CN115993412A - HPLC detection method for colchicine impurity F - Google Patents
HPLC detection method for colchicine impurity F Download PDFInfo
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- CN115993412A CN115993412A CN202211561955.5A CN202211561955A CN115993412A CN 115993412 A CN115993412 A CN 115993412A CN 202211561955 A CN202211561955 A CN 202211561955A CN 115993412 A CN115993412 A CN 115993412A
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Abstract
The invention discloses an HPLC detection method of colchicine impurity F (nor colchicine). The invention has higher system applicability, and simultaneously has incomparable advantages in specificity, quantitative limit, detection limit, linear range and repeatability, and higher precision.
Description
Technical Field
The invention belongs to the field of chemical drug analysis and detection, and particularly relates to an HPLC detection method for colchicine impurity F.
Background
Colchicine is an alkaloid extracted from bulb of Iphigenia indica Iphigenia indica Kunthet benth of Liliaceae, and is mainly used for treating gout. The product is loaded in Chinese pharmacopoeia, european pharmacopoeia and United states pharmacopoeia, and impurity F is loaded in colchicine raw material drug standard of the pharmacopoeia of each country, and is checked by ferric iron chromogenic method. The structural formula is as follows:
the color development method has poor specificity, cannot give specific impurity content values, and cannot be used for measuring the impurity F due to the interference of auxiliary materials in the tablet; and in addition, pharmacopoeia related substances are adopted to check chromatographic conditions, the impurity F peak shape is poor, and the integration is inaccurate. The invention adopts HPLC method to measure the content of impurity F in colchicine. The peak shape is good, the specificity is strong, the interference of other impurities is avoided, the method is reliable, and the specific content value of the impurities can be calculated.
Disclosure of Invention
The invention aims to: the invention aims to provide an HPLC detection method for impurity F in colchicine and tablets thereof, which completely accords with the standard in the aspects of specificity, quantitative limit, detection limit, linear range, repeatability, accuracy and the like and has higher precision.
In order to achieve the above purpose, the present invention provides the following technical solutions: an HPLC detection method of colchicine impurity F is characterized in that the content of the colchicine and the impurity F in the tablet thereof is detected by a liquid chromatography;
the impurity F is nor colchicine, the chemical name is N- (10-hydroxy-1, 2, 3-trimethoxy-9-oxo-5, 6,7, 9-tetrahydrobenzo [ a ] heptadiene-7-yl) acetamide, and the structural formula is as follows:
specifically, the detection method adopts a chromatographic column with octyl silane bonded silica gel as a filler, adopts a mixed solution of solution A-methanol as a diluent and a mobile phase of a sample solution to perform isocratic elution, wherein the solution A is a mixed solution of edetate disodium (also called disodium edetate or EDTA-2 Na) -phosphoric acid-water.
Specifically, the HPLC detection method for impurity F in colchicine provided by the invention comprises the following steps:
a) Preparing raw material medicine sample solution
Precisely weighing colchicine raw material medicine, placing into a measuring flask, and preparing a sample solution containing 0.25mg colchicine raw material medicine in each 1ml by using a diluent;
b) Preparing test solution of tablet
Taking colchicine tablets, grinding, precisely weighing a proper amount, placing into a measuring flask, and adding a diluent to prepare a tablet sample solution containing 0.25mg colchicine in each 1 ml;
c) Preparing reference substance solution
Taking a proper amount of impurity F reference substance, adding a diluent for dissolving and quantitatively diluting to prepare a solution with the concentration of about 2.5 mug in each 1 ml;
d) Precisely measuring the sample solution and the reference solution respectively, injecting into a liquid chromatograph, recording the chromatogram, and calculating the content of impurity F in the sample solution by using an external standard method and peak area.
Specifically, the diluents in the steps a), b) and c) are as follows: solution A-methanol (55:45).
