CN117330680A - Method for evaluating florfenicol bulk drug - Google Patents
Method for evaluating florfenicol bulk drug Download PDFInfo
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- CN117330680A CN117330680A CN202311207186.3A CN202311207186A CN117330680A CN 117330680 A CN117330680 A CN 117330680A CN 202311207186 A CN202311207186 A CN 202311207186A CN 117330680 A CN117330680 A CN 117330680A
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- 229960003760 florfenicol Drugs 0.000 title claims abstract description 167
- AYIRNRDRBQJXIF-NXEZZACHSA-N (-)-Florfenicol Chemical compound CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CF)NC(=O)C(Cl)Cl)C=C1 AYIRNRDRBQJXIF-NXEZZACHSA-N 0.000 title claims abstract description 166
- 239000003814 drug Substances 0.000 title claims abstract description 73
- 229940079593 drug Drugs 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 39
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 claims abstract description 27
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 24
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 claims abstract description 18
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 18
- 239000012535 impurity Substances 0.000 claims abstract description 17
- 238000004949 mass spectrometry Methods 0.000 claims abstract description 7
- 238000010998 test method Methods 0.000 claims abstract description 5
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical group CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 111
- 238000012360 testing method Methods 0.000 claims description 97
- 239000000523 sample Substances 0.000 claims description 39
- 239000000243 solution Substances 0.000 claims description 37
- 239000010949 copper Substances 0.000 claims description 29
- 239000012488 sample solution Substances 0.000 claims description 28
- 238000002360 preparation method Methods 0.000 claims description 25
- 238000001514 detection method Methods 0.000 claims description 24
- 239000011550 stock solution Substances 0.000 claims description 24
- 239000000126 substance Substances 0.000 claims description 24
- 150000002500 ions Chemical class 0.000 claims description 22
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 21
- 229910052785 arsenic Inorganic materials 0.000 claims description 21
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 claims description 21
- 229910052793 cadmium Inorganic materials 0.000 claims description 21
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 claims description 21
- 229910052802 copper Inorganic materials 0.000 claims description 21
- 238000001819 mass spectrum Methods 0.000 claims description 21
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 claims description 21
- 229910052753 mercury Inorganic materials 0.000 claims description 21
- 238000001228 spectrum Methods 0.000 claims description 18
- 239000013582 standard series solution Substances 0.000 claims description 18
- 238000007865 diluting Methods 0.000 claims description 14
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 229940088679 drug related substance Drugs 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000012086 standard solution Substances 0.000 claims description 10
- 239000001307 helium Substances 0.000 claims description 9
- 229910052734 helium Inorganic materials 0.000 claims description 9
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 238000005070 sampling Methods 0.000 claims description 8
- 230000002378 acidificating effect Effects 0.000 claims description 7
- 238000010813 internal standard method Methods 0.000 claims description 7
- 238000010828 elution Methods 0.000 claims description 6
- 239000003623 enhancer Substances 0.000 claims description 6
- 239000007789 gas Substances 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000000945 filler Substances 0.000 claims description 5
- 239000002904 solvent Substances 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 238000011002 quantification Methods 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 238000000889 atomisation Methods 0.000 claims description 3
- 229910052732 germanium Inorganic materials 0.000 claims description 3
- GNPVGFCGXDBREM-UHFFFAOYSA-N germanium atom Chemical compound [Ge] GNPVGFCGXDBREM-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 238000004458 analytical method Methods 0.000 abstract description 9
- 238000002203 pretreatment Methods 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000002156 mixing Methods 0.000 description 15
- 238000005303 weighing Methods 0.000 description 11
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 239000002994 raw material Substances 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000009616 inductively coupled plasma Methods 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 6
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- AACILMLPSLEQMF-UHFFFAOYSA-N 2,2-dichloroethenyl 2-ethylsulfinylethyl methyl phosphate Chemical compound CCS(=O)CCOP(=O)(OC)OC=C(Cl)Cl AACILMLPSLEQMF-UHFFFAOYSA-N 0.000 description 4
- 238000012417 linear regression Methods 0.000 description 4
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 3
- 229960005091 chloramphenicol Drugs 0.000 description 3
- 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 3
- 238000010812 external standard method Methods 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 239000001257 hydrogen Substances 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 239000001301 oxygen Substances 0.000 description 3
- 229910052760 oxygen Inorganic materials 0.000 description 3
- 238000004451 qualitative analysis Methods 0.000 description 3
- 238000012113 quantitative test Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000004885 tandem mass spectrometry Methods 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- OTVAEFIXJLOWRX-NXEZZACHSA-N thiamphenicol Chemical class CS(=O)(=O)C1=CC=C([C@@H](O)[C@@H](CO)NC(=O)C(Cl)Cl)C=C1 OTVAEFIXJLOWRX-NXEZZACHSA-N 0.000 description 2
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 239000012490 blank solution Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- -1 florfenicol ion Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229960003053 thiamphenicol Drugs 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- G01N30/06—Preparation
-
- 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
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
Abstract
The invention discloses a method for evaluating a florfenicol bulk drug, which relates to the technical field of drug analysis and comprises the following steps: analyzing the main content of the florfenicol bulk drug by a high performance liquid chromatography test method, establishing automatic association scanning by liquid chromatography-tandem mass spectrometry to qualitatively compare the main content, and finally measuring impurities by inductively coupled plasma mass spectrometry. The pretreatment method is simple, low in cost, accurate and efficient in liquid chromatography, accurate in comparison of liquid chromatography-tandem mass spectrometry and three-stage mass spectrometry, capable of eliminating interference of false positive, high in sensitivity of the inductively coupled plasma mass spectrometry analyzer and capable of accurately analyzing impurities.
