CN111521714A - Method for separating and measuring capecitabine and impurities thereof - Google Patents
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Abstract
A method for separating and measuring capecitabine and impurities thereof comprises the following steps of 1) taking capecitabine or a preparation containing capecitabine, and adding a diluent to dissolve the capecitabine or the preparation containing capecitabine to obtain a sample solution with the concentration of 0.5 mg/ml; 2) taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution to obtain a control solution with the concentration of 5 mu g/ml; 3) injecting equal volumes of the sample solution in the step 1) and the contrast solution in the step 2) into a high performance liquid chromatograph, performing gradient elution by using a mobile phase A and a mobile phase B as mobile phases, recording a chromatogram, and calculating the content of impurities according to a self contrast method. The separation and determination method is simple and convenient to operate, and can effectively separate and determine the capecitabine and impurities thereof and effectively control the quality of the capecitabine and products thereof.
Description
Technical Field
The invention relates to the field of analytical chemistry, in particular to a method for separating and determining capecitabine and impurities thereof.
Background
Capecitabine (Capecitabine) is a fluoropyrimidine deoxynucleoside carbamate antitumor drug developed by Roche of Switzerland. Approved by the U.S. food and drug safety administration in 1998 for marketing in the U.S. for the treatment of metastatic colorectal cancer in combination with docetaxel for the treatment of metastatic breast cancer. The medicine is marketed in China in 2001, and 8 months in 2008, the treatment of advanced gastric cancer is approved by the China food and drug administration. Capecitabine has been widely used at home and abroad in many countries for the treatment of several of the above cancers. The chemical name of capecitabine is: 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine, the chemical structure of which is as follows:
the route of capecitabine synthesis currently used for production is as follows:
in the process of synthesizing the target compound, there are several steps of important intermediates and unknown impurities, which are known substances (i.e., impurities) commonly known to be involved in the quality control of drugs, and degradation products that are generated during the process, which may affect the purity and quality of the drugs due to incomplete removal. Eleven known impurities that are primarily controlled for the synthesis of capecitabine are: impurity A-SM1, impurity A-Z1, impurity A-Z2, impurity A-Z3, impurity A-Z5, impurity A-Z8, impurity A-Z9, impurity A-Z10, impurity A-Z11, impurity A-Z13 and A-intermediate A1, and the structural formula of the compound is shown in the following formula (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) and (k).
Therefore, the capecitabine has more impurities and similar structure, and brings difficulty to separation. In addition, the polarity of each impurity is different, and on the premise of satisfying the separation of capecitabine and each impurity, the separation between the impurities is also satisfied, which results in the increase of the detection difficulty. The capecitabine is difficult to be effectively separated from impurities and impurities by adopting a conventional detection method, so that the quality control of the capecitabine is influenced.
In order to accurately control the quality of capecitabine and its preparation products, it is necessary to research a method for separating and detecting capecitabine and its preparation-related substances simply, rapidly and accurately.
Disclosure of Invention
The invention aims to provide a method for separating and measuring capecitabine and impurities thereof, which is simple and convenient to operate and can effectively realize separation and measurement of capecitabine and impurities thereof and impurities, thereby realizing the purpose of controlling the quality of capecitabine and products thereof.
The technical scheme of the invention is as follows: a method for separating and determining capecitabine and impurities thereof comprises the following steps,
1) preparation of sample solution
Taking capecitabine or a preparation containing capecitabine, and adding a diluent to dissolve the capecitabine or the preparation containing capecitabine to obtain a sample solution with the concentration of 0.5 mg/ml;
2) preparation of control solutions
Taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution to obtain a control solution with the concentration of 5 mu g/ml;
3) injecting equal volumes of the sample solution in the step 1) and the contrast solution in the step 2) into a high performance liquid chromatograph, performing gradient elution by using a mobile phase A and a mobile phase B as mobile phases, recording a chromatogram, calculating the content of impurities according to a self contrast method,
the chromatographic column stationary phase filler of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, and the mobile phase A is methanol, acetonitrile and 0.1% acid solution in a volume ratio of 4:1:15, and the mobile phase B is methanol, acetonitrile, 0.1% acid solution according to a volume ratio of 18:1:1, the mobile phase enters a chromatographic column by adopting a gradient elution mode for 0 minute, the volume percentage of the mobile phase A is 90-100 percent, and the volume percentage of the mobile phase B is 0-10 percent; 0 to 5 minutes, the volume percentage of the mobile phase A is 90 to 100 percent, and the volume percentage of the mobile phase B is 0 to 10 percent; from 5 minutes to 60 minutes, the volume percentage of mobile phase a decreased linearly to 50% -55%, and the volume percentage of mobile phase B increased linearly to 45% -50%; from 60 minutes to 61 minutes, the volume percentage of mobile phase a increased linearly to 90% -100%, the volume percentage of mobile phase B decreased linearly to 0% -10%; the volume percentage of the mobile phase A is 90-100 percent and the volume percentage of the mobile phase B is 0-10 percent in 61-70 minutes.