Specifically, the calculation formula in the step d) is as follows:
wherein:
wherein: f-response factor; m is m S -the sample amount of impurity B in the control solution; c S -content of impurity B; v (V) S -total dilution volume of impurity B in control solution; a is that S Peak area f of impurity B in control solution chromatogram Average of -an average value of the response factors; a is that i -peak area of impurity B in the test sample; m is m i -sample weighing of the test sample; v (V) i -sample dilution volume.
Preferably, the detection conditions of the liquid chromatography are as follows:
chromatographic column: welch Xtime C8 chromatographic column with specification of 150mm×4.6mm,5 μm;
mobile phase: solution A-methanol (55:45);
elution mode: isocratic elution;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
detection wavelength: 245nm;
sample injection amount: 20. Mu.L.
Specifically, the preparation method of the solution A comprises the steps of taking 100mg of edetate disodium and 2.0ml of phosphoric acid, adding water for dissolution, diluting to 1000ml, and shaking uniformly.
Preferably, the detection method can quantitatively detect 0.01% of impurity F in the test sample at the lowest.
In particular, the method is applicable to colchicine and colchicine-containing related formulations, preferably colchicine tablets, which require detection of colchicine impurity F.
The beneficial effects are that: (1) According to the invention, phosphoric acid and edetate disodium are added in the solvent and the mobile phase, so that the peak shape of the impurity F is optimized, and meanwhile, the complexing of the impurity F with metal ions polluted in the solvent and a chromatographic system is avoided, so that the regression coefficient of the standard curve of the impurity F reference substance is more than 0.999. (2) The invention has incomparable advantages in specificity, quantitative limit, detection limit, linear range, accuracy and precision.
Drawings
FIG. 1 is a chromatogram of impurity F under mobile phase 1 (water-methanol) system;
FIG. 2 is a chromatogram of impurity F in mobile phase 2 (0.2% phosphoric acid solution-methanol) system;
FIG. 3 is a chromatogram of impurity F in mobile phase 3 (solution A-methanol) system;
fig. 4 is a standard graph of the impurity F control.
Detailed Description
The present invention will be further described in detail with reference to the following examples and drawings for the purpose of enhancing the understanding of the present invention, which examples are provided for the purpose of illustrating the present invention only and are not to be construed as limiting the scope of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
According to the detection method disclosed by the invention, the detection of the impurity F is mainly performed by an external standard method.
The instrument and reagent information used in the embodiments of the detection method of the present invention are shown in the following table:
example 1
This example provides a method for HPLC detection of impurity F in colchicine.
1. Detection method
(1) Preparing diluent
100mg of edetate disodium and 2.0ml of phosphoric acid are taken, dissolved in water and diluted to 1000ml, and the mixture is shaken uniformly to prepare solution A, and solution A-methanol (55:45) is prepared.
(2) Preparing raw material medicine sample solution
About 25mg of colchicine is taken, precisely weighed, placed in a 100ml measuring flask, added with a diluent for dissolution and dilution to a scale, and shaken well.
(3) Preparing test solution of tablet
25 tablets (about equivalent to 25mg containing colchicine) of the product are taken, put into a 100ml measuring flask, added with a diluent for dissolution and dilution to a scale, shaken uniformly, filtered, and the subsequent filtrate is taken.
(4) Preparing reference substance solution
Taking a proper amount of impurity F reference substance, adding a diluent for dissolving and quantitatively diluting to prepare a solution with the concentration of about 2.5 mug in each 1 ml.
(5) Precisely measuring the sample solution and the reference solution respectively, injecting into a liquid chromatograph, recording the chromatogram, and calculating the content of impurity F in the sample solution by using an external standard method and peak area.
2. Chromatographic conditions:
chromatographic column: welch Xtime C8 chromatographic column with specification of 150mm×4.6mm,5 μm;
mobile phase: solution A-methanol (55:45);
elution mode: isocratic elution;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
detection wavelength: 245nm;
sample injection amount: 20. Mu.L.