Description
Technical Field
The invention relates to the technical field of medicine analysis, in particular to a method for evaluating a florfenicol bulk drug.
Background
Florfenicol is widely used as a novel chloramphenicol broad-spectrum antibiotic special for animals, especially in animal husbandry and aquaculture. Florfenicol, also known as florfenicol, is a monofluoro derivative of thiamphenicol and is a novel broad-spectrum antibiotic for animals. The florfenicol has the advantages of high antibacterial activity, no potential aplastic anemia and the like, plays an important role in veterinary clinic compared with chloramphenicol and thiamphenicol, and becomes a main substitute after the forbidding of chloramphenicol. The florfenicol preparation used at present in China mainly comprises soluble powder, injection, premix and the like. The application purposes of different preparations are different, and the curative effects are different to a certain extent, so that the purity of each product is higher, the impurities are fewer, the curative effect is better, and the effect is more thoroughly exerted.
The florfenicol is difficult to dissolve in water and difficult to detect, so that the development of an evaluation technology for accurately and reliably using the florfenicol as a drug raw material is necessary, the safety of animal food is ensured, and the quality of animal food in China is improved.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for evaluating a florfenicol bulk drug, which has the advantages of simple pretreatment method, low cost, accurate and high-efficiency liquid chromatography, accurate comparison of liquid chromatography-tandem mass spectrometry and three-stage mass spectrometry, capability of eliminating false positive interference, high sensitivity of an inductively coupled plasma mass spectrometry analyzer and capability of accurately analyzing impurities.
The invention aims at providing a method for evaluating a florfenicol bulk drug.
The method for evaluating the florfenicol bulk drug comprises the following steps:
analyzing the main content of the florfenicol bulk drug by a high performance liquid chromatography test method, establishing automatic association scanning by liquid chromatography-tandem mass spectrometry to qualitatively compare the main content, and finally measuring impurities by inductively coupled plasma mass spectrometry.
Preferably, the method comprises:
(1) Standard solution preparation: dissolving a florfenicol standard substance to a constant volume to prepare a standard series solution with gradient concentration;
(2) Sample solution preparation: dissolving a florfenicol bulk drug sample, and then carrying out pretreatment and volume fixing to obtain a sample solution;
(3) High performance liquid chromatography test:
(3-1) respectively performing high performance liquid chromatography test on the standard series solutions with gradient concentration in the step (1), and establishing a florfenicol standard curve according to the test result;
(3-2) performing high performance liquid chromatography test on the sample solution obtained in the step (2), and taking the test result into the florfenicol standard curve obtained in the step (3-1) to obtain the main content of the florfenicol bulk drug;
(4) Liquid chromatography-tandem mass spectrometry test:
(4-1) further diluting the standard series solution with the gradient concentration in the step (1), respectively carrying out liquid chromatography-tandem mass spectrometry test, establishing a florfenicol standard curve according to a liquid chromatography test result, and establishing a three-level spectrum library according to a mass spectrometry test result;
(4-2) further diluting the sample solution obtained in the step (2), then carrying out liquid chromatography-tandem mass spectrometry test, and carrying the test result into the florfenicol standard curve obtained in the step (4-1) to obtain the main content of the florfenicol bulk drug; comparing the mass spectrum test result with the three-level spectrum chart library obtained in the step (4-1), and qualitatively comparing the main content of the florfenicol bulk drug;
(5) Inductively coupled plasma mass spectrometry testing: and quantitatively testing the impurity content in the florfenicol bulk drug sample by adopting an internal standard method.
Preferably, the step (1) includes:
(1-1) preparation of standard stock solution: dissolving, diluting and fixing the florfenicol standard substance to prepare a standard stock solution with the concentration of 100 mug/mL;
(1-2) diluting the standard stock solution prepared in the step (1-1) again, and fixing the volume to prepare a standard series solution with gradient concentration.
Preferably, the method comprises the steps of,
in step (1):
the solvent for dissolving the florfenicol standard is acetonitrile.
Preferably, the method comprises the steps of,
in the step (2):
the solvent for dissolving the florfenicol crude drug sample is acetonitrile solution, preferably, the concentration of the acetonitrile solution is 10-25wt%; and/or the number of the groups of groups,
the pretreatment is an organic filter membrane filtration, preferably the pore size of the organic filter membrane is 0.22-0.45 μm.
Preferably, the method comprises the steps of,
in the step (3):
the high performance liquid chromatography test conditions are as follows: chromatographic column: TC-C18 250X 4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25% acetonitrile; and/or the number of the groups of groups,
the detection limit of the high performance liquid chromatography test is 0.05 mug/mL, and/or the quantification limit is 0.15 mug/mL.
Preferably, the method comprises the steps of,
in the step (4):
the liquid chromatography-tandem mass spectrometry conditions were: chromatographic column with octadecylsilane chemically bonded silica as filler; taking an acidic aqueous solution as a mobile phase A and an acidic acetonitrile solution as a mobile phase B, and performing gradient elution; column temperature 40 ℃; flow rate: 0.35mL/min; sample injection amount: 2. Mu.L;
the mass spectrum test conditions of the liquid chromatography-tandem mass spectrum are as follows: ion source: ESI, negative ion mode; ion source temperature: 550 ℃; air curtain 35psi, sprayer 55psi; scanning mode: multi-reaction detection MRM; and on the basis of multi-reaction monitoring MRM, establishing an automatic association scan IDA, triggering an enhancer ion scan EPI by the multi-reaction monitoring MRM, formulating a plurality of parent ions with strongest triggering to execute the enhancer ion scan EPI, and executing dynamic background subtraction, wherein the range of the automatic association scan IDA is set to be 50-500Da, and the sectional scanning is respectively 50-71.518Da,71.518-122.175Da,122.175-233.162Da and 233.162-500Da.
