CN114324663A - Method for detecting lornoxicam related substances - Google Patents
Method for detecting lornoxicam related substances Download PDFInfo
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Abstract
The invention belongs to the technical field of substance detection, and particularly relates to a method for detecting related substances of lornoxicam. Detecting by liquid chromatography, wherein the mobile phase is a mixture of a mobile phase A and a mobile phase B, the mobile phase A is 0.025mol/L ammonium acetate solution, and the pH value is adjusted to 8.0 by ammonia water; the mobile phase B is methanol. According to the method, through screening and optimization of chromatographic conditions, the impurity detection sensitivity and the separation degree among impurities can be met, good detection and effective separation of each impurity in the lornoxicam sample are ensured, and the method can be used for quantification of each impurity in the lornoxicam sample; the invention has good applicability and durability.
Description
Technical Field
The invention belongs to the technical field of substance detection, and particularly relates to a method for detecting related substances of lornoxicam.
Background
Lornoxicam (Lornoxicam, chloronicicam) chemical name: 6-chloro-4-hydroxy-2-methyl-N-2-pyridyl-2H-thieno [2,3-e ] -1, 2-thiazine-3-formamide-1, 1-dioxide, is a novel non-steroidal anti-inflammatory drug (NSAID) and has the effects of analgesia, anti-inflammation, antipyresis and the like. The solubility of the lornoxicam raw material in a solvent is poor, at present, the commercially available lornoxicam is determined according to a method of related substances in the lornoxicam raw material of 2020 edition of Chinese pharmacopoeia, the method has low detection sensitivity, and the impurity separation effect of the raw material for injection is poor, and the known impurities and the unknown impurities are interfered with each other, so that the calculation of the content of each impurity is not facilitated. The method for detecting related substances in lornoxicam raw materials in the second part of the China pharmacopoeia of 2020 edition cannot meet the effective separation of impurities.
Therefore, it is highly desirable to develop a method for measuring related substances in lornoxicam, which satisfies the sensitivity of detecting impurities and the degree of separation between impurities, and enhances the applicability and durability of the method.
Disclosure of Invention
The invention aims to provide a method for detecting related substances of lornoxicam, which can ensure good detection and effective separation of each impurity in lornoxicam samples through screening and optimization of chromatographic conditions and can be used for quantification of each impurity in lornoxicam; the invention has good applicability and durability.
The method for detecting related substances of lornoxicam adopts liquid chromatography for detection, wherein the mobile phase is a mixture of a mobile phase A and a mobile phase B, the mobile phase A is 0.025mol/L ammonium acetate solution, and the pH value is adjusted to 8.0 by ammonia water; the mobile phase B is methanol.
Wherein:
during detection, the following gradient elution procedure is adopted for elution:
| time/minute | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 78 | 22 |
| 13 | 55 | 45 |
| 35 | 35 | 65 |
| 45 | 35 | 65 |
| 50 | 78 | 22 |
| 65 | 78 | 22 |
。
During detection, the sample injection concentration of the test solution is 2 mg/mL. The wavelength is 280 nm; the sample injection amount is 10 mu L; the column temperature is 30 +/-2 ℃; the flow rate is 0.9-1.1 ml/min; the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, 4.6X 250mm, 5 μm.
The preferred column temperature is 30 ℃; a flow rate of 1.0ml/min is preferred.
During detection, the diluent is a mixture of 0.025mol/L ammonium acetate solution and methanol, and the volume ratio of the ammonium acetate solution to the methanol is 50: 50; the ammonium acetate solution was adjusted to pH 8.0 with ammonia.
The blank solution is a mixture of 0.025mol/L ammonium acetate solution and methanol, and the volume ratio of the ammonium acetate solution to the methanol is 50: 50; the ammonium acetate solution was adjusted to pH 8.0 with ammonia.
Control solutions were prepared by weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxy-3-thiophenesulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridin-2-ylurea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, and a diluent were added to prepare a mixed solution containing 2. mu.g of each substance per 1 ml.
The preparation process of the test solution comprises the steps of weighing 20mg of the lornoxicam test sample, placing the sample in a 10ml volumetric flask, adding a diluent for ultrasonic dissolution assistance, diluting to a scale, and shaking up to obtain the lornoxicam test sample.