The impurities referred to herein refer to impurities introduced during synthesis or impurities resulting from degradation of a drug substance, including starting materials and the like, and also include impurities of known structure and impurities of unknown structure. The separation and determination of capecitabine and impurities thereof refers to the separation and determination of capecitabine bulk drug and impurities containing capecitabine or related substances, and the impurities with known structures comprise: : 5-fluorocytosine (impurity A-SM1 for short), 5 '-deoxy-5-fluorocytosine nucleoside (impurity A-Z1 for short), 5' -deoxy-5-fluorouracil nucleoside (impurity A-Z2 for short), 5 '-deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine (impurity A-Z3 for short), 3' -O- (5 '-deoxy-. beta. -D-ribofuranosyl) capecitabine (impurity A-Z5 for short), pentyl 5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate (impurity A-Z10 for short), 2',3 '-O-carbonyl-5' -deoxy-5-fluoro-N4- [ (pentyloxy) carbonyl ] cytosine Nucleoside (impurity A-Z11 for short), 1- (5-deoxy-. beta. -D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidinecarbamic acid methyl ester (impurity A-Z13 for short), 2',3 '-di-O-acetyl-5' -deoxy-5-fluorocytidine (referred to as impurity A-intermediate A1), 5 '-deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-2' -diacetate (referred to as impurity A-Z8), and 5 '-deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-3' -diacetate (referred to as impurity A-Z9).
Further, in the step 1) and the step 2), the diluent is methanol, acetonitrile and water according to a volume ratio of 4:1:15, and (3) mixing.
Preferably, the chromatographic column of the high performance liquid chromatograph in the step 3) is Agilent ZORBAX Eclipse PlusC18, 5 μm, 4.6mm × 250 mm.
Preferably, the mobile phase in the step 3) enters the chromatographic column by adopting a gradient elution mode, wherein the volume percentage of the mobile phase A is 98 percent, and the volume percentage of the mobile phase B is 2 percent for 0 minute; from 0 minute to 5 minutes, with a volume percent of mobile phase a of 98% and a volume percent of mobile phase B of 2%; from 5 minutes to 60 minutes, the volume percent of mobile phase a decreased linearly to 52%, and the volume percent of mobile phase B increased linearly to 48%; from 60 minutes to 61 minutes, the volume percent of mobile phase a increased linearly to 98%, and the volume percent of mobile phase B decreased linearly to 2%; from 61 minutes to 70 minutes, the volume percent of mobile phase a was 98% and the volume percent of mobile phase B was 2%.
Further, the acid solution in step 3) is perchloric acid, acetic acid or trifluoroacetic acid.
Further, the detection wavelength of the high performance liquid chromatograph in the step 3) is 220-280nm, and the temperature of the chromatographic column is 35-45 ℃.
Further, the flow rate of the mobile phase of the high performance liquid chromatograph in the step 3) is 0.5-1.5ml/min, and the sample solution and the control solution have the volume of 20 mu L of sample introduction.
