CN113552239A - Method for determining formamide content in temozolomide bulk drug - Google Patents
Method for determining formamide content in temozolomide bulk drug Download PDFInfo
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- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 title claims abstract description 208
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229960004964 temozolomide Drugs 0.000 title claims abstract description 56
- 239000003814 drug Substances 0.000 title claims abstract description 50
- 238000000034 method Methods 0.000 title claims abstract description 31
- 229940079593 drug Drugs 0.000 title claims abstract description 15
- 239000000243 solution Substances 0.000 claims abstract description 45
- 238000005303 weighing Methods 0.000 claims abstract description 40
- 239000002994 raw material Substances 0.000 claims abstract description 35
- 238000007865 diluting Methods 0.000 claims abstract description 34
- 239000012085 test solution Substances 0.000 claims abstract description 26
- 239000012088 reference solution Substances 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 230000014759 maintenance of location Effects 0.000 claims abstract description 11
- 239000013558 reference substance Substances 0.000 claims abstract description 6
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- 239000011550 stock solution Substances 0.000 claims description 36
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 238000009210 therapy by ultrasound Methods 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 13
- 239000000523 sample Substances 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 10
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000002347 injection Methods 0.000 claims description 3
- 239000007924 injection Substances 0.000 claims description 3
- 238000004811 liquid chromatography Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 3
- 239000000741 silica gel Substances 0.000 claims description 3
- 229910002027 silica gel Inorganic materials 0.000 claims description 3
- 238000005259 measurement Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 9
- 239000012086 standard solution Substances 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 6
- 238000011084 recovery Methods 0.000 description 5
- 238000001228 spectrum Methods 0.000 description 4
- 238000010200 validation analysis Methods 0.000 description 4
- DVNYTAVYBRSTGK-UHFFFAOYSA-N 5-aminoimidazole-4-carboxamide Chemical compound NC(=O)C=1N=CNC=1N DVNYTAVYBRSTGK-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 206010018338 Glioma Diseases 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- IKZLMSPFYNDYIL-UHFFFAOYSA-N (5E)-5-diazoimidazole-4-carboxamide Chemical compound NC(=O)C1=NC=NC1=[N+]=[N-] IKZLMSPFYNDYIL-UHFFFAOYSA-N 0.000 description 1
- 208000003174 Brain Neoplasms Diseases 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 239000002295 alkylating antineoplastic agent Substances 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006193 diazotization reaction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 210000004914 menses Anatomy 0.000 description 1
- HAMGRBXTJNITHG-UHFFFAOYSA-N methyl isocyanate Chemical compound CN=C=O HAMGRBXTJNITHG-UHFFFAOYSA-N 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
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- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
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Abstract
The invention discloses a method for measuring formamide content in a temozolomide raw material medicine, which is characterized by comprising the following steps of: preparing a reference solution: precisely weighing formamide, and diluting with a mobile phase to prepare a reference substance solution; preparing a test solution: precisely weighing temozolomide raw material medicine, and diluting with a mobile phase to prepare a test solution; precisely measuring a reference solution and a test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of formamide in the temozolomide bulk drug by peak area according to an external standard method if a peak which is consistent with the retention time of the formamide peak exists in the chromatogram of the test solution; the method has the characteristics of simple process, low use cost, good precision, high accuracy and the like, and can quickly determine the content of formamide in the temozolomide bulk drug.
Description
Technical Field
The invention belongs to the technical field of medicines, relates to the field of medicine impurity detection and analysis, and particularly relates to a method for determining formamide content in a temozolomide raw material medicine.
Background
Temozolomide (Temozolomide) is an alkylating anticancer drug, pioneered by Can research venturs, uk, developed by Schering-Plough, approved by the FDA for 8 menses in 1999, approved for marketing in the united states and in many countries in europe. Has broad-spectrum antitumor activity [ L.H.Tsang, et al.cancer Chemother Pharmacol.27 (1991): 342-346], especially for gliomas (brain cancer) and melanomas (skin cancer). Temozolomide capsules have been approved in the united states and europe for the treatment of malignant gliomas.