The method for detecting the lornoxicam related substances comprises the following steps: and (3) respectively taking 10 mu L of the blank solution, the reference solution and the sample solution, injecting the blank solution, the reference solution and the sample solution into a liquid chromatograph to obtain a result, and calculating the content of related substances in the sample solution according to an area normalization method.
The method for detecting the related substances of the lornoxicam further comprises the steps of taking 10 mu L of the mixed solution of the test sample and the impurities and injecting the mixed solution of the test sample and the impurities into a liquid chromatograph to obtain an HPLC (high performance liquid chromatography) spectrum.
Preparing a test article and impurity mixed solution: weighing 20mg of lornoxicam test sample, precisely transferring 1ml of impurity mixed storage solution, putting the impurity mixed storage solution into a 10ml volumetric flask together, adding a diluent for ultrasonic dissolution assistance, diluting to a scale, and shaking up to obtain the lornoxicam test sample.
Preparing an impurity mixed storage solution: weighing tenoxicam, 5-chloro-3-sulfino-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-methyl formate-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1 respectively, 1-dioxide and 6-chloro-4-hydroxy-2-methyl-N- (pyridine-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-formamide-1, 1-dioxide are placed in a 10ml volumetric flask, added with a diluent for ultrasonic dissolution assistance and diluted to the scale, shaken well to prepare a mixed solution containing 20 mu g of each substance per 1 ml.
The invention has the following beneficial effects:
the method for detecting related substances in Lornoxicam raw materials in 2020 edition of Chinese pharmacopoeia has low detection sensitivity, the impurity separation effect of the raw materials is poor, and the effective separation of impurities cannot be met under the condition that known impurities and unknown impurities are mutually interfered. The method can meet the impurity detection sensitivity and the separation degree among impurities through screening and optimizing of chromatographic conditions, ensures good detection and effective separation of each impurity in the lornoxicam sample, can be used for quantifying each impurity in the lornoxicam sample, and has good precision; the invention has good applicability, repeatability and durability.
Drawings
FIG. 1 is an HPLC profile of a blank solution of the present invention;
FIG. 2 is an HPLC chromatogram of a control solution of the present invention;
FIG. 3 is an HPLC chromatogram of a test solution of the present invention;
FIG. 4 is an HPLC chromatogram of a test sample + impurity mixed solution of the present invention;
FIG. 5 is an HPLC chromatogram of a test sample + impurity mixed solution detected according to the second standard method of the Chinese pharmacopoeia 2020 edition;
FIG. 6 is an HPLC chromatogram of a mixed stock solution for impurity detection according to the second standard method of the Chinese pharmacopoeia 2020 edition;
FIG. 7 is an HPLC chromatogram of the lornoxicam test sample + impurity mixed solution in method 1;
FIG. 8 is an HPLC chromatogram of the lornoxicam test sample + impurity mixed solution in method 2;
FIG. 9 is an HPLC chromatogram of three solutions of a blank solution, a test solution and a reference solution in a specificity test;
wherein, the HPLC curve of the blank solution, the HPLC curve of the sample solution and the HPLC curve of the reference solution are sequentially arranged from top to bottom;
FIG. 10 is a linear standard curve for impurities Y0-Z2 in a linear experiment;
FIG. 11 is a linear standard curve for impurities Y0-Z3 in a linear experiment;
FIG. 12 is a linear standard curve for lornoxicam in a linear test;
FIG. 13 is a linear standard curve for impurities Y0-Z4 in a linear experiment;
FIG. 14 is a linear calibration curve for the impurity 2-aminopyridine in a linear experiment;
fig. 15 is a linear standard curve for the impurity tenoxicam in a linear test;
FIG. 16 is a linear standard curve for impurity Y1 in a linear test;
FIG. 17 is a linear standard curve for impurities Y0-Z6 in a linear experiment;
FIG. 18 is a linear standard curve for impurities Y0-Z5 in a linear experiment;
FIG. 19 is a linear standard curve for impurities Y1-Z2 in a linear experiment.
Detailed Description
The present invention is further illustrated by the following specific examples.