Preferably, the method comprises the steps of,
the gradient elution conditions are as follows: 0 to 1min:80% A-20% B; 1-4 min:5%A-95% B; 4-5.5 min:5%A-95% B;5.5 to 7 minutes: 80% A-20% B.
Preferably, the method comprises the steps of,
in the step (5):
the impurity is at least one of arsenic, cadmium, mercury, lead and copper; and/or the number of the groups of groups,
the arsenic, copper used 72 germanium as an internal standard, and/or the cadmium used 115In as an internal standard, and/or the mercury, lead used 209Bi as an internal standard.
Preferably, the method comprises the steps of,
in the step (5):
the inductively coupled plasma mass spectrometry test conditions are as follows: radio frequency power: 1600W; sampling depth: 5.0mm; atomization chamber temperature: room temperature; ni sampling cone; ni truncated cone; plasma gas flow: 15.0L/min; atomizing air flow rate: 1.0L/min; auxiliary air flow: 1.20L/min; sample washing: 50s; reading delay: 15s; lead, cadmium, arsenic, copper test mode: helium collision mode; mercury test mode: standard mode.
The method specifically adopts the following scheme:
(1) Standard solution preparation
Florfenicol standard stock solution preparation (100 μg/mL): 1mL of florfenicol standard substance 1000 mug/mL is accurately weighed by a pipette, dissolved by acetonitrile and fixed to a scale, and uniformly mixed, thus obtaining 100 mug/mL of florfenicol standard stock solution.
Standard series of solutions: accurately transferring 5, 10, 20, 50, 100, 200 and 500 mu L of florfenicol standard stock solution (100 mu g/mL) into 7 10mL volumetric flasks, dissolving with 25wt% acetonitrile, fixing the volume to a scale, and uniformly mixing to prepare a standard series solution with working series solution of 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 mu g/mL;
(2) Sample solution preparation: weighing about 20mg of florfenicol crude drug sample, dissolving in a 100mL volumetric flask, fixing the volume to a scale with 10-25wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.22-0.45 μm, diluting for 100 times, and then analyzing by liquid chromatography;
(3) High performance liquid chromatography test:
(3-1) establishing a florfenicol standard curve: performing high performance liquid chromatography test on the standard series solutions with gradient concentration in the step (1), and performing linear regression by taking the standard solution concentrations of 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 mug/mL florfenicol as an abscissa and the peak area as an ordinate to obtain a linear equation;
(3-2) performing high performance liquid chromatography test on the sample solution obtained in the step (2), and taking the test result into the florfenicol standard curve obtained in the step (3-1) to obtain the main content of the florfenicol bulk drug;
the liquid chromatography test conditions adopted are as follows: chromatographic column: TC-C18 250X 4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25wt% acetonitrile;
wherein, selection of detection wavelength:
carrying out ultraviolet spectrum full-wavelength scanning on the florfenicol standard solution by using a DAD detector, wherein the full-wavelength scanning finds that the florfenicol standard solution has stronger absorption between 210 and 245nm, the strongest peak 210nm and the second strongest peak 223nm are selected as detection wavelengths, and the background interference is reduced;
selection of mobile phase:
the influence of different methanol and acetonitrile reagent concentrations on the separation of florfenicol is examined, and the experiment sets the influence of 20wt%, 40wt%, 60wt%, 80wt%, 100wt% methanol concentration and 25wt%, 50wt%, 75wt% and 100wt% acetonitrile concentration on the separation of florfenicol, and determines the optimal mobile phase concentration; the research shows that the methanol solution is used as the mobile phase, the florfenicol chromatographic peak is too fast to reach the better separation degree, and the acetonitrile solution is used as the mobile phase, so that the separation degree is better, wherein the separation effect of the 25wt% acetonitrile solution is optimal;
selection of sample injection chromatographic column:
as florfenicol is a weak polar compound, a chromatographic column should select a nonpolar or weak polar stationary phase, the invention intends to select nonpolar C18 type reversed phase chromatographic columns with different fillers, which can realize higher florfenicol separation degree under proper conditions, and select TC-C18 (250 multiplied by 4.6mm 5 μm) and eclipse-C18 (150 multiplied by 4.6mm 3.5 μm) two C18 type reversed phase chromatographic columns for test, and the result shows that the comparison result of TC-C18 (250 multiplied by 4.6mm 5 μm) shows that the florfenicol has sharp spectrum peak shape on the TC-C18, no tail and solvent effect, and better separation degree, therefore, TC-C18 (250 multiplied by 4.6mm 5 μm) is selected as the chromatographic column;
(4) Liquid chromatography-tandem mass spectrometry test:
florfenicol standard stock solution preparation (100 μg/mL): accurately weighing 1000 mug/mL of 1mL of florfenicol standard substance by a pipette, dissolving the 1 mug/mL of florfenicol standard substance in a 10mL volumetric flask, fixing the volume to a scale by acetonitrile, and uniformly mixing to obtain 100 mug/mL of florfenicol standard stock solution;
standard intermediate solution (1. Mu.g/mL): accurately weighing 100 mug/mL of 0.1mL of florfenicol standard stock solution in a 10mL volumetric flask by using a pipette, dissolving by using acetonitrile, fixing the volume to a scale, and uniformly mixing to obtain 1 mug/mL of florfenicol standard intermediate solution;