The safety and effectiveness of medicines are important problems generally concerned by people at present, and the existing temozolomide synthesis process takes 5-amino-imidazole-4-formamide or hydrochloride thereof as a raw material, synthesizes 5-diazo-imidazole-4-formamide through diazotization, and then reacts with methyl isocyanate in the presence of a polar solvent DMSO to synthesize temozolomide. The synthesis process of the raw material 5-amino-imidazole-4-formamide is as follows:
formamide is used in the synthesis process of the raw material 5-amino-imidazole-4-formamide, and the raw material medicine of temozolomide has the possibility of formamide which belongs to a toxic substance. Temozolomide is an anticancer drug, as prescribed by the ICH M7 guidelines, and therefore the maximum daily intake limit for MIC given in terms of 1-10 years of its dosing cycle can be calculated as 10 μ g, and the maximum daily dose of temozolomide calculated as 400mg, is equivalent to a limit of 25ppm (0.0025%), so that the formamide limit in temozolomide is required to be no more than 25ppm (0.0025%). However, in the legal standard of temozolomide in China, a formamide inspection method in temozolomide is not adopted, the standard is lacked, no relevant literature reports exist at home and abroad, the medicine quality cannot be evaluated, and potential safety hazards exist.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide the method for detecting formamide in the temozolomide raw material medicine, which has the characteristics of simple process, low use cost, good precision, high accuracy and the like, and can quickly determine the content of formamide in the temozolomide raw material medicine.
The invention is realized by the following technical scheme:
preparing a reference solution: precisely weighing formamide, and diluting with a mobile phase to prepare a reference substance solution;
preparing a test solution: precisely weighing temozolomide raw material medicine, and diluting with a mobile phase to prepare a test solution;
precisely measuring a reference solution and a test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of formamide in the temozolomide bulk drug by peak area according to an external standard method if a peak consistent with the retention time of the formamide peak exists in the chromatogram of the test solution.
The invention further improves the scheme as follows:
the preparation process of the reference solution comprises the following steps: precisely weighing 60-70 mg of formamide, placing the formamide in a 100ml volumetric flask, diluting the formamide to a scale with a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide in the 100ml volumetric flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to serve as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml volumetric flask, adding the mobile phase to dilute to a scale, and shaking up to obtain the reference stock solution.
Further, the preparation process of the test solution is as follows: precisely weighing 25-35 mg of the temozolomide raw material medicine, placing the temozolomide raw material medicine in a 10ml volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 5min to dissolve the temozolomide raw material medicine, diluting the temozolomide raw material medicine to a scale with the mobile phase, and shaking up to obtain a sample solution.
Further, the mobile phase is acetonitrile and 0.01mol/L potassium dihydrogen phosphate solution with the mass ratio of 2: 98.
Further, the chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica gel column (250X 4.6mm, 5 μm) was used as an analytical column; mixing acetonitrile: 0.01mol/L potassium dihydrogen phosphate solution 2:98 is used as a mobile phase, the detection wavelength is 200nm, the flow rate is 0.5ml/min, the column temperature is 30 ℃, and the sample injection amount is 20 mul.
The invention has the beneficial effects that:
the method provided by the invention adopts liquid chromatography for separation and detection, has high equipment popularization rate and low use cost, and thus, the application occasion and the application range of the method can be effectively enlarged.
In the detection method provided by the invention, all the used reagents are pollution-free, the treatment processes of the reference substance and the test sample are simple, the use amount of the reference substance and the test sample is small, and the reference substance solution, the test sample solution and the self reference solution can be conveniently and quickly prepared.
Under the chromatographic condition provided by the invention, the separation degree between impurities and main components is good, the system stability and repeatability are good, the accuracy is high, the peak-off time is short, the analysis time is short, and the experiment cost is saved.
The method provided by the invention can be used for rapidly, effectively and simply measuring the impurity amount in the temozolomide, can be used for evaluating or evaluating the components of the medicine, avoids potential safety hazards, and can provide reliable data monitoring for the quality safety of the raw material medicine.