Example 1
1. Chromatographic conditions
2. Procedure of experiment
(1) Diluent agent: 0.025mol/L ammonium acetate solution (pH adjusted to 8.0 with ammonia water) and methanol at a volume ratio of 50: 50.
(2) Preparing a test solution: weighing 20mg of lornoxicam test sample, placing the sample in a 10ml volumetric flask, adding a diluent for ultrasonic dissolution assistance, diluting to a scale, and shaking up to obtain the lornoxicam test sample.
(3) Preparation of a reference solution: respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, placed in a 10ml volumetric flask, added with diluent to assist in ultrasonic dissolution and diluted to the scale, shaken well to make a mixed solution containing 2. mu.g of each substance per 1 ml.
(4) Blank solution: 0.025mol/L ammonium acetate solution (pH adjusted to 8.0 with ammonia water) and methanol at a volume ratio of 50: 50.
(5) Preparing a test article and impurity mixed solution: weighing 20mg of lornoxicam test sample, precisely transferring 1ml of impurity mixed storage solution, putting the impurity mixed storage solution into a 10ml volumetric flask together, adding a diluent for ultrasonic dissolution assistance, diluting to a scale, and shaking up to obtain the lornoxicam test sample.
Preparing an impurity mixed storage solution: weighing tenoxicam, 5-chloro-3-sulfino-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-methyl formate-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1 respectively, 1-dioxide and 6-chloro-4-hydroxy-2-methyl-N- (pyridine-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-formamide-1, 1-dioxide are placed in a 10ml volumetric flask, added with a diluent for ultrasonic dissolution assistance and diluted to the scale, shaken well to prepare a mixed solution containing 20 mu g of each substance per 1 ml.
(6) Injecting 10 μ L of each of the blank solution (blank solvent), the reference solution (mixed standard solution), the sample solution, and the sample + impurity mixed solution (sample added standard solution) into a liquid chromatograph, recording chromatogram, specifically shown in fig. 1-4, and calculating the content of related substances in the sample solution according to area normalization method.
By comparing spectrograms of the reference solution and the mixed solution of the test sample and the impurities, the substances in the mixed solution of the test sample and the impurities can be well detected and effectively separated, so that the chromatographic conditions of the invention meet the impurity detection sensitivity and the separation degree between the impurities, and the content of the related impurities in the solution of the test sample can be accurately detected.
The invention makes the following researches on the chromatographic conditions during detection, and the specific process is as follows.
1. According to the related substance detection method published in the second part of the lornoxicam Chinese pharmacopoeia 2020 edition, the impurity mixed stock solution, the test sample + impurity mixed solution are injected into the liquid chromatograph according to the chromatographic conditions in table 1, and chromatograms are recorded, as shown in fig. 5 and fig. 6.
TABLE 1 Lornoxicam Chinese pharmacopoeia 2020 edition relevant substance method chromatographic condition
As can be seen from fig. 5-6, the results obtained by performing the test according to the related substance detection method published in the 2020 edition of lornoxicam chinese pharmacopoeia, cannot satisfy the effective separation of impurities, and the poor separation degree affects the chromatographic peak integration, thereby affecting the result accuracy of the impurity content determination; the initial proportion of methanol in isocratic elution of the method is 42%, the proportion is high, so that impurities with high polarity (impurities Y0-Z2 and impurities Y0-Z3) are not reserved, a blank solvent interferes with the detection of the impurities with high polarity, the calculation result of the impurities can be influenced, and a reasonable basis cannot be provided for the determination of the content of each substance in a product; moreover, the method has a low concentration of the sample, resulting in a poor detection capability of the impurity at the limiting concentration. Therefore, the method suitable for measuring the related substances of the variety is redeveloped and optimized to meet the requirements of impurity detection sensitivity and separation between impurities and achieve the aims of enhancing the applicability and durability of the method.