standard series of solutions: accurately transferring 5, 10, 20, 50, 100, 200 and 500 mu L of florfenicol standard stock solution (1 mu g/mL) into 7 10mL volumetric flasks, dissolving with 25wt% acetonitrile, fixing the volume to a scale, and uniformly mixing to prepare a standard series solution with working series solution of 10, 20, 50, 100 and 200 ng/mL;
sample solution preparation: weighing about 20mg of florfenicol crude drug sample, dissolving in a 100mL volumetric flask, fixing the volume to a scale with 10-25wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.22-0.45 μm, diluting 1000 times, and analyzing by liquid chromatography-tandem mass spectrometry;
(4-1) respectively carrying out liquid chromatography-tandem mass spectrometry on the standard series solutions with the gradient concentration, establishing a florfenicol standard curve according to a liquid chromatography test result, and establishing a three-level spectrum library according to a mass spectrometry test result;
(4-2) carrying out liquid chromatography-tandem mass spectrometry test on the sample solution, and taking the test result into the florfenicol standard curve obtained in the step (4-1) to obtain the main content of the florfenicol bulk drug; comparing the mass spectrum test result with the three-level spectrum chart library obtained in the step (4-1), and qualitatively comparing the main content of the florfenicol bulk drug;
wherein, the liquid chromatography test conditions are as follows: chromatographic column with octadecylsilane chemically bonded silica as filler; taking an acidic aqueous solution as a mobile phase A and an acidic acetonitrile solution as a mobile phase B, and performing gradient elution: 0 to 1min:80% A-20% B; 1-4 min:5%A-95% B; 4-5.5 min:5%A-95% B;5.5 to 7 minutes: 80% A-20% B; column temperature 40 ℃; flow rate: 0.35mL/min; sample injection amount: 2. Mu.L;
the mass spectrum test conditions are as follows: ion source: ESI, negative ion mode; ion source temperature: 550 ℃; air curtain 35psi, sprayer 55psi; scanning mode: multi-reaction detection MRM; the florfenicol ion pair m/z 356 is more than m/z 335.8, m/z 356 is more than m/z 184.9, DP is 80V,Collision Energy and is 12V and 23V respectively; an automatic association scan (IDA) is established on the basis of multi-reaction monitoring (MRM), the MRM triggers enhancer ion scanning (EPI), a plurality of parent ions with strongest trigger are formulated to execute EPI, dynamic background subtraction is executed, the IDA scanning range is set to be 50-500Da, and the sectional scanning ranges are respectively 50-71.518Da,71.518-122.175Da,122.175-233.162Da and 233.162-500Da.
(5) Inductively coupled plasma mass spectrometry testing: quantitatively testing the impurity content in the florfenicol bulk drug sample by adopting an internal standard method;
establishing a determination method for determining arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) and copper (Cu) In florfenicol bulk drug by using a full-automatic digestion-inductively coupled plasma mass spectrometry (ICP-MS) method, quantitatively testing by using an internal standard method In order to reduce matrix interference and improve detection precision and accuracy, wherein arsenic (As), copper (Cu) adopt 72 germanium As an internal standard substance and cadmium (Cd) adopt 115In As an internal standard substance, and mercury (Hg) and lead (Pb) adopt 209Bi As internal standard substances; the test conditions of the NexION 2000G inductively coupled plasma combined mass spectrometer are as follows: 1600W; sampling depth: 5.0mm; atomization chamber temperature: room temperature; ni sampling cone; ni truncated cone; plasma gas flow: 15.0L/min; atomizing air flow rate: 1.0L/min; auxiliary air flow: 1.20L/min; sample washing: 50s; reading delay: 15s; lead, cadmium, arsenic, copper test mode: helium collision mode; mercury test mode: standard mode.
The invention establishes a method for evaluating the florfenicol bulk drug, which selects liquid chromatography as a method for detecting the main content of the florfenicol bulk drug, and optimizes the liquid chromatography conditions by researching the conditions such as sample pretreatment method, chromatographic column separation, qualitative wavelength, flow and the like, so that a target object and a sample matrix interference peak can be well separated, and the quantification is more accurate; establishing automatic association scanning through liquid chromatography-tandem mass spectrometry three-stage mass spectrometry to qualitatively compare main content, and eliminating false positive interference; finally, the impurity determination is carried out through an inductively coupled plasma mass spectrometer, and then the comprehensive evaluation analysis is carried out on the florfenicol bulk drug, the detection sensitivity of the method is high, the accuracy is good, the operation is simple and convenient, the main content and the impurity detection of the florfenicol bulk drug can be used for the evaluation of the florfenicol bulk drug, the method has important significance in the aspect of drug quality control, and the method can also provide thought for the main content evaluation of other bulk drugs.
Drawings
FIG. 1 is a liquid chromatographic standard of the florfenicol standard solution of example 1 at a concentration of 5 μg/mL;
FIG. 2 is a standard three-stage spectrum of the florfenicol standard series of solutions of example 1.
Detailed Description
The present invention is described in detail below with reference to the specific drawings and examples, and it is necessary to point out that the following examples are given for further illustration of the present invention only and are not to be construed as limiting the scope of the present invention, since numerous insubstantial modifications and adaptations of the invention to those skilled in the art will still fall within the scope of the present invention.