Further, in step 3), before the sample solution and the control solution are introduced, 5-fluorocytosine, 5 '-deoxy-5-fluorocytosine nucleoside, 5' -deoxy-5-fluorouracil nucleoside, 5 '-deoxy-5-fluoro-N- [ (2-methylbut) carbonyl ] cytidine, 3' -O- (5 '-deoxy-. beta. -D-ribofuranosyl) capecitabine, pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate, 2',3 '-O-carbonyl-5' -deoxy-5-fluoro-N4- [ (pentyloxy) carbonyl ] cytidine, 1- (5-deoxy-. beta. -D-ribosyl) -5-fluoro- Methyl 1, 2-dihydro-2-oxo-4-pyrimidinecarbamate, 2',3' -di-O-acetyl-5 ' -deoxy-5-fluorocytidine, 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-2 ' -diacetate, and 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-3 ' -diacetate were dissolved in a diluent to 0.4 to 0.6mg/mL, respectively, to obtain impurity control solutions, a part of each sample solution was taken and mixed with each of the impurity control solutions to obtain an impurity-added solution, the impurity-added solution was injected into a high performance liquid chromatograph, a chromatogram was recorded, and the degree of separation was confirmed, wherein the separation degree of 5 '-deoxy-5-fluorouracil nucleoside from 1- (5-deoxy-beta-D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidinecarbamic acid methyl ester is not less than 2.0, and the separation degree of 5' -deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine from capecitabine is not less than 2.0.
Adopt above-mentioned technical scheme to have following beneficial effect:
1. the separation and determination method provided by the invention is used for determining capecitabine and related substances thereof under the same liquid phase condition, the main peak retention time (about 44 min) of capecitabine is appropriate, impurities can be effectively separated, the separation degree is high (the separation degree of capecitabine and adjacent nearest impurities is 4.6), the specificity is strong, the detection structure is accurate and reliable, and the method is a simple, convenient and reasonable detection method.
2. The separation and determination method adopts a gradient elution mode of a mobile phase A (methanol, acetonitrile and 0.1% acid solution are mixed according to a volume ratio of 4:1:15) and a mobile phase B (methanol, acetonitrile and 0.1% acid solution are mixed according to a volume ratio of 18:1:1) to determine impurities of capecitabine and a preparation thereof. The mobile phase A and the mobile phase B are mixed solutions of three solvents, so that the separation degree is increased, and impurities are effectively separated; gradient elution is adopted to ensure that capecitabine is effectively separated from impurities and impurities are effectively separated from impurities.
3. The diluent used by the separation and determination method of the invention is methanol, acetonitrile and water according to the volume ratio of 4:1:15, the interference of solvent peaks and solvent effects can be effectively eliminated.
4. The separation and determination method of the invention prepares the impurity sample adding solution and performs determination under the same liquid phase condition, the separation degree of the impurity 5 '-deoxy-5-fluorouracil nucleoside from the impurity 1- (5-deoxy-beta-D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidine methyl carbamate is not less than 2.0, the separation degree of the impurity 5' -deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine from capecitabine is not less than 2.0, and the separation degrees of the main peak, adjacent impurities and impurities meet the requirements.
The following further description is made with reference to the accompanying drawings and detailed description.
Drawings
FIG. 1 is a liquid chromatogram of a diluent according to an embodiment;
FIG. 2 is a liquid chromatogram of an impurity loading solution of example one;
FIG. 3 is a liquid chromatogram of a control solution of the example;
FIG. 4 is a liquid chromatogram of a sample solution of example one.
Detailed Description
Example one
Apparatus and conditions
According to high performance liquid chromatography (China pharmacopoeia 2015 edition regulation 0512), using Agilent ZORBAXeclipse Plus C18, 5 μm, 4.6 × 250mm as chromatographic column, methanol-acetonitrile-0.1% trifluoroacetic acid solution (4:1:15) as mobile phase A; methanol-acetonitrile-0.1% trifluoroacetic acid solution (18:1:1) as mobile phase B, gradient elution was performed according to Table 1 at a flow rate of 1.0mL per minute, a column temperature of 40 ℃ and a detection wavelength of 250 nm.
TABLE 1
| Time (min) | Mobile phase a (% by volume) | Mobile phase B (% by volume) |
| 0 | 98 | 2 |
| 0-5 | 98 | 2 |
| 5-60 | 52 | 48 |
| 60-61 | 98 | 2 |
| 61-70 | 98 | 2 |
Preparing sample solution, control solution and impurity sample-adding solution
An appropriate amount of capecitabine (purchased from san Jose, Inc., Chongqing) was dissolved in a diluent (methanol: acetonitrile: water 4:1:15) and diluted to a solution having a concentration of 0.5mg/ml, to obtain a sample solution. An appropriate amount of the sample solution was precisely measured, and diluted with a diluent (methanol: acetonitrile: water 4:1:15) to a solution having a concentration of 5. mu.g/ml, to obtain a control solution.