Table 2 summary of known impurity retention times
2. Screening the related substances of lornoxicam under the detection condition. Since the polarity difference between the impurities is large, the impurities cannot be separated well in an isocratic mode, and the method needs to be investigated in a gradient elution mode according to the polarity difference of the impurities, so that the method is optimized by performing the gradient elution. Selecting an acetate (ammonia water for adjusting pH) -methanol system for liquid quality analysis, selecting a 0.025mol/L ammonium acetate solution with a stable system, and adjusting the pH to 8.0 by using ammonia water as a mobile phase A in order to keep the lornoxicam in a molecular state in the solution and properly improve the pH value of the mobile phase; compared with methanol and acetonitrile, the acetonitrile has stronger elution capability, and because the polarity of part of impurities is relatively close, the use of acetonitrile as a mobile phase is possibly not beneficial to improving the separation degree among the impurities; gradient elution is selected and applied to separate the chromatographic peak, so that the chromatographic peak has good shape and the retention time is relatively stable; methanol with moderate elution capacity is selected as the mobile phase B, and elution is carried out in a gradient mode.
(1) The method comprises the following steps: the chromatographic conditions are shown in table 3 below, and the results of injecting the lornoxicam test sample + impurity mixed solution into the liquid chromatograph are shown in fig. 7.
TABLE 3 chromatographic conditions for method 1 for the substances
As can be seen from fig. 7, the separation of each known impurity is significantly improved, and only when the operation time reaches 20min, the separation of two impurities (impurities Y0-Z5 and impurities Y0-Z6) is poor, indicating that the proportion of the organic phase in the time period is slightly higher, so the experiment is carried out by adjusting the proportion of the organic phase in the time period; if the impurity which is eluted first peaks at 4.3 minutes, in order to relieve the proportion of the organic phase in the 20min period, the initial proportion of the organic phase can be slightly increased, so that the proportion of the organic phase in the 20min period can be slowly increased, and the separation degree of the impurities in the period is further improved.
(2) The method 2 comprises the following steps: the chromatographic conditions are shown in table 4 below, and the results of injecting the lornoxicam test sample + impurity mixed solution into the liquid chromatograph are shown in fig. 8.
TABLE 4 chromatographic conditions for method 2 for the substances
As can be seen from fig. 8, the separation degree of the two impurities (impurities Y0-Z5 and impurities Y0-Z6) with poor separation degree in the original method 1 is significantly improved in the method 2, and the separation degree between the other impurities, that is, the separation degree between the main peak and the adjacent peak, satisfies the requirement. In addition, although the separation degree of the impurities Y0-Z5 and Y0-Z6 is obviously improved and meets the detection requirement, the risk still exists.
(3) The method 3 comprises the following steps: on the basis of the method 2, the initial proportion of methanol is adjusted, the chromatographic conditions are shown in the following table 5, the lornoxicam test sample and the impurity mixed solution are injected into a liquid chromatograph, and the measuring result spectrum of the lornoxicam test sample and the impurity mixed solution is shown in fig. 4.
TABLE 5 chromatographic conditions for substance method 3
As can be seen from FIG. 4, the conditions in method 3 all ensure that the detection of known impurities is not disturbed, and the sample injection precision is good, and the reproducibility of the peak area and the retention time is good.
The invention carries out specificity test, detection limit test, repeatability test, linear test and sample injection precision test, and the specific contents are as follows:
1. specificity test
Solution preparation:
(1) diluent (blank solution): 0.025mol/L ammonium acetate solution (pH adjusted to 8.0 with ammonia water) and methanol at a volume ratio of 50: 50.
(2) Preparation of control solutions (mixed standard solutions): respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, placed in a 10ml volumetric flask, added with diluent to assist in ultrasonic dissolution and diluted to the scale, shaken well to make a mixed solution containing 2. mu.g of each substance per 1 ml.
(3) Preparing a test solution: weighing 20mg of lornoxicam test sample, placing the sample in a 10ml volumetric flask, adding a diluent for ultrasonic dissolution assistance, diluting to a scale, and shaking up to obtain the lornoxicam test sample.
The blank solution, the sample solution and the control solution were each taken 10. mu.l and injected into a liquid chromatograph, and a chromatogram was recorded, as shown in FIG. 9. It can be seen from fig. 9 that the blank solvent does not interfere with the sample detection.
2. Sample introduction precision test
2.1 preparation of the solution
Control stock solution (mixed standard solution): respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-formic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl 6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, placed in a 10ml volumetric flask, added with diluent to assist in ultrasonic dissolution and diluted to the scale, shaken well to make a mixed solution containing 2. mu.g of each substance per 1 ml. Sampling, and continuously injecting the sample for 6 times into a liquid chromatograph for detection.