3. Impurity F content calculation formula
(1)
Wherein: f-response factor;
ms is the sample weighing amount of impurity F in the reference substance solution;
cs-content of impurity F;
vs—total dilution volume of impurity F in control solution;
as-peak area of impurity F in control solution chromatogram.
(2)
Wherein: faverage-average of response factors;
ai-peak area of impurity F in the sample;
mi-sample weighing amount of a test sample;
vi-dilution volume of test sample.
Example 2
The present example provides a spectrum difference between pure water, 0.2% phosphoric acid and solution a as the aqueous phase of impurity F in colchicine in the mobile phase, and at the same time, the separation from colchicine was examined.
1. Chromatographic conditions:
chromatographic column: welch Xtime C8 column (150 mm. Times.4.6 mm,5 μm)
Mobile phase 1: water-methanol (65:35)
Mobile phase 2:0.2% phosphoric acid-methanol (55:45)
Mobile phase 3: solution A-methanol (55:45)
Flow rate: 1.0ml/min
Column temperature: 25 DEG C
Detection wavelength: 245nm;
sample injection amount: 10 μl of
2. Test solution: taking appropriate amounts of colchicine and impurity F, dissolving and diluting with mobile phases 1 and 2 respectively to obtain solutions containing about 0.25mg of colchicine and appropriate amounts of impurity F in each 1 ml.
3. Impurity F control solution: taking an impurity F reference substance, and preparing a gradient concentration with the concentration of 0.125-3.75 mug/ml by using a mobile phase 2 and a mobile phase 3 respectively as each linear solution.
Solutions prepared in mobile phase 1, mobile phase 2 and mobile phase 3 were measured according to the three chromatographic conditions of example 1 and chromatograms were recorded, see figures 1-3 and table 2 of the specification.
Table 2: difference of impurity F in different aqueous phases
Results: 1. under the 3 chromatographic conditions, colchicine as a main component does not interfere with detection of impurity F, and has good specificity.
2. Under the condition of mobile phase 1, the retention time of the impurity F is longer, and the peak shape is poor; the analysis shows that the impurity F contains enol groups, shows weak acidity and can be partially dissociated, so that the peak shape is influenced. The addition of phosphoric acid to mobile phase 2 and the simultaneous increase of the organic phase ratio reduced the retention time of impurity F to about 8 minutes, with a greater improvement in peak shape, indicating that the acidic dissociation of impurity F was inhibited. However, under the condition of mobile phase 2, the standard curve regression coefficient r of the impurity F is only about 0.98, and part of the impurity F in the analysis solution can be complexed with the solvent or metal ions in the chromatographic system, so that the peak area is affected. And adding strong complexing agent edetate disodium into the mobile phase 3 to competitively inhibit the complexing of the impurity F and metal ions, wherein as a result, the standard curve regression coefficient r of the impurity F is 0.9995, and meets the requirement of not less than 0.999.
Example 3: quantitative limit and detection limit test
The detection Limit (LOD) and the quantification Limit (LOQ) are determined according to the signal-to-noise ratio method. Gradually diluting the reference substance solution, comparing the measured signal with the baseline noise, and calculating the lowest concentration which can be reliably detected, wherein the concentration is a quantitative limit concentration when the signal-to-noise ratio is about 10:1; the concentration is the detection limit concentration at a signal to noise ratio of about 3:1. The results are shown in Table 3.
Table 3: quantitative limit and detection limit results
Note that: sensitivity = impurity F limit of quantitation or limit of detection/sample concentration (0.25 mg/ml)
The quality control limit of the impurity F in the bulk drug and the tablet is respectively 0.15% and 1.0%, and the sensitivity of the quantitative limit and the detection limit is lower than 1/10 of the quality control limit of the impurity F, so that the sensitivity of the invention is proved to be good.