Respectively taking 5-fluorocytosine (referred to as impurity A-SM1), 5 '-deoxy-5-fluorocytosine nucleoside (referred to as impurity A-Z1), 5' -deoxy-5-fluorouracil nucleoside (referred to as impurity A-Z2), 5 '-deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine (referred to as impurity A-Z3), 3' -O- (5 '-deoxy-beta-D-ribofuranosyl) capecitabine (referred to as impurity A-Z5), (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) pentyl carbamate (referred to as impurity A-Z10), 2',3 '-O-carbonyl-5' -deoxy-5-fluoro-N4- [ (pentyloxy) carbonyl ] Cytosine nucleoside (referred to as impurity A-Z11), methyl 1- (5-deoxy-. beta. -D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidinecarbamate (referred to as impurity A-Z13), 2',3' -di-O-acetyl-5 ' -deoxy-5-fluorocytidine (referred to as impurity A-intermediate A1), 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-2 ' -diacetate (referred to as impurity A-Z8), 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-3 ' -diacetate (referred to as impurity A-Z9) (these impurities are all available from san Jongqing industries, Ltd.), precisely weighing, and dissolving with diluent (methanol: acetonitrile: water 4:1:15) to concentration of 0.40-0.60mg/ml to obtain impurity reference solutions; and mixing a proper amount of sample solution with the impurity reference substance solutions to obtain an impurity sample adding solution.
Measurement Process
And (3) respectively injecting 20 mu L of diluent (methanol: acetonitrile: water is 4:1:15), impurity sample adding solution, control solution and sample solution into a high performance liquid chromatograph, and recording corresponding chromatograms, wherein according to a self-control method, the impurities A-Z1 are not more than 0.3%, the impurities A-Z2 are not more than 0.3%, the impurities A-Z11 are not more than 0.05%, other single unknown impurities are not more than 0.05%, and the total impurities are not more than 0.5%, and the results are respectively shown in figure 1, figure 2, figure 3 and figure 4. As shown in the attached figure 2, the impurities A-SM1, A-Z1, A-Z2, A-Z13, A-intermediate A1, A-Z10, A-Z3, A-Z5, A-Z11, A-Z8 and A-Z9 are respectively in the order of peak appearance.
FIG. 1 shows that the diluent and chromatographic system do not interfere with the assay;
fig. 2 shows that the separation and determination method of the present invention can effectively separate impurities with unknown structures and impurities with known structures which may exist in capecitabine, and the detection sensitivity of each impurity can meet the requirements, the separation degree of the impurity a-Z2 and the impurity a-Z13 is not less than 2.0, the separation degree of the impurity a-Z3 and the capecitabine is not less than 2.0, and the separation degree of the main peak, the adjacent impurities and each impurity meets the requirements, i.e., the separation and determination method of the present invention can be used for the determination of impurities of capecitabine and preparations thereof.
FIGS. 3 and 4 show that the capecitabine sample tested contained 0.035% for impurity A-Z1, 0.045% for impurity A-Z2, 0.004% for impurity A-Z13, 0.005% for impurity A-Z10, 0.028% for impurity A-Z3, 0.018% for impurity A-Z11, 0.011% for impurity A-Z8, 0.014% for impurity A-Z9, and the remaining known impurities were not detected, 0.014% for the other single maximum impurity, and 0.199% for total impurities.
