CN117686628A - Method for detecting thiamphenicol genotoxic impurities - Google Patents
Method for detecting thiamphenicol genotoxic impurities Download PDFInfo
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- CN117686628A CN117686628A CN202311666085.2A CN202311666085A CN117686628A CN 117686628 A CN117686628 A CN 117686628A CN 202311666085 A CN202311666085 A CN 202311666085A CN 117686628 A CN117686628 A CN 117686628A
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- 229960003053 thiamphenicol Drugs 0.000 title claims abstract description 47
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 title claims abstract description 47
- 239000012535 impurity Substances 0.000 title claims abstract description 40
- 231100000024 genotoxic Toxicity 0.000 title claims abstract description 36
- 230000001738 genotoxic effect Effects 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 32
- 239000000243 solution Substances 0.000 claims abstract description 48
- 238000012360 testing method Methods 0.000 claims abstract description 33
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000012488 sample solution Substances 0.000 claims abstract description 19
- 239000013558 reference substance Substances 0.000 claims abstract description 15
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007864 aqueous solution Substances 0.000 claims abstract description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims abstract description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 6
- 238000010828 elution Methods 0.000 claims abstract description 5
- 238000010812 external standard method Methods 0.000 claims abstract description 4
- 239000000945 filler Substances 0.000 claims abstract description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 3
- 239000000741 silica gel Substances 0.000 claims abstract 2
- 229910002027 silica gel Inorganic materials 0.000 claims abstract 2
- 239000000523 sample Substances 0.000 claims description 41
- 238000002347 injection Methods 0.000 claims description 13
- 239000007924 injection Substances 0.000 claims description 13
- 238000001514 detection method Methods 0.000 claims description 12
- 239000012088 reference solution Substances 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 3
- 239000003814 drug Substances 0.000 abstract description 7
- 229940079593 drug Drugs 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- JXTHNDFMNIQAHM-UHFFFAOYSA-N dichloroacetic acid Chemical compound OC(=O)C(Cl)Cl JXTHNDFMNIQAHM-UHFFFAOYSA-N 0.000 description 40
- 239000003085 diluting agent Substances 0.000 description 23
- 229960005215 dichloroacetic acid Drugs 0.000 description 20
- 239000011550 stock solution Substances 0.000 description 11
- 238000005303 weighing Methods 0.000 description 11
- 239000012085 test solution Substances 0.000 description 10
- 238000007865 diluting Methods 0.000 description 7
- 239000012467 final product Substances 0.000 description 7
- 229960005091 chloramphenicol Drugs 0.000 description 5
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 5
- 238000011084 recovery Methods 0.000 description 5
- 239000012490 blank solution Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000037396 body weight Effects 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
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- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010057190 Respiratory tract infections Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 208000001848 dysentery Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 231100000590 oncogenic Toxicity 0.000 description 1
- 230000002246 oncogenic effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000011003 system suitability test Methods 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 208000019206 urinary tract infection Diseases 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for detecting thiamphenicol genotoxic impurities, which comprises the steps of respectively preparing thiamphenicol test sample solution and genotoxic impurity reference substance solution; respectively injecting thiamphenicol test sample solution and genotoxic impurity reference substance solution into a liquid chromatograph, and calculating the content of genotoxic impurities in thiamphenicol by adopting an external standard method; the liquid chromatography conditions were: the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler, the mobile phase adopts a gradient elution method, the mobile phase A is phosphoric acid aqueous solution, the mobile phase B is acetonitrile or methanol, and the volume percentage of the mobile phase A and the mobile phase B is always kept 100%. The method has the advantages of high sensitivity, good repeatability, high accuracy and the like, so as to control the quality of the thiamphenicol bulk drug, and has great application value and economic value.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a method for detecting dichloroacetic acid which is a genotoxic impurity in thiamphenicol.
Background
Thiamphenicol has the chemical name: d-threo-2-dichloroacetamido-1- (4-methylsulfonylphenyl) -1, 3-propanediol having the structural formula:
the chemical formula: c (C) 12 H 15 C l2 NO 5 S
Molecular weight: 356.222
Thiamphenicol is chloramphenicol antibiotic, has antibacterial effect 3-5 times greater than chloramphenicol, and has immunosuppression 6 times greater than chloramphenicol, and is clinically used for treating respiratory tract infection, urinary tract infection, liver and gall infection, intestinal tract infection, surgical infection, typhoid fever, dysentery, meningitis, etc. Toxicity similar to that of chloramphenicol which generates aplastic anemia after long-term administration has not been found in clinical long-term use. The medicine is orally taken and absorbed well, and the blood concentration can reach peak after 2 hours, and the medicine is higher and lasting than chloramphenicol, and has slight side effect.