Example 4: linearity and range detection
Taking a proper amount of impurity F, precisely weighing, dissolving with a solvent, and diluting to a proper concentration to obtain an impurity F stock solution; taking impurity F stock solution for quantitative dilution, and respectively preparing solutions with the concentration of 0.05%, 0.2%, 0.6%, 1.2% and 1.5% of the sample concentration, wherein the solutions are used as sample solutions with linear gradient concentrations. The linear relationship is plotted as a function of measured peak area versus analyte concentration, and linear regression is performed using the least squares method, requiring that the value of the linear regression coefficient r be not less than 0.999, the results are shown in Table 4 and FIG. 4.
Table 4: results of the linear measurement
Note that: intercept ratio = linear equation intercept/1.0% concentration peak area
As shown by the results of the table, the detection method provided by the invention proves that the linear correlation coefficient r of the impurity F is 0.9995 within the range of 0.05-1.5% relative to the concentration of the sample, and the detection method has good linear relation.
Example 5: precision detection of reference substance solution sample injection
The impurity F reference solution was taken and continuously measured 6 times to examine the relative standard deviation of the peak area. The results are shown in Table 5.
Table 5: results of precision test of control solution injection
As shown by the results of the table, the detection method provided by the invention aims at determining the sample injection precision of the impurity F reference substance solution, the peak area RSD is less than 2%, and the precision is good.
Example 6: stability detection of control and test solution
Taking impurity F reference substance solution, crude drug and tablet sample solution, respectively feeding 20 μl in 0h, 2h, 4h, 6h, 8h, 10h and 12h, recording chromatogram, calculating relative standard deviation of impurity F peak area, and testing the results in Table 6.
Table 6: stability test results of reference and test sample solutions
| Time | 1.0% control | Crude drug test article | Tablet test article |
| 0h | 216819 | <LOQ | <LOQ |
| 2h | 221798 | <LOQ | <LOQ |
| 4h | 220599 | <LOQ | <LOQ |
| 6h | 219004 | <LOQ | <LOQ |
| 8h | 220105 | <LOQ | <LOQ |
| 10h | 217378 | <LOQ | <LOQ |
| 12h | 215520 | <LOQ | <LOQ |
| Average of | 218746 | / | / |
| RSD | 1.03％ | / | / |
As shown in the table, the peak area RSD of the reference solution is less than 2.0%, the impurity F detected by the sample solution is less than the quantitative limit, and the solution stability is good after the sample solution is left at room temperature for 12 hours.
Example 7: repeatability detection
Colchicine drug substance and tablet samples were taken and the test method according to example 1 of the present invention was repeated 6 times to verify the good precision of the method, and the results are shown in table 7.
Table 7: repeatability test results
| Numbering device | Crude drug test article | Tablet test article |
| A1 | ＜LOQ | ＜LOQ |
| A2 | ＜LOQ | ＜LOQ |
| A3 | ＜LOQ | ＜LOQ |
| A4 | ＜LOQ | ＜LOQ |
| A5 | ＜LOQ | ＜LOQ |
| A6 | ＜LOQ | ＜LOQ |
| Mean value of | / | / |
| RSD(％) | / | / |
As shown in the table above, the impurity F is detected for 6 times to be lower than the detection limit, and the method is proved to have good precision.
Example 8: intermediate precision detection
Samples of the crude drug and tablet were taken and measured by different personnel at different times and on different instruments in the same manner as the repeatability of example 7, and the difference between the measured results of the two persons was compared, and the results are shown in Table 8.
Table 8: results of intermediate precision test
As shown in the table, the impurity F in twelve sample solutions measured by different personnel at different times and on different instruments is lower than the quantitative limit, and the intermediate precision of the detection method is good.
Example 9: accuracy detection
The ratio (recovery rate) between the actual measured amount and the theoretical amount of the impurity F in the labeled sample is measured by a sample adding recovery method, expressed as percent, and the recovery rate is required to be between 90 and 110 percent so as to prove that the method has good accuracy, and the result is shown in Table 9.
Table 9: accuracy test results
The table shows that the recovery rate of the impurity F in the bulk drug and the tablet is between 90.0% and 110.0%, which meets the verification requirement, and the method is proved to have good accuracy; the recovery rate RSD value is less than 10%, and the repeatability is good.