The raw materials used in the examples of the present invention are uniform and commercially available products.
Example 1
Liquid chromatography analysis of main content of florfenicol bulk drug (Anhui Libo pharmaceutical Co., ltd., batch No. FF 220107002):
sample solution preparation: weighing about 20mg of florfenicol crude drug sample, dissolving in a 10mL volumetric flask, fixing the volume to a scale with 25wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.45 mu m, diluting by 100 times, and then analyzing by liquid chromatography;
florfenicol standard stock solution preparation (100 μg/mL): accurately weighing 1000 mug/mL of 1mL of florfenicol standard substance by a pipette, dissolving the 1 mug/mL of florfenicol standard substance in a 10mL volumetric flask, fixing the volume to a scale by acetonitrile, and uniformly mixing to obtain 100 mug/mL of florfenicol standard stock solution;
standard series of solutions were prepared: accurately transferring 5, 10, 20, 50, 100, 200 and 500 mu L of florfenicol standard stock solution (100 mu g/mL) into 7 10mL volumetric flasks, dissolving with 25wt% acetonitrile solution, fixing the volume to a scale, and uniformly mixing to prepare standard series solutions with the series solutions of 0.05, 0.1, 0.2, 0.5, 1, 2 and 5 mu g/mL; linear regression was performed with 0.05, 0.1, 0.2, 0.5, 1, 2, 5 μg/mL standard solution concentrations of florfenicol as abscissa and peak area as ordinate, yielding the linear equation: y= 44983.4x-315.083, correlation coefficient r= 0.99975;
the adopted high performance liquid chromatography test conditions are as follows: instrument: shimadzu LC2030 high performance liquid chromatography, chromatographic column: TC-C18 250X 4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25wt% acetonitrile; the main content of florfenicol in the florfenicol bulk drug is 99.72-99.93% calculated by an external standard method through comparison with a standard substance, and parallel test results are shown in the following table 1:
TABLE 1
Detection limit and quantification limit: when the florfenicol sample solution is diluted to the concentration of 0.05 mug/mL, the signal to noise ratio is more than 3 (the test result is shown in table 2), and the requirement of the signal to noise ratio of the detection limit is met, and the florfenicol sample solution can be used as the detection limit. When the concentration is 0.15 mug/mL, the RSD of 6 sampling is 1.51% (the test result is shown in Table 3), the signal to noise ratio is more than 10, and the signal to noise ratio requirement of the quantitative limit is met. Thus, the detection limit of the florfenicol high performance liquid chromatography test method is 0.05 mug/mL, and the quantitative limit is 0.15 mug/mL.
TABLE 2
| Numbering device | Peak area | Signal to noise ratio |
| 1 | 2299 | 3.13 |
TABLE 3 Table 3
Repeatability: repeating test by adding florfenicol standard substance into blank solution, preparing 0.5 μg/mL standard intermediate solution as sample solution, sampling for 6 times, counting peak area, and calculating RSD. The results showed that the repeatability test RSD was 0.64% (test results are shown in table 4).
TABLE 4 Table 4
Recovery rate: three different concentrations were selected for labeling, and three parallel samples of each concentration were prepared for the test solution, the samples were taken, and the recovery was calculated, showing that the recovery of florfenicol was 88.7% -100% at the three different concentrations labeled level (test results are shown in table 5).
TABLE 5
Florfenicol raw material liquid medicine phase chromatography-tandem mass spectrometry qualitative analysis:
florfenicol standard stock solution preparation (100 μg/mL): accurately weighing 1000 mug/mL of 1mL of florfenicol standard substance by a pipette, dissolving the 1 mug/mL of florfenicol standard substance in a 10mL volumetric flask, fixing the volume to a scale by acetonitrile, and uniformly mixing to obtain 100 mug/mL of florfenicol standard stock solution;
standard intermediate solution (1. Mu.g/mL): accurately weighing 100 mug/mL of 0.1mL of florfenicol standard stock solution in a 10mL volumetric flask by using a pipette, dissolving by using acetonitrile, fixing the volume to a scale, and uniformly mixing to obtain 1 mug/mL of florfenicol standard intermediate solution;
standard series of solutions: accurately transferring 5, 10, 20, 50, 100, 200 and 500 mu L of florfenicol standard stock solution (1 mu g/mL) into 7 10mL volumetric flasks, dissolving with 25wt% acetonitrile, fixing the volume to a scale, and uniformly mixing to prepare a standard series solution with working series solution of 10, 20, 50, 100 and 200 ng/mL;
sample solution preparation: weighing about 20mg of florfenicol crude drug sample, dissolving in a 100mL volumetric flask, fixing the volume to a scale with 25wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.45 mu m, diluting 1000 times, and then analyzing by liquid chromatography-tandem mass spectrometry; taking the standard solution concentration of the florfenicol of 10, 20, 50, 100 and 200ng/mL as an abscissa and the peak area as an ordinate, carrying out linear regression to obtain a linear equation: y=3125.7070701x+3906.57, and the correlation coefficient r= 0.99975.