2.2 the results are shown in Table 6.
TABLE 6 results of sample introduction precision measurement
3. Limit of detection test
The control solution of the invention is taken and diluted by diluent, the corresponding concentration when the signal to noise ratio is 3:1 is determined as the detection limit, and the specific test result data is shown in Table 7.
TABLE 7 detection Limit test results
4. Repeatability test
The repeatability is verified by preparing 6 test solutions and testing, each solution being injected 1 time. The relative standard deviation of the known impurity content of the results of 6 times of measurement is required to meet the relevant requirements in the four-part general rule 9101 of the 2020 edition in Chinese pharmacopoeia to confirm that the method has good precision.
4.1 solution preparation
(1) Diluent (blank solution): 0.025mol/L ammonium acetate solution (pH adjusted to 8.0 with ammonia water) and methanol at a volume ratio of 50: 50.
(2) Control stock solution: respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo- (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate 1, 1-dioxide and 6-chloro-4-hydroxy-2-methyl-N- (pyridine-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-formamide-1, 1-dioxide are placed in a 10ml volumetric flask, added with a diluent for ultrasonic dissolution assistance and diluted to the scale, shaken well to prepare a mixed solution containing 20 mu g of each substance per 1 ml.
(3) Adding a standard solution into a test sample: weighing 20mg of lornoxicam test sample, placing the sample in a 10ml volumetric flask, adding a diluent for ultrasonic dissolution assistance, adding 1ml of the reference substance storage solution, placing the reference substance storage solution in the volumetric flask, diluting the diluent to a scale, and shaking up to obtain the lornoxicam test sample. 6 parts are prepared in parallel. The 6 parts of test sample are added with the standard solution for detection, and the specific data are shown in table 8.
TABLE 8 repeatability test results-sample spiking solution
5. Linear test
And (5) observing the linear change condition of the measured substance in a limited range.
5.1 solution preparation
Quantitative limiting storage solution: respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, placed in a 10ml volumetric flask, added with diluent to assist in ultrasonic dissolution and diluted to the scale, shaken well to make a mixed solution containing 2. mu.g of each substance per 1 ml.
Control stock solution: respectively weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxyl-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-formic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-yl urea, methyl 6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide and 6-chloro-4-hydroxy-2-methyl-N- (pyridine-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-formamide-1, 1-dioxide are placed in a 10ml volumetric flask, added with a diluent for ultrasonic dissolution assistance and diluted to the scale, shaken well to prepare a mixed solution containing 20 mu g of each substance per 1 ml.
Linear solution 1 (limit of quantitation): and (3) placing 2ml of the precise quantitative limiting storage solution into a 10ml measuring flask, adding a diluent to dilute to a scale, and shaking up to obtain the product.
Linear solution 2 (50%): precisely measuring 0.5ml of the reference stock solution, placing in a 10ml measuring flask, diluting with diluent to scale, and shaking.
Linear solution 3 (80%): precisely measuring 0.8ml of the reference stock solution, placing in a 10ml measuring flask, diluting with diluent to scale, and shaking.
Linear solution 4 (100%): precisely measuring 1ml of the reference stock solution, placing in a 10ml measuring flask, diluting with diluent to scale, and shaking up.
Linear solution 5 (120%): precisely measuring 1.2ml of the reference stock solution, placing in a 10ml measuring flask, diluting with diluent to scale, and shaking.
Linear solution 6 (150%): precisely measuring 1.5ml of the reference stock solution, placing in a 10ml measuring flask, diluting with diluent to scale, and shaking.
Linear solution 7 (200%): precisely measuring 2ml of the reference substance storage solution, placing the reference substance storage solution in a 10ml measuring flask, diluting the reference substance storage solution to a scale with a diluent, and shaking up to obtain the reagent.
Precisely measuring 10 μ l of each of the linear solutions 1-7, injecting into a liquid chromatograph, and recording chromatogram.
5.2 the results are shown in tables 9-18.