Tests are used for verifying the detection method provided by the invention, and the results show that the method has strong specificity, good precision and high accuracy, and can quickly determine the amount of formamide in the temozolomide bulk drug.
Drawings
FIG. 1 is a spectrum obtained in example 1;
FIG. 2 is a spectrum obtained in example 2;
FIG. 3 is a spectrum obtained in example 3;
FIG. 4 is a standard curve for formamide;
due to the limited space, the spectrum of the validation part of the method is not described in the specification.
Detailed Description
Instrumentation, reagents and sources of reagents
Examples 1 to 3
Liquid chromatography conditions:
octadecylsilane chemically bonded silica gel column (250X 4.6mm, 5 μm) was used as an analytical column; mixing acetonitrile: 0.01mol/L potassium dihydrogen phosphate solution 2:98 is used as a mobile phase, the detection wavelength is 200nm, the flow rate is 0.5ml/min, the column temperature is 30 ℃, and the sample injection amount is 20 mul.
Preparing a reference solution: precisely weighing 60-70 mg of formamide, placing the formamide in a 100ml volumetric flask, diluting the formamide to a scale with a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide in the 100ml volumetric flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to serve as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml volumetric flask, adding the mobile phase to dilute to a scale, and shaking up to obtain the reference stock solution.
Preparing a test solution: precisely weighing 25-35 mg of the temozolomide raw material medicine, placing the temozolomide raw material medicine in a 10ml volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 5min to dissolve the temozolomide raw material medicine, diluting the temozolomide raw material medicine to a scale with the mobile phase, and shaking up to obtain a sample solution.
Precisely measuring a reference solution and a test solution, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of formamide in the temozolomide bulk drug by peak area according to an external standard method if a peak consistent with the retention time of the formamide peak exists in the chromatogram of the test solution.
Sampling different batches of test solution for analysis, obtaining spectrograms as shown in figures 1 to 3, calculating the content of formamide, and obtaining the result shown in the following table.
TABLE 1 measurement of formamide in the samples
| Serial number | Batch number | Formamide (less than or equal to 0.022%) |
| Example 1 | 20191101 | Not detected out |
| Example 2 | 20191102 | Not detected out |
| Example 3 | 20191103 | Not detected out |
The experimental results show that formamide was not detected in any of the three batches.
Analytical method validation
1. Specificity
(1) Blank solvent: the mobile phase was taken as the blank solvent.
(2) Formamide positioning solution: taking about 66mg of formamide, accurately weighing, placing in a 100ml measuring flask, diluting to scale with a mobile phase, shaking up, accurately weighing 1ml, placing in a 100ml measuring flask, adding the mobile phase to dilute to scale, shaking up, and using as a reference stock solution (positioning solution).
(3) Control solution: precisely measuring 1ml of reference stock solution, placing in a 10ml measuring flask, adding mobile phase to dilute to scale, and shaking to obtain reference solution;
(4) test solution: taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, diluting to scale with the mobile phase, and shaking up to obtain a sample solution.
(5) Adding a standard solution into a test sample: taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding appropriate amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with mobile phase, shaking uniformly, and adding standard solution as sample.
Precisely measuring the solutions by 20 μ l each, injecting into a liquid chromatograph, and recording chromatogram, wherein the formamide peak retention time and the theoretical plate number are shown in Table 2.
TABLE 2 formamide Peak Retention time and theoretical plate number
| Solutions of | Retention time/min | Number of theoretical plate |
| Positioning solution | 5.535 | 10741 |
| Control solution | 5.533 | 12089 |
| Test solution | — | — |
| Sample adding solution | 5.539 | 12079 |
The results show that: the blank solvent and the test solution have no interference to the formamide determination, which indicates that the formamide determination method has good specificity.
2. Linearity and range
Precisely weighing formamide about 66mg, placing in a 100ml measuring flask, diluting with mobile phase to scale, shaking, precisely weighing 1ml, placing in a 100ml measuring flask, adding mobile phase to dilute to scale, shaking, and storing as reference stock solution.