The liquid chromatography-tandem mass spectrometry conditions were: instrument: AB Sciex QTRAP 4500 liquid mass spectrometer; chromatographic column with octadecylsilane chemically bonded silica as filler; in an acidic aqueous solution: aqueous formic acid (formic acid: aladine, lot number F112032, new region of shanghai purdong) at a concentration of 0.1wt% d was used as mobile phase a, and acetonitrile (formic acid: aladine, lot number F112032, new region of shanghai purdong; acetonitrile: merck, lot number AX0155, new region of shanghai purdong) at a concentration of 0.1wt% was used as mobile phase B for gradient elution: 0 to 1min:80% A-20% B; 1-4 min:5%A-95% B; 4-5.5 min:5%A-95% B;5.5 to 7 minutes: 80% A-20% B; column temperature 40 ℃; flow rate: 0.35mL/min; sample injection amount: 2. Mu.L;
the mass spectrum test conditions of the liquid chromatography-tandem mass spectrum are as follows: ion source: ESI, negative ion mode; ion source temperature: 550 ℃; air curtain 35psi, sprayer 55psi; scanning mode: multi-reaction detection MRM; an automatic association scan (IDA) is established on the basis of multi-reaction monitoring (MRM), the MRM triggers enhancer ion scanning (EPI), a plurality of parent ions with strongest trigger are formulated to execute EPI, dynamic background subtraction is executed, the IDA scanning range is set to be 50-500Da, and the sectional scanning ranges are respectively 50-71.518Da,71.518-122.175Da,122.175-233.162Da and 233.162-500Da. Establishing a three-level spectrum library according to mass spectrum test results of standard series solutions, and qualitatively comparing main contents of the florfenicol bulk drug in the sample solution;
respectively carrying out liquid chromatography-tandem mass spectrometry on the standard series solutions with the gradient concentration, establishing a florfenicol standard curve according to a liquid chromatography test result, and then carrying the test result of the sample solution into the florfenicol standard curve to obtain the main content of the florfenicol bulk drug; establishing a florfenicol three-level spectrum library according to a mass spectrum test result, comparing the test result of the sample solution with the three-level spectrum library, and qualitatively comparing the main content of the florfenicol raw material medicine; the three-level spectrum chart library is compared, the matching degree of the florfenicol reaches 96.386 percent, and the florfenicol is identified to eliminate false positive interference.
Impurity analysis of florfenicol bulk drug by inductively coupled plasma mass spectrometry (ICP-MS):
a full-automatic digestion-determination method for determining arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) and copper (Cu) In florfenicol bulk drug is established, in order to reduce matrix interference and improve detection precision and accuracy, an internal standard method is adopted for quantitative test, 115In is adopted As an internal standard substance for arsenic (As), copper (Cu) and cadmium (Cd), and 209Bi is adopted As an internal standard substance for mercury (Hg) and lead (Pb). The NexION 2000G inductively coupled plasma combined mass spectrometer test element can select two different modes, namely a collision mode and a standard mode, wherein collision gas adopted in the collision mode comprises helium, oxygen, methane and hydrogen. The impact mode can effectively eliminate the influence on the element test caused by mass spectrum interference, but the corresponding value of the element is reduced when the element is tested by adopting the impact mode. Therefore, lead, cadmium, copper and arsenic in the sample are tested in a helium collision mode, and mercury is tested in a standard mode. The results show that the florfenicol bulk drug sample is tested, wherein the content of copper, cadmium and mercury are not detected, the lead content in the calculated sample is respectively 0.0259mg/kg-0.0339mg/kg, and the arsenic content is 0.0461-0.0557mg/kg (the test results are shown in Table 6).
TABLE 6
Example 2
Main content liquid chromatography analysis of florfenicol drug substance (commercially available, huameixing Hao biopharmaceutical Co., ltd., lot number 20230330):
sample solution preparation: weighing about 20mg of florfenicol crude drug sample, dissolving in a 10mL volumetric flask, fixing the volume to a scale with 25wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.45 mu m, diluting for 20 times, and then analyzing by liquid chromatography;
florfenicol standard stock solution preparation (100 μg/mL): as in example 1;
standard series of solutions were prepared: as in example 1;
the adopted high performance liquid chromatography test conditions are as follows: instrument: shimadzu high performance liquid chromatography column: TC-C18250×4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25wt% acetonitrile; the main content of florfenicol in the florfenicol bulk drug is calculated to be 19.48-19.72% by comparing with a standard substance by an external standard method, and the parallel test result is shown in the following table 7:
TABLE 7
Florfenicol raw material liquid medicine phase chromatography-tandem mass spectrometry qualitative analysis:
florfenicol standard stock solution preparation (100 μg/mL): as in example 1;
standard intermediate solution (1. Mu.g/mL): as in example 1;
standard series of solutions: as in example 1;
sample solution preparation: weighing about 20mg of florfenicol bulk drug sample, dissolving in a 100mL volumetric flask, fixing the volume to a scale with 10wt% acetonitrile solution, uniformly mixing, filtering with an organic filter membrane of 0.22 mu m, diluting by 200 times, and then analyzing by liquid chromatography-tandem mass spectrometry; taking the standard solution concentration of the florfenicol of 10, 20, 50, 100 and 200ng/mL as an abscissa and the peak area as an ordinate, carrying out linear regression to obtain a linear equation: y=3146.81421x+3904.88, correlation coefficient r= 0.99984.
The liquid chromatography-tandem mass spectrometry conditions were: as in example 1;
the mass spectrum test conditions of the liquid chromatography-tandem mass spectrum are as follows: as in example 1;
respectively carrying out liquid chromatography-tandem mass spectrometry on the standard series solutions with the gradient concentration, establishing a florfenicol standard curve according to a liquid chromatography test result, and then carrying the test result of the sample solution into the florfenicol standard curve to obtain the main content of the florfenicol bulk drug; establishing a florfenicol three-level spectrum library according to a mass spectrum test result, comparing the test result of the sample solution with the three-level spectrum library, and qualitatively comparing the main content of the florfenicol raw material medicine; the three-level spectrum chart library is compared, the matching degree of the florfenicol reaches 97.123 percent, and the florfenicol is identified to eliminate false positive interference.