According to the synthetic process route of thiamphenicol raw material, there is a risk of generating genotoxic impurity dichloroacetic acid by hydrolysis, and no report on a detection method of dichloroacetic acid impurity in thiamphenicol is seen so far, so that it is highly required to develop an analysis method for rapidly and effectively detecting genotoxic impurity dichloroacetic acid so as to control the quality of thiamphenicol raw material.
Disclosure of Invention
The invention aims to provide a method for separating genotoxic impurity dichloroacetic acid from thiamphenicol, so as to rapidly and effectively detect potential genotoxic impurities in thiamphenicol bulk drugs, control the quality of the thiamphenicol bulk drugs and have larger application value and economic value.
According to the synthetic process route of thiamphenicol raw material, there is risk of hydrolysis to produce genotoxic impurity dichloroacetic acid, and its structural formula is as follows:
according to ICH-M7, the acceptable intake of a compound specificity can be calculated from rodent oncogenic potency data, such as TD50 (the dose administered resulting in 50% tumor incidence). The TD50 value is extrapolated linearly to an incidence of one-ten-thousandth (i.e., life risk level) by simply dividing it by 50000. This method is similar to the derivation method used by TTC. The TD50 of dichloroacetic acid was found to be 161mg/kg body weight/day (rat) and 119mg/kg body weight/day (mouse). Lower mouse values (i.e., more conservative) were used in calculating acceptable intake. To derive the dose of carcinogens in one ten thousandth, this value is divided by 50000:119 mg/kg/50000 = 2.38 μg/kg; total daily intake was deduced as: 2.38 μg/kg×50kg=119 μg (per 50kg body weight); thus, daily intake of dichloroacetic acid during life was 119. Mu.g/day; according to the clinical dosage in the specification of the thiamphenicol preparation, the maximum daily oral dosage of the thiamphenicol preparation is 3g. The maximum limit of dichloroacetic acid in thiamphenicol is as follows: 119 μg/3 g=39.7 ppm.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method for detecting genetic toxin impurities of thiamphenicol adopts high performance liquid chromatography to detect the genetic toxin impurities of dichloroacetic acid in thiamphenicol, and the detection method comprises the following steps:
(1) Respectively preparing thiamphenicol test sample solution and genotoxic impurity reference substance solution;
(2) Injecting the thiamphenicol test sample solution and the genotoxic impurity reference substance solution obtained in the step (1) into a liquid chromatograph in a distributed manner to finish the determination of genotoxic impurities; calculating the content of genotoxic impurities in thiamphenicol by adopting an external standard method;
further, the genotoxic impurity is dichloroacetic acid;
further, the liquid chromatography conditions of the step (2) are as follows:
chromatographic column: octadecylsilane chemically bonded silica is adopted as a filler;
the mobile phase adopts a gradient elution method, the mobile phase A is phosphoric acid aqueous solution, the mobile phase B is acetonitrile or methanol,
further, the volume percentage of the mobile phase A and the mobile phase B is always kept 100%;
as a preferred embodiment, the phosphoric acid aqueous solution in the mobile phase a is an aqueous solution with a volume fraction of 0.08% to 0.12%, and most preferably is an aqueous solution of 0.1% phosphoric acid.
Further, mobile phase B was acetonitrile.
Further, the flow rate of the mobile phase in the method is 0.5 to 1.5ml/min, and most preferably 1.0ml/min.
Further, in the method, the column temperature of the chromatographic column is 30-35 ℃, and most preferably 30 ℃; the detector wavelength was 210nm.
Further, the diluent is 50% to 100% acetonitrile aqueous solution, most preferably 60% acetonitrile aqueous solution.
In the step (2), octadecylsilane chemically bonded silica is adopted as a filler in a chromatographic column for high performance liquid chromatography, and the length of the chromatographic column is 150-250 mm, preferably 250mm; the inner diameter is 3.0-4.6 mm, preferably 4.6mm; the particle size is 2.7 to 5. Mu.m, preferably 5. Mu.m.
Further, the gradient elution procedure in the method is as follows:
| time (min) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 85 | 15 |
| 10 | 85 | 15 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 30 | 85 | 15 |
Further, in order to ensure the quality of the product, the limit of impurities is kept below 35ppm, and the concentration of the sample solution of the dichloroacetic acid reference substance is prepared to be 0.4-1.0 mug/ml, and most preferably 0.6 mug/ml. The preparation concentration of the thiamphenicol test sample is 10-25 mg/ml. Most preferably 20mg/ml. The final limit was set at 30ppm.