Precisely measuring the reference stock solutions 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.2ml, 1.5ml and 2.0ml, respectively placing in 10ml measuring flasks, diluting to scale with mobile phase, and shaking to obtain solutions of Linear 1, Linear 2, Linear 3, Linear 4, Linear 5, Linear 6 and Linear 7.
Precisely measuring the above solution and the limit solution by 20 μ l each, injecting into a liquid chromatograph, and recording chromatogram. The linear concentration was used as the abscissa and the peak area was used as the ordinate to plot a standard curve, as shown in fig. 4, and a regression equation was calculated. The results are shown in Table 3.
TABLE 3 results of formamide linearity measurement
| — | Concentration (μ g/ml) | Peak area |
| Limit of quantification | 0.013776 | 884 |
| Linear 1 | 0.13776 | 5756 |
| Linearity 2 | 0.34440 | 15270 |
| Line 3 | 0.55104 | 26999 |
| Linearity 4 | 0.68880 | 33856 |
| Linear 5 | 0.82656 | 41158 |
| Linear 6 | 1.03320 | 51348 |
| Line 7 | 1.37760 | 69317 |
The results show that the linear relationship of formamide is good in the concentration range of 0.013776 mu g/ml-1.37760 mu g/ml. The regression equation: 50772x-988.35, correlation index: r2＝0.9992，
3. Detection limit and quantification limit
(1) Limit of quantification
And determining the quantitative limit by using a signal-to-noise ratio method, and determining the quantitative limit by using the corresponding concentration when the signal-to-noise ratio is 10: 1. Precisely weighing about 66mg of formamide, placing the formamide into a 100ml measuring flask, diluting the formamide to a scale by using a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide into the 100ml measuring flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to be used as a reference stock solution; precisely measuring 1ml of reference stock solution, placing in a 10ml measuring flask, diluting to scale with mobile phase, and shaking to obtain reference solution; precisely measuring 1ml, placing in a 50ml measuring flask, adding mobile phase to dilute to scale, shaking up, and using as formamide quantitative limiting solution with the concentration of 0.013776 μ g/ml, which is 2.09% of the limit.
(2) Detection limit
And determining the detection limit by using a signal-to-noise ratio method, and determining the detection limit by using the corresponding concentration when the signal-to-noise ratio is 3: 1. Precisely measuring 5ml of formamide quantitative limiting solution, placing the solution into a 15ml measuring flask, adding a mobile phase to dilute the solution to a scale, shaking the solution uniformly to serve as formamide detection limiting solution, wherein the concentration of the formamide detection limiting solution is 0.004592 mu g/ml and is 0.70 percent of the limit.
4. Accuracy of
Precisely weighing formamide about 66mg, placing in a 100ml measuring flask, diluting with mobile phase to scale, shaking, precisely weighing 1ml, placing in a 100ml measuring flask, adding mobile phase to dilute to scale, shaking, and storing as reference stock solution.
Precisely measuring 1ml of reference stock solution, placing the reference stock solution in a 50ml measuring flask, diluting the reference stock solution to a scale with a mobile phase, and shaking up to be used as formamide quantitative limit stock solution;
precisely measuring 1ml of reference stock solution, placing in a 10ml measuring flask, diluting to scale with mobile phase, and shaking to obtain reference solution;
taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, diluting to scale with the mobile phase, and shaking up to obtain blank test solution.
Taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 0.7ml of quantitative limit stock solution, diluting to scale with the mobile phase, shaking up, and taking as quantitative limit recovery solution (three parts are prepared in parallel);
taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 0.8ml of reference stock solution, diluting to scale with the mobile phase, shaking up, and taking as 80% recovery rate solution (preparing three parts in parallel);
taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with the mobile phase, shaking up, and taking as 100% recovery rate solution (preparing three parts in parallel);
taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1.2ml of reference stock solution, diluting to scale with the mobile phase, shaking up, and taking as 120% recovery rate solution (preparing three parts in parallel);
precisely measuring the above solutions at 20 μ l each, injecting into liquid chromatograph, and recording chromatogram. The formamide recovery and the content RSD (%) were calculated and the results are shown in Table 4.
