid chromatography, and the method comprises the following specific steps:
a. preparing a test solution: adding acetonitrile aqueous solution to dissolve a tedizolid phosphate intermediate to be detected to obtain the intermediate;
b. preparation of system suitability solution: dissolving related substance reference substance in acetonitrile water solution to obtain related substance solution; dissolving a tedizolid phosphate intermediate reference substance with a related substance solution, adding an acetonitrile aqueous solution, and uniformly mixing to obtain the tedizolid phosphate intermediate reference substance;
c. respectively sucking a test solution and a system applicability solution, and injecting the test solution and the system applicability solution into a chromatograph under the following chromatographic conditions:
a chromatographic column: octadecylsilane chemically bonded silica is used as a filling agent; mobile phase: taking 0.02 to 0.08 percent of inorganic acid aqueous solution as a mobile phase A and acetonitrile as a mobile phase B; the gradient elution procedure was: 0-15min, 10-75% of mobile phase B; 15-20min, 75% of mobile phase B; 20-21min, 75-10% of mobile phase B; 21-33min, 10% mobile phase B.
2. The detection method according to claim 1, characterized in that: the mass volume ratio of the tedizolid phosphate intermediate to be detected to the acetonitrile water solution in the step a) is 0.5 to 1.5mg:1ml; the volume ratio of acetonitrile to water in the acetonitrile water solution is (50 to 70): (30 to 50).
3. The detection method according to claim 2, characterized in that: the volume ratio of acetonitrile to water in the acetonitrile water solution is 60:40.
4. the detection method according to claim 1, characterized in that: and each 1ml of the system applicability solution in the step b) contains 0.01-0.1mg of related substances and 0.5-1.5 mg of tedizolid phosphate intermediate.
5. The detection method according to claim 1 or 4, characterized in that: the tedizolid phosphate intermediate is 2- (2-methyl-2H-tetrazol-5-yl) -5- (4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl) pyridine; the related substance is 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine.
6. The detection method according to claim 1, characterized in that: the chromatographic column in the step C) is Agilent 5 TC-C18 (2), the specification is 4.6 multiplied by 250mm, and the size is 5 mu m; the mobile phase A is 0.05 percent of perchloric acid aqueous solution in v/v; the detection wavelength in the chromatographic condition is 254nm, the sample injection amount is 10ul, and the flow rate is 0.5-1.5 ml/min; the column temperature is 30 to 40 ℃.
7. The detection method according to claim 6, characterized in that: the flow rate was 1.0ml/min and the column temperature was 30 ℃.
8. The detection method according to claim 1, wherein: and a chromatographic peak corresponding to the retention time of a chromatographic peak of a related substance in the system applicability solution chromatogram is presented in the chromatogram of the test solution, namely the test solution contains the corresponding related substance.
9. A method for measuring the content of related substances of a tedizolid phosphate intermediate is characterized by comprising the following steps: the specific operation steps are as follows:
1) Detected according to the method of any one of claims 1~8;
2) And (3) calculating the content of related substances according to the following calculation formula:x ⅰ %=A ⅰ ×F/A×100%；
wherein ,x ⅰ percent: the content of the relevant substances in the test sample,A ⅰ : the peak area of the relevant substance in the test solution,A: the sum of all peak areas in the test solution,Fcorrection factors for the substances of interest.
10. The content measurement method according to claim 9, wherein: the related substance is 5-bromo-2- (2-methyl-2H-tetrazol-5-yl) pyridine, and the correction factor is 0.57.
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