And further, in the step 2), the peak areas of the thiamphenicol test sample and the dichloroacetic acid reference substance are used as the basis, and if the peak area of the thiamphenicol test sample in the detection is smaller than the peak area of the dichloroacetic acid reference substance, the test sample is judged to be qualified. The content formula for calculating genotoxic impurities in thiamphenicol by an external standard method is as follows:
A sam : peak area of the test solution;
A std : peak area of control solution;
C std : concentration of control solution, mg/mL;
W s : sample weight of the test sample, mg;
V s : dilution volume of test solution, mL.
The limit of detection and the limit of quantification of the genotoxic impurities meet the limit requirements (LOQ is 10ppm LOD is 5 ppm).
The beneficial effects are that:
the method of the invention adopts a high-sensitivity and high-durability chromatographic method, and can effectively separate the genotoxic impurity dichloroacetic acid in the thiamphenicol from other substances in the test sample by adopting simple chromatographic conditions, thereby having the advantages of short analysis time, high sensitivity, good repeatability, high accuracy and the like, and being capable of rapidly and effectively detecting the genotoxic impurity in the thiamphenicol bulk drug, thereby solving the problem that the genotoxic impurity is difficult to separate and measure, and further ensuring the quality controllability of the thiamphenicol bulk drug in mass production.
Drawings
Fig. 1: blank solvent liquid chromatogram;
fig. 2: a control solution liquid chromatogram;
fig. 3: liquid chromatogram of the sample solution;
fig. 4:100% adding standard sample solution liquid chromatogram;
fig. 5: a linear graph.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
Example 1
System suitability test
Instrument parameter settings are recorded by a list, see table 1.
TABLE 1
Blank solution/diluent: 60% acetonitrile in water;
control solution: taking about 50.0mg of dichloroacetic acid reference substance, precisely weighing, placing into a 50ml brown measuring flask, dissolving the diluent, fixing volume, and shaking uniformly. Precisely weighing 1.0ml, placing into a 100ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference stock solution. Precisely measuring 3.0ml of reference stock solution into a 50ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference solution. 2 parts are arranged in parallel.
The sample injection sequence is shown in Table 2.
TABLE 2
| Solution name | Number of sample injection needles |
| Blank space | ≥1 |
| Control solution 1 | 5 |
| Control solution 2 | 2 |
System applicability requirements:
the blank solution has no interference to the peak of the sample; the blank solvent chromatogram is shown in FIG. 1.
The RSD of the continuous 5-needle main peak area of the reference substance solution 1 is not more than 6%; the reference solution is shown in figure 2.
The recovery rate of the response factor between the control solution 2 and the first needle control solution 1 should be [85.0%,110.0% ].
The system applicability results are shown in Table 3.
TABLE 3 Table 3
Conclusion: the system applicability meets the requirements. The chromatograms are shown in FIGS. 1-3, respectively.
Example 2
Detection and quantification limit
Preparing a solution: quantitative limiting solution: taking about 50.0mg of dichloroacetic acid reference substance, precisely weighing, placing into a 50ml brown measuring flask, dissolving the diluent, fixing volume, and shaking uniformly. Precisely weighing 1.0ml, placing into a 100ml brown volumetric flask, dissolving the diluent, fixing volume, and shaking. (0.01 mg/mL), precisely measuring 1.0mL of the pre-solution, placing into a 50mL brown volumetric flask, diluting to scale with a diluent, and shaking uniformly to obtain the final product. (10 ppm)
Detection limit solution: diluting the LOQ solution by 2 times, and sampling to obtain the concentration of the LOD with the S/N of more than or equal to 3.
The quantitative limit was sampled in the order of table 4 below.
TABLE 4 Table 4
| Solution name | Number of sample injection needles |
| Blank space | ≥1 |
| LOQ solution | 6 |
The detection limits were sampled in the order of Table 5 below.
TABLE 5
| Solution name | Number of sample injection needles |
| Blank space | ≥1 |
| LOD solution | 1 |
The requirements are:
the quantitative limit S/N is more than or equal to 10;
the peak area RSD in the 6-needle quantitative limiting solution is less than or equal to 12 percent.
The detection limit S/N is more than or equal to 3
TABLE 6
Conclusion:
the detection limit was 0.1ug/ml (5 ppm), S/N=7.20
The limit of quantitation was 0.2ug/ml (10 ppm) and RSD was 8.67%
As is clear from the above, the detection limit and the quantitative limit of the method are both lower than the limit (30 ppm) and the sensitivity is high.