TABLE 4 formamide accuracy measurement results
The result shows that the accuracy of the formamide content measuring method is good.
5. Precision degree
(1) Precision of the instrument
Precisely measuring 20 μ l of the reference solution, repeatedly injecting sample for 6 times, and recording chromatogram. The results of calculation of the formamide retention time and the RSD (%) of the peak area are shown in tables 5 to 7.
TABLE 5 measurement of precision of the apparatus JQ1322
| Control solution | Retention time/min | Peak area |
| 1 | 5.538 | 34158 |
| 2 | 5.541 | 33997 |
| 3 | 5.541 | 34049 |
| 4 | 5.539 | 34072 |
| 5 | 5.538 | 34144 |
| 6 | 5.539 | 34085 |
| Mean value of | 5.539 | 34084 |
| SD | 0.001 | 60.02 |
| RSD％ | 0.02 | 0.18 |
TABLE 6 measurement of precision of apparatus-JQ 1323
| Control solution | Retention time/min | Peak area |
| 1 | 5.065 | 41170 |
| 2 | 5.066 | 41125 |
| 3 | 5.066 | 41096 |
| 4 | 5.066 | 41034 |
| 5 | 5.066 | 40798 |
| 6 | 5.066 | 40897 |
| Mean value of | 5.066 | 41020 |
| SD | 0.000 | 144.16 |
| RSD％ | 0.01 | 0.35 |
TABLE 7 measurement of precision of the apparatus JQ1312
| Control solution | Retention time/min | Peak area |
| 1 | 6.565 | 30468 |
| 2 | 6.564 | 30566 |
| 3 | 6.565 | 30567 |
| 4 | 6.564 | 30512 |
| 5 | 6.564 | 30510 |
| 6 | 6.566 | 30371 |
| Mean value of | 6.565 | 30499 |
| SD | 0.001 | 73.14 |
| RSD％ | 0.01 | 0.24 |
The result shows that the instrument precision of the method is good.
(2) Repeatability of
Precisely weighing about 66mg of formamide, placing the formamide into a 100ml measuring flask, diluting the formamide to a scale by using a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide into the 100ml measuring flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to be used as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml measuring flask, adding mobile phase to dilute to scale, and shaking to obtain reference solution.
Taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with the mobile phase, shaking up to obtain a repetitive solution (test sample and standard solution), and preparing 6 parts in parallel.
Precisely measuring the above solutions each 20 μ l, injecting into liquid chromatograph, and recording chromatogram. The results are shown in Table 8.
TABLE 8 results of reproducibility measurement
The result shows that the formamide measuring and analyzing method has good repeatability.
(3) Intermediate precision
Precisely weighing about 66mg of formamide, placing the formamide into a 100ml measuring flask, diluting the formamide to a scale by using a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide into the 100ml measuring flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to be used as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml measuring flask, adding mobile phase to dilute to scale, and shaking to obtain reference solution.
Taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with the mobile phase, shaking uniformly, and preparing 6 parts in parallel as intermediate precision solution (test sample and standard solution).
Precisely measuring the above solutions at 20 μ l each, injecting into liquid chromatograph, and recording chromatogram. The RSD (%) of the formamide content of the test sample plus standard solution was determined by different analysts at different times and in different instruments according to the formamide in temozolomide assay. The results are shown in Table 9.
TABLE 9 results of intermediate precision measurement
The result shows that the intermediate precision of the formamide content measuring method is good.
6. Stability of solution
Taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding appropriate amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with mobile phase, and shaking up to obtain stable solution.
Precisely measuring 20 mu l of stable solution, injecting into a liquid chromatograph at intervals of 1h within 24 hours continuously, recording a chromatogram, and calculating the RSD of the formamide peak area. The results are shown in Table 10.