Impurity analysis of florfenicol bulk drug by inductively coupled plasma mass spectrometry (ICP-MS):
a full-automatic digestion-determination method for determining arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) and copper (Cu) In florfenicol bulk drug is established, in order to reduce matrix interference and improve detection precision and accuracy, an internal standard method is adopted for quantitative test, 115In is adopted As an internal standard substance for arsenic (As), copper (Cu) and cadmium (Cd), and 209Bi is adopted As an internal standard substance for mercury (Hg) and lead (Pb). The NexION 2000G inductively coupled plasma combined mass spectrometer test element can select two different modes, namely a collision mode and a standard mode, wherein collision gas adopted in the collision mode comprises helium, oxygen, methane and hydrogen. The impact mode can effectively eliminate the influence on the element test caused by mass spectrum interference, but the corresponding value of the element is reduced when the element is tested by adopting the impact mode. Therefore, lead, cadmium, copper and arsenic in the sample are tested in a helium collision mode, and mercury is tested in a standard mode. The results show that the florfenicol bulk drug sample is tested, wherein the content of copper, cadmium, mercury and lead is not detected, and the arsenic content in the calculated sample is 0.0429-0.0469mg/kg (the test results are shown in Table 8).
TABLE 8
Example 3
Liquid chromatography analysis of main content of florfenicol bulk drug (commercially available, living animal pharmaceutical Co., ltd., batch No. xh-zsy 057) in the case of fertilizer:
sample solution preparation: as in example 1;
florfenicol standard stock solution preparation (100 μg/mL): as in example 1;
standard series of solutions were prepared: as in example 1;
the adopted high performance liquid chromatography test conditions are as follows: instrument: shimadzu high performance liquid chromatography column: TC-C18250×4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25wt% acetonitrile; the main content of florfenicol in the florfenicol bulk drug is calculated to be 19.64-19.92% by comparing with a standard substance through an external standard method, and parallel test results are shown in the following table 9:
TABLE 9
Florfenicol raw material liquid medicine phase chromatography-tandem mass spectrometry qualitative analysis:
florfenicol standard stock solution preparation (100 μg/mL): as in example 1;
standard intermediate solution (1. Mu.g/mL): as in example 1;
standard series of solutions: as in example 1;
sample solution preparation: as in example 1.
The liquid chromatography-tandem mass spectrometry conditions were: as in example 1;
the mass spectrum test conditions of the liquid chromatography-tandem mass spectrum are as follows: as in example 1;
respectively carrying out liquid chromatography-tandem mass spectrometry on the standard series solutions with the gradient concentration, establishing a florfenicol standard curve according to a liquid chromatography test result, and then carrying the test result of the sample solution into the florfenicol standard curve to obtain the main content of the florfenicol bulk drug; establishing a florfenicol three-level spectrum library according to a mass spectrum test result, comparing the test result of the sample solution with the three-level spectrum library, and qualitatively comparing the main content of the florfenicol raw material medicine; the three-level spectrum chart library is compared, the matching degree of the florfenicol reaches 98.403 percent, and the florfenicol is identified to eliminate false positive interference.
Impurity analysis of florfenicol bulk drug by inductively coupled plasma mass spectrometry (ICP-MS):
a full-automatic digestion-determination method for determining arsenic (As), cadmium (Cd), mercury (Hg), lead (Pb) and copper (Cu) In florfenicol bulk drug is established, in order to reduce matrix interference and improve detection precision and accuracy, an internal standard method is adopted for quantitative test, 115In is adopted As an internal standard substance for arsenic (As), copper (Cu) and cadmium (Cd), and 209Bi is adopted As an internal standard substance for mercury (Hg) and lead (Pb). The NexION 2000G inductively coupled plasma combined mass spectrometer test element can select two different modes, namely a collision mode and a standard mode, wherein collision gas adopted in the collision mode comprises helium, oxygen, methane and hydrogen. The impact mode can effectively eliminate the influence on the element test caused by mass spectrum interference, but the corresponding value of the element is reduced when the element is tested by adopting the impact mode. Therefore, lead, cadmium, copper and arsenic in the sample are tested in a helium collision mode, and mercury is tested in a standard mode. The results show that the florfenicol bulk drug sample is tested, wherein the content of copper, cadmium, mercury and the like is not detected, the lead content in the sample is calculated to be 0.0229-0.0241mg/kg, and the arsenic content is calculated to be 0.0321-0.0339mg/kg (the test results are shown in Table 10).
Table 10
The results show that: the method for evaluating the florfenicol bulk drug is simple and convenient to operate, quick, low in reagent consumption, weak in matrix interference, high in accuracy and low in cost, and the main content of the florfenicol bulk drug is analyzed by a high-performance liquid chromatography test method according to conditions such as a sample pretreatment method, chromatographic column separation, qualitative wavelength and flow; establishing automatic association scanning through liquid chromatography-tandem mass spectrometry three-stage mass spectrometry to qualitatively compare main content, and eliminating false positive interference; finally, the impurity is measured by an inductively coupled plasma mass spectrometer, so that the florfenicol bulk drug is evaluated and analyzed, the method has important significance in the aspect of quality control of the florfenicol bulk drug, and a thought can be provided for main content evaluation of other bulk drugs.