Example 3
Linear range test
Linear stock: taking about 50.0mg of dichloroacetic acid reference substance, precisely weighing, placing into a 50ml brown measuring flask, dissolving the diluent, fixing volume, and shaking uniformly. Precisely weighing 1.0ml, placing into a 100ml brown volumetric flask, dissolving the diluent, fixing volume, and shaking. (0.01 mg/ml)
60% linear solution: precisely measuring 1.0mL of linear stock solution, placing into a 50mL brown volumetric flask, diluting to scale with diluent, and shaking to obtain the final product. (10 ppm)
80% linear solution: precisely measuring 2.0mL of linear stock solution, placing into a 50mL brown volumetric flask, diluting to scale with diluent, and shaking to obtain the final product. (20 ppm)
100% linear solution: precisely measuring 3.0mL of linear stock solution, placing into a 50mL brown volumetric flask, diluting to scale with diluent, and shaking to obtain the final product. (30 ppm)
120% linear solution: precisely measuring 4.0mL of linear stock solution, placing into a 50mL brown volumetric flask, diluting to scale with diluent, and shaking to obtain the final product. (40 ppm)
140% linear solution: precisely measuring 5.0mL of linear stock solution, placing into a 50mL brown volumetric flask, diluting to scale with diluent, and shaking to obtain the final product. (50 ppm)
The sample injection was performed in the following sample injection order of table 7.
TABLE 7
The requirements are:
listing the linear equation, the linear correlation coefficient R 2 ≥0.990；
The data results are shown in Table 8 below.
TABLE 8
| Concentration (mg/ml) | Peak area |
| 0.00022 | 2.61 |
| 0.00043 | 4.72 |
| 0.00065 | 7.09 |
| 0.00086 | 9.09 |
| 0.00108 | 11.41 |
Conclusion: the linear equation is: y=10209 x+0.3917, linear correlation coefficient R 2 =0.999, the correlation coefficient is high in the linear range.
Repeatability test
Control solution: taking about 50.0mg of dichloroacetic acid reference substance, precisely weighing, placing into a 50ml brown measuring flask, dissolving the diluent, fixing volume, and shaking uniformly. Precisely weighing 1.0ml, placing into a 100ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference stock solution. Precisely measuring 3.0ml of reference stock solution into a 50ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference solution. 2 parts are arranged in parallel.
Test solution: about 200.0mg of the sample is taken, precisely weighed, placed in a 10ml brown measuring flask, dissolved by a diluent, fixed in volume and shaken uniformly to obtain the product. 6 parts were prepared in parallel.
(note: because the sample is unstable, the sample should be prepared at present)
The sample injection was performed in the sample injection order of table 9 below.
TABLE 9
| Solution name | Number of sample injection needles |
| Blank solution | ≥1 |
| Control solution 1 | 5 |
| Control solution 2 | 2 |
| Sample solution 1 | 1 |
| Sample solution 2 | 1 |
| Sample solution 3 | 1 |
| Sample solution 4 | 1 |
| Sample solution 5 | 1 |
| Sample solution 6 | 1 |
| Control solution 1 | 1 |
The requirements are:
the RSD of the content measurement result of 6 parts of the test solution should not exceed 16.0%. The chromatogram of the test solution is shown in figure 3.
The data results are shown in Table 10 below.
Table 10
Wherein:
the content formula of genotoxic impurities in thiamphenicol is as follows:
A sam : peak area of the test solution;
A std : peak area of control solution;
C std : concentration of control solution, mg/mL;
W s : sample weight of the test sample, mg;
V s : dilution volume of test solution, mL.
Conclusion:
the RSD of the content measurement result of 6 parts of test sample solution is 14.95%
Thus, the method has good repeatability in the range of the test concentration.
Example 4
Precision testing
Control solution: taking about 50.0mg of dichloroacetic acid reference substance, precisely weighing, placing into a 50ml brown measuring flask, dissolving the diluent, fixing volume, and shaking uniformly. Precisely weighing 1.0ml, placing into a 100ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference stock solution. Precisely measuring 3.0ml of reference stock solution into a 50ml brown volumetric flask, dissolving the diluent, fixing the volume, and shaking to obtain reference solution. 2 parts are arranged in parallel.
Test solution: taking about 200.0mg of thiamphenicol test sample, precisely weighing, placing into a 10ml brown measuring flask, dissolving the diluent, fixing the volume, and shaking uniformly to obtain the final product. 3 parts were prepared in parallel.
80% of the standard sample solution: about 200.0mg of the sample is taken, precisely weighed, placed in a 10ml brown measuring flask, and dissolved in 80% linear solution, fixed in volume and shaken uniformly to obtain the product. 3 parts were prepared in parallel.