TABLE 10 results of solution stability measurements
The results show that the stability of the stabilizing solution is good within 24 hours.
7. Durability
Precisely weighing about 66mg of formamide, placing the formamide into a 100ml measuring flask, diluting the formamide to a scale by using a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide into the 100ml measuring flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to be used as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml measuring flask, adding mobile phase to dilute to scale, and shaking to obtain reference solution.
Taking about 30mg of temozolomide raw material medicine (batch number: 20191201), precisely weighing, placing in a 10ml measuring flask, adding a proper amount of mobile phase, performing ultrasonic treatment for 5min to dissolve, precisely adding 1ml of reference stock solution, diluting to scale with mobile phase, shaking uniformly, and using as durable solution (test sample and standard solution).
Precisely measuring the sample solution and the reference solution by 20 μ l each, injecting into a liquid chromatograph, and recording chromatogram. The chromatogram was recorded by varying the flow rate (0.5. + -. 0.1ml/min), the column temperature (30. + -. 5 ℃ C.) and the column, according to the formamide determination method. Calculating the content of the formamide (RSD%) in the sample and standard solution. The results are shown in Table 11.
TABLE 11 results of durability measurement
The results show that the formamide determination analysis method is very robust.
Summary of the methodological validation of the formamide content determination analysis
TABLE 12 summary of formamide assay methodological validation
Claims (5)
1. The method for measuring the formamide content of the temozolomide bulk drug is characterized by comprising the following steps:
preparing a reference solution: precisely weighing formamide, and diluting with a mobile phase to prepare a reference substance solution;
preparing a test solution: precisely weighing temozolomide raw material medicine, and diluting with a mobile phase to prepare a test solution;
precisely measuring a reference solution and a test solution by adopting a liquid chromatography, respectively injecting the reference solution and the test solution into a liquid chromatograph, recording a chromatogram, and calculating the content of formamide in the temozolomide bulk drug by using a peak area according to an external standard method if a peak which is consistent with the retention time of the formamide peak exists in the chromatogram of the test solution.
2. The method for determining the formamide content of a temozolomide bulk drug according to claim 1, which is characterized in that: the preparation process of the reference solution comprises the following steps: precisely weighing 60-70 mg of formamide, placing the formamide in a 100ml volumetric flask, diluting the formamide to a scale with a mobile phase, shaking up, precisely weighing 1ml of formamide, placing the formamide in the 100ml volumetric flask, adding the mobile phase to dilute the formamide to the scale, and shaking up to serve as a reference stock solution; precisely measuring 1ml of the reference stock solution, placing in a 10ml volumetric flask, adding the mobile phase to dilute to a scale, and shaking up to obtain the reference stock solution.
3. The method for determining the formamide content of a temozolomide bulk drug according to claim 1, which is characterized in that: the preparation process of the test solution comprises the following steps: precisely weighing 25-35 mg of the temozolomide raw material medicine, placing the temozolomide raw material medicine in a 10ml volumetric flask, adding a proper amount of mobile phase, carrying out ultrasonic treatment for 5min to dissolve the temozolomide raw material medicine, diluting the temozolomide raw material medicine to a scale with the mobile phase, and shaking up to obtain a sample solution.
4. The method for determining the formamide content of a temozolomide bulk drug according to any one of claims 1, 2 or 3, characterized in that: the mobile phase is acetonitrile and 0.01mol/L potassium dihydrogen phosphate solution with the mass ratio of 2: 98.
5. The method for determining the formamide content of a temozolomide bulk drug according to claim 1, which is characterized in that: the chromatographic conditions of the liquid chromatograph are as follows: octadecylsilane chemically bonded silica gel column (250X 4.6mm, 5 μm) was used as an analytical column; mixing acetonitrile: 0.01mol/L potassium dihydrogen phosphate solution 2:98 is used as a mobile phase, the detection wavelength is 200nm, the flow rate is 0.5ml/min, the column temperature is 30 ℃, and the sample injection amount is 20 mul.
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