Claims (8)
1. A method for separating and measuring capecitabine and impurities thereof is characterized by comprising the following steps,
1) preparation of sample solution
Taking capecitabine or a preparation containing capecitabine, and adding a diluent to dissolve the capecitabine or the preparation containing capecitabine to obtain a sample solution with the concentration of 0.5 mg/ml;
2) preparation of control solutions
Taking the sample solution obtained in the step 1), and adding a diluent to dilute the sample solution to obtain a control solution with the concentration of 5 mu g/ml;
3) injecting equal volumes of the sample solution in the step 1) and the contrast solution in the step 2) into a high performance liquid chromatograph, performing gradient elution by using a mobile phase A and a mobile phase B as mobile phases, recording a chromatogram, calculating the content of impurities according to a self contrast method,
the chromatographic column stationary phase filler of the high performance liquid chromatograph is octadecylsilane chemically bonded silica, and the mobile phase A is methanol, acetonitrile and 0.1% acid solution in a volume ratio of 4:1:15, and the mobile phase B is methanol, acetonitrile, 0.1% acid solution according to a volume ratio of 18:1:1, the mobile phase enters a chromatographic column by adopting a gradient elution mode for 0 minute, the volume percentage of the mobile phase A is 90-100 percent, and the volume percentage of the mobile phase B is 0-10 percent; 0 to 5 minutes, the volume percentage of the mobile phase A is 90 to 100 percent, and the volume percentage of the mobile phase B is 0 to 10 percent; from 5 minutes to 60 minutes, the volume percentage of mobile phase a decreased linearly to 50% -55%, and the volume percentage of mobile phase B increased linearly to 45% -50%; from 60 minutes to 61 minutes, the volume percentage of mobile phase a increased linearly to 90% -100%, the volume percentage of mobile phase B decreased linearly to 0% -10%; the volume percentage of the mobile phase A is 90-100 percent and the volume percentage of the mobile phase B is 0-10 percent in 61-70 minutes.
2. The method according to claim 1, wherein the diluent in the steps 1) and 2) is methanol, acetonitrile, and water in a volume ratio of 4:1:15, and (3) mixing.
3. The method of claim 1, wherein the column of the high performance liquid chromatograph of step 3) is Agilent ZORBAX Eclipse Plus C18, 5 μm, 4.6mm x 250 mm.
4. The method according to claim 1, wherein the mobile phase in step 3) enters the chromatographic column by adopting a gradient elution mode, wherein the volume percentage of the mobile phase A is 98 percent and the volume percentage of the mobile phase B is 2 percent in 0 minute; from 0 minute to 5 minutes, with a volume percent of mobile phase a of 98% and a volume percent of mobile phase B of 2%; from 5 minutes to 60 minutes, the volume percent of mobile phase a decreased linearly to 52%, and the volume percent of mobile phase B increased linearly to 48%; from 60 minutes to 61 minutes, the volume percent of mobile phase a increased linearly to 98%, and the volume percent of mobile phase B decreased linearly to 2%; from 61 minutes to 70 minutes, the volume percent of mobile phase a was 98% and the volume percent of mobile phase B was 2%.
5. The method according to claim 1, wherein the acid solution of step 3) is perchloric acid, acetic acid or trifluoroacetic acid.
6. The method as claimed in claim 1, wherein the detection wavelength of the HPLC of step 3) is 220-280nm, and the temperature of the chromatographic column is 35-45 ℃.
7. The method of claim 1, wherein the flow rate of the mobile phase of the high performance liquid chromatograph in step 3) is 0.5-1.5ml/min, and the sample solution and the control solution have a volume of 20 μ L.
8. The method according to claim 1, wherein the sample solution and the control solution are precisely weighed in step 3) before the sample solution and the control solution, and 5-fluorocytosine, 5 '-deoxy-5-fluorocytosine nucleoside, 5' -deoxy-5-fluorouracil nucleoside, 5 '-deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine, 3' -O- (5 '-deoxy- β -D-ribofuranosyl) capecitabine, pentyl (5-fluoro-2-oxo-1, 2-dihydropyrimidin-4-yl) carbamate, 2',3 '-O-carbonyl-5' -deoxy-5-fluoro-N4- [ (pentyloxy) carbonyl ] cytosine nucleoside, and the like, 1- (5-deoxy-. beta. -D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidinecarbamic acid methyl ester, 2',3' -di-O-acetyl-5 ' -deoxy-5-fluorocytidine, 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-2 ' -diacetate, 5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl ] cytidine-3 ' -diacetate were dissolved in a diluent to 0.4 to 0.6mg/mL, respectively, to obtain impurity control solutions, a part of the sample solution was taken and mixed with the impurity control solutions to obtain impurity-added solutions, injecting the impurity sample solution into a high performance liquid chromatograph, recording a chromatogram, and confirming the separation degree, wherein the separation degree of 5 '-deoxy-5-fluorouracil and 1- (5-deoxy-beta-D-ribosyl) -5-fluoro-1, 2-dihydro-2-oxo-4-pyrimidine carbamate is more than or equal to 2.0, and the separation degree of 5' -deoxy-5-fluoro-N- [ (2-methylbutane) carbonyl ] cytidine and capecitabine is more than or equal to 2.0.
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