Claims (10)
1. The method for evaluating the florfenicol bulk drug is characterized by comprising the following steps:
analyzing the main content of the florfenicol bulk drug by a high performance liquid chromatography test method, establishing automatic association scanning by liquid chromatography-tandem mass spectrometry to qualitatively compare the main content, and finally measuring impurities by inductively coupled plasma mass spectrometry.
2. The method for evaluating a florfenicol drug substance according to claim 1, characterized in that the method comprises:
(1) Standard solution preparation: dissolving a florfenicol standard substance to a constant volume to prepare a standard series solution with gradient concentration;
(2) Sample solution preparation: dissolving a florfenicol bulk drug sample, and then carrying out pretreatment and volume fixing to obtain a sample solution;
(3) High performance liquid chromatography test:
(3-1) respectively performing high performance liquid chromatography test on the standard series solutions with gradient concentration in the step (1), and establishing a florfenicol standard curve according to the test result;
(3-2) performing high performance liquid chromatography test on the sample solution obtained in the step (2), and taking the test result into the florfenicol standard curve obtained in the step (3-1) to obtain the main content of the florfenicol bulk drug;
(4) Liquid chromatography-tandem mass spectrometry test:
(4-1) further diluting the standard series solution with the gradient concentration in the step (1), respectively carrying out liquid chromatography-tandem mass spectrometry test, establishing a florfenicol standard curve according to a liquid chromatography test result, and establishing a three-level spectrum library according to a mass spectrometry test result;
(4-2) further diluting the sample solution obtained in the step (2), then carrying out liquid chromatography-tandem mass spectrometry test, and carrying the test result into the florfenicol standard curve obtained in the step (4-1) to obtain the main content of the florfenicol bulk drug; comparing the mass spectrum test result with the three-level spectrum chart library obtained in the step (4-1), and qualitatively comparing the main content of the florfenicol bulk drug;
(5) Inductively coupled plasma mass spectrometry testing: and quantitatively testing the impurity content in the florfenicol bulk drug sample by adopting an internal standard method.
3. The method for evaluating a florfenicol drug substance according to claim 2, wherein the step (1) comprises:
(1-1) preparation of standard stock solution: dissolving, diluting and fixing the florfenicol standard substance to prepare a standard stock solution with the concentration of 100 mug/mL;
(1-2) diluting the standard stock solution prepared in the step (1-1) again, and fixing the volume to prepare a standard series solution with gradient concentration.
4. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in step (1):
the solvent for dissolving the florfenicol standard is acetonitrile.
5. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in the step (2):
the solvent for dissolving the florfenicol crude drug sample is acetonitrile solution, preferably, the concentration of the acetonitrile solution is 10-25wt%; and/or the number of the groups of groups,
the pretreatment is an organic filter membrane filtration, preferably the pore size of the organic filter membrane is 0.22-0.45 μm.
6. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in the step (3):
the high performance liquid chromatography test conditions are as follows: chromatographic column: TC-C18 250X 4.6mm,5 μm; detection wavelength: 223nm; a detector: a DAD detector; column temperature: 40 ℃; flow rate: 1.0mL/min; sample injection amount: 20. Mu.L; mobile phase: 25% acetonitrile; and/or the number of the groups of groups,
the detection limit of the high performance liquid chromatography test is 0.05 mug/mL, and/or the quantification limit is 0.15 mug/mL.
7. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in the step (4):
the liquid chromatography-tandem mass spectrometry conditions were: chromatographic column with octadecylsilane chemically bonded silica as filler; taking an acidic aqueous solution as a mobile phase A and an acidic acetonitrile solution as a mobile phase B, and performing gradient elution; column temperature 40 ℃; flow rate: 0.35mL/min; sample injection amount: 2. Mu.L;
the mass spectrum test conditions of the liquid chromatography-tandem mass spectrum are as follows: ion source: ESI, negative ion mode; ion source temperature: 550 ℃; air curtain 35psi, sprayer 55psi; scanning mode: multi-reaction detection MRM; and on the basis of multi-reaction monitoring MRM, establishing an automatic association scan IDA, triggering an enhancer ion scan EPI by the multi-reaction monitoring MRM, formulating a plurality of parent ions with strongest triggering to execute the enhancer ion scan EPI, and executing dynamic background subtraction, wherein the range of the automatic association scan IDA is set to be 50-500Da, and the sectional scanning is respectively 50-71.518Da,71.518-122.175Da,122.175-233.162Da and 233.162-500Da.
8. The method for evaluating a florfenicol drug substance according to claim 7, wherein:
the gradient elution conditions are as follows: 0 to 1min:80% A-20% B; 1-4 min:5%A-95% B; 4-5.5 min:5%A-95% B;5.5 to 7 minutes: 80% A-20% B.
9. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in the step (5):
the impurity is at least one of arsenic, cadmium, mercury, lead and copper; and/or the number of the groups of groups,
the arsenic, copper used 72 germanium as an internal standard, and/or the cadmium used 115In as an internal standard, and/or the mercury, lead used 209Bi as an internal standard.
10. The method for evaluating a florfenicol drug substance according to claim 2, wherein:
in the step (5):
the inductively coupled plasma mass spectrometry test conditions are as follows: radio frequency power: 1600W; sampling depth: 5.0mm; atomization chamber temperature: room temperature; ni sampling cone; ni truncated cone; plasma gas flow: 15.0L/min; atomizing air flow rate: 1.0L/min; auxiliary air flow: 1.20L/min; sample washing: 50s; reading delay: 15s; lead, cadmium, arsenic, copper test mode: helium collision mode; mercury test mode: standard mode.
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