100% of standard test solution: about 200.0mg of the sample is taken, precisely weighed, placed in a 10ml brown measuring flask, dissolved in 100% linear solution, fixed in volume and shaken uniformly to obtain 3 parts of parallel preparation.
120% of labeled test sample solution: about 200.0mg of the sample is taken, precisely weighed, placed in a 10ml brown measuring flask, and dissolved in 120% linear solution, fixed in volume and shaken uniformly to obtain the product. 3 parts were prepared in parallel.
(note: because the sample is unstable, the sample should be prepared at present)
The sample injection was performed in the sample injection order of table 11 below.
TABLE 11
| Solution name | Number of sample injection needles |
| Blank solution | ≥1 |
| Control solution 1 | 5 |
| Control solution 2 | 2 |
| Test solution-1 | 1 |
| Test solution-2 | 1 |
| Test solution-3 | 1 |
| 80% labeled test sample solution-1 | 1 |
| 80% labeled test sample solution-2 | 1 |
| 80% labeled test sample solution-3 | 1 |
| Control solution 1 | 1 |
| 100% labeled test sample solution-1 | 1 |
| 100% labeled test sample solution-2 | 1 |
| 100% labeled test sample solution-3 | 1 |
| 120% labeled test sample solution-1 | 1 |
| 120% labeled test sample solution-2 | 1 |
| 120% labeled test sample solution-3 | 1 |
| Control solution 1 | 1 |
The requirements are:
three solutions with the concentration of 80%, 100% and 120%, wherein three needles are used for each concentration, and the recovery rate of each needle is 85% -110%; RSD should not exceed 10.0%. The chromatogram of the 100% labeled test solution is shown in FIG. 4.
The data results are shown in Table 12 below.
Table 12
Conclusion:
concentration is 80 percent, standard adding recovery rate is 85 percent to 110 percent, and RSD is: 3.80%;
the concentration is 100 percent, the standard adding recovery rate is 85 percent to 110 percent, and the RSD is: 3.20%;
concentration is 120 percent, standard adding recovery rate is 85 percent to 110 percent, and RSD is: 0.49%;
from this, the accuracy of the method is high.
Claims (9)
1. The method for detecting the thiamphenicol genotoxic impurities is characterized by comprising the following steps of:
(1) Respectively preparing thiamphenicol test sample solution and genotoxic impurity reference substance solution;
(2) Respectively injecting the thiamphenicol test sample solution and the genotoxic impurity reference substance solution obtained in the step (1) into a liquid chromatograph, and calculating the content of genotoxic impurities in the thiamphenicol by adopting an external standard method;
wherein, the liquid chromatography conditions are as follows: the chromatographic column adopts octadecylsilane chemically bonded silica gel as filler, the mobile phase adopts a gradient elution method, the mobile phase A is phosphoric acid aqueous solution, the mobile phase B is acetonitrile or methanol, and the volume percentage of the mobile phase A and the mobile phase B is always kept 100%.
2. The method for detecting thiamphenicol genotoxic impurities according to claim 1, wherein the aqueous solution of mobile phase a phosphoric acid is an aqueous solution with a volume fraction of 0.08% to 0.12%.
3. The method for detecting thiamphenicol genotoxic impurities according to claim 1, wherein the gradient elution method is as follows:
4. the method for detecting thiamphenicol genotoxic impurities according to claim 1, wherein the chromatographic column has a length of 150-250 mm, an inner diameter of 3.0-4.6 mm, a particle diameter of 2.7-5 μm, a detection wavelength of 210nm, a column temperature of 30-35 ℃ and a flow rate of 0.5-1.5 ml/min.
5. The method for detecting a thiamphenicol genotoxic impurity according to claim 1, wherein the mobile phase a is acetonitrile.
6. The method for detecting a thiamphenicol genotoxic impurity according to claim 5, wherein the chromatographic column has a length of 250mm, an inner diameter of 4.6mm, a particle diameter of 5 μm, a detection wavelength of 210nm, a column temperature of 30℃and a flow rate of 1.0ml/min.
7. The method for detecting thiamphenicol genotoxic impurities according to claim 1, wherein the concentration of the thiamphenicol test sample solution is 10-25 mg/ml.
8. The method for detecting a genotoxic impurity by thiamphenicol according to claim 1, wherein the solubility of the genotoxic impurity reference solution is 0.4 to 1.0. Mu.g/ml.
9. The method for detecting a thiamphenicol genotoxic impurity according to claim 1, wherein the sample injection amount is 20. Mu.l.
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