TABLE 9 Linear-impurities Y0-Z2
TABLE 10 Linear-impurities Y0-Z3
TABLE 11 Linear-lornoxicam
TABLE 12 Linear-impurities Y0-Z4
TABLE 13 Linear-impurity 2-aminopyridine
TABLE 14 Linear-impurity tenoxicam
TABLE 15 Linear-impurity Y1
TABLE 16 Linear-impurities Y0-Z6
TABLE 17 Linear-impurities Y0-Z5
TABLE 18 Linear-impurities Y1-Z2
The above results show that: the known impurities and lornoxicam have good linear relation within the range of quantitative limit to 4 mu g, the response factors RSD are all less than 10%, the correlation coefficients r are all more than 0.9900, and the y-axis intercept deviation% is all less than 25%.
Claims (10)
1. A method for detecting related substances of lornoxicam adopts liquid chromatography for detection, and is characterized in that: the mobile phase is a mixture of a mobile phase A and a mobile phase B, the mobile phase A is 0.025mol/L ammonium acetate solution, and the pH value is adjusted to 8.0 by ammonia water; the mobile phase B is methanol.
2. The method for detecting a lornoxicam-related substance according to claim 1, wherein: during detection, the following gradient elution procedure is adopted for elution:
。
3. The method for detecting a lornoxicam-related substance according to claim 1, wherein: during detection, the sample injection concentration of the test solution is 2 mg/mL.
4. The method for detecting a lornoxicam-related substance according to claim 1, wherein: during detection, the wavelength is 280 nm; the sample injection amount is 10 mu L; the column temperature is 30 +/-2 ℃; the flow rate is 0.9-1.1 ml/min; the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, 4.6X 250mm, 5 μm.
5. The method for detecting a lornoxicam-related substance according to claim 4, wherein: the column temperature is 30 ℃; the flow rate was 1.0 ml/min.
6. The method for detecting a lornoxicam-related substance according to claim 1, wherein: during detection, the diluent is a mixture of 0.025mol/L ammonium acetate solution and methanol, and the volume ratio of the ammonium acetate solution to the methanol is 50: 50; the ammonium acetate solution was adjusted to pH 8.0 with ammonia.
7. The method for detecting a lornoxicam-related substance according to claim 1, wherein: during detection, the blank solution is a mixture of 0.025mol/L ammonium acetate solution and methanol, and the volume ratio of the ammonium acetate solution to the methanol is 50: 50; the ammonium acetate solution was adjusted to pH 8.0 with ammonia.
8. The method for detecting a lornoxicam-related substance according to claim 6, wherein: in the detection, the reference solution is prepared by weighing lornoxicam, tenoxicam, 5-chloro-3-sulfinyl-2-thiophene-carboxylic acid, 5-chloro-2-carboxy-3-thiophene sulfonic acid, 2-aminopyridine, 6-chloro-4-hydroxy-2-methyl-2H-thiophene [2,3-e ] -1, 2-thiazine-3-carboxylic acid methyl ester-1, 1-dioxide, oxo (2-pyridylamino) -acetic acid, 1, 3-bipyridine-2-ylurea, methyl-6-chloro-4-methoxy-2-methyl-2H-thiophene [2,3-e ] [1,2] thiazine-3-carboxylate-1, 1-dioxide, 6-chloro-4-hydroxy-2-methyl-N- (pyridin-2-yl) -2H-thieno [2,3-e ] [1,2] thiazine-3-carboxamide-1, 1-dioxide, and a diluent were added to prepare a mixed solution containing 2. mu.g of each substance per 1 ml.
9. The method for detecting a lornoxicam-related substance according to claim 6, wherein: during detection, the preparation process of the test solution is as follows, 20mg of the lornoxicam test sample is weighed and placed in a 10ml volumetric flask, a diluent is added for ultrasonic dissolution assistance, and the mixture is diluted to a scale and shaken up, thus obtaining the lornoxicam test solution.
10. The method for detecting a lornoxicam-related substance according to any one of claims 1 to 9, wherein: and (3) respectively taking 10 mu L of the blank solution, the reference solution and the sample solution, injecting the blank solution, the reference solution and the sample solution into a liquid chromatograph to obtain a result, and calculating the content of related substances in the sample solution according to an area normalization method.
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| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
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| WO2008127291A2 (en) * | 2006-10-10 | 2008-10-23 | Los Alamos National Security, Llc | Advanced drug development and manufacturing |
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