In summary, in the specific test, the main component peak of the sample and the impurity thereof do not affect the detection of impurity F.
In the limit assay, the impurity F limit concentration is far below the quality control limit (0.15%); the detection limit concentration of the impurity F is lower than 1/10 (0.01%) of the quality control limit.
In the test of the linear range, the linear relation of the impurity F is good in the range of 0.05-1.5% relative to the concentration of the test sample, the correlation coefficient r is 0.9995, and the percentage of the y-axis intercept relative to the peak area of the limiting concentration reference substance is far less than 25%.
In the accuracy test, the recovery rate of the impurity F is between 90% and 110%, and the RSD value is less than 10%.
In the reproducibility test, 6 samples were less than LOQ for impurity F.
In the test of intermediate precision, the impurity F content of 6 samples respectively measured on different instruments by different testers on different dates is smaller than LOQ.
Therefore, the invention adopts an acidic disodium edentate-containing mobile phase system, optimizes the peak shape of the impurity F, inhibits the complexation of the impurity F in the solution with the solvent and metal ions in the chromatographic system, effectively separates the impurity F from colchicine and impurities thereof, has good peak symmetry, is not interfered by trace metal ion pollutants in the chromatographic system to cause asymmetric peak shape and peak area with abnormal numerical value, and is beneficial to the detection of the impurity F. Meanwhile, the method has the advantages of no comparability in the aspects of specificity, quantitative limit, detection limit, linear range and repeatability, and has higher precision.
The foregoing is merely a preferred embodiment of the invention, and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are also intended to be considered as protective scope of the invention.
Claims (7)
1. An HPLC detection method of colchicine impurity F is characterized in that the content of the impurity F in colchicine or colchicine tablets is detected by chromatography;
the impurity F is nor colchicine, and the structural formula is as follows:
2. the method according to claim 1, wherein the method uses a column packed with octyl silane bonded silica gel, and uses a mixed solution of solution a-methanol as a diluent and mobile phase of a sample solution, wherein the solution a is a mixed solution of disodium edetate-phosphoric acid-water.
3. The method according to claim 2, characterized in that it comprises in particular the following steps:
a) Preparing colchicine raw material medicine sample solution
Precisely weighing colchicine raw material medicine, placing the colchicine raw material medicine into a measuring flask, and preparing colchicine raw material medicine sample solution by using a diluent;
b) Preparing colchicine tablet test solution
Taking colchicine tablets, grinding, precisely weighing a proper amount, placing into a measuring flask, and adding a diluent to prepare colchicine tablet test sample solution;
c) Preparing reference substance solution
Taking a proper amount of impurity F, adding a diluent to dissolve the impurity F to prepare a reference substance solution;
d) Precisely measuring the sample solution and the reference solution respectively, injecting into a liquid chromatograph, recording the chromatogram, and calculating the content of impurity F in the sample solution by using an external standard method and peak area.
4. A method according to claim 3, wherein the detection conditions of the liquid chromatograph are:
chromatographic column: welch Xtime C8 chromatographic column with specification of 150mm×4.6mm,5 μm;
mobile phase: a mixed solution of solution A and methanol in a volume ratio of 55:45;
elution mode: isocratic elution;
flow rate: 1.0ml/min;
column temperature: 25 ℃;
detection wavelength: 245nm;
sample injection amount: 20. Mu.L.
5. The method according to claim 4, wherein the solution A is prepared by dissolving disodium edentate 100mg and phosphoric acid 2.0ml in water, diluting to 1000ml, and shaking.
6. The method according to claim 4, wherein the detection method is capable of quantitatively detecting 0.01% of impurity F in the sample at the minimum.
7. The HPLC method of colchicine impurity F according to any one of claims 1-6, which is suitable for colchicine bulk drugs and colchicine-containing related formulations, preferably colchicine tablets, which are required for the detection of colchicine impurity F.
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