CN117214368A - HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor - Google Patents
HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor Download PDFInfo
- Publication number
- CN117214368A CN117214368A CN202311182678.1A CN202311182678A CN117214368A CN 117214368 A CN117214368 A CN 117214368A CN 202311182678 A CN202311182678 A CN 202311182678A CN 117214368 A CN117214368 A CN 117214368A
- Authority
- CN
- China
- Prior art keywords
- tacrolimus
- ascomycin
- standard
- mobile phase
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- QJJXYPPXXYFBGM-LFZNUXCKSA-N Tacrolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1\C=C(/C)[C@@H]1[C@H](C)[C@@H](O)CC(=O)[C@H](CC=C)/C=C(C)/C[C@H](C)C[C@H](OC)[C@H]([C@H](C[C@H]2C)OC)O[C@@]2(O)C(=O)C(=O)N2CCCC[C@H]2C(=O)O1 QJJXYPPXXYFBGM-LFZNUXCKSA-N 0.000 title claims abstract description 142
- 229960001967 tacrolimus Drugs 0.000 title claims abstract description 141
- QJJXYPPXXYFBGM-SHYZHZOCSA-N tacrolimus Natural products CO[C@H]1C[C@H](CC[C@@H]1O)C=C(C)[C@H]2OC(=O)[C@H]3CCCCN3C(=O)C(=O)[C@@]4(O)O[C@@H]([C@H](C[C@H]4C)OC)[C@@H](C[C@H](C)CC(=C[C@@H](CC=C)C(=O)C[C@H](O)[C@H]2C)C)OC QJJXYPPXXYFBGM-SHYZHZOCSA-N 0.000 title claims abstract description 141
- ZDQSOHOQTUFQEM-XCXYXIJFSA-N ascomycin Natural products CC[C@H]1C=C(C)C[C@@H](C)C[C@@H](OC)[C@H]2O[C@@](O)([C@@H](C)C[C@H]2OC)C(=O)C(=O)N3CCCC[C@@H]3C(=O)O[C@H]([C@H](C)[C@@H](O)CC1=O)C(=C[C@@H]4CC[C@@H](O)[C@H](C4)OC)C ZDQSOHOQTUFQEM-XCXYXIJFSA-N 0.000 title claims abstract description 125
- 238000000855 fermentation Methods 0.000 title claims abstract description 52
- 230000004151 fermentation Effects 0.000 title claims abstract description 52
- 238000000034 method Methods 0.000 title claims abstract description 38
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 23
- ZDQSOHOQTUFQEM-PKUCKEGBSA-N ascomycin Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C\C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-PKUCKEGBSA-N 0.000 title claims abstract 31
- 239000012071 phase Substances 0.000 claims abstract description 66
- 238000001514 detection method Methods 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 150000007522 mineralic acids Chemical class 0.000 claims abstract description 20
- 239000003960 organic solvent Substances 0.000 claims abstract description 17
- 238000010812 external standard method Methods 0.000 claims abstract description 13
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 13
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 12
- 239000002904 solvent Substances 0.000 claims abstract description 12
- 239000012074 organic phase Substances 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 239000000945 filler Substances 0.000 claims abstract description 7
- 239000000377 silicon dioxide Substances 0.000 claims abstract description 7
- 238000010829 isocratic elution Methods 0.000 claims abstract description 6
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims abstract description 6
- 239000012224 working solution Substances 0.000 claims description 63
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 57
- 239000003085 diluting agent Substances 0.000 claims description 43
- 239000011550 stock solution Substances 0.000 claims description 36
- 239000012488 sample solution Substances 0.000 claims description 35
- 238000005303 weighing Methods 0.000 claims description 34
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 238000002360 preparation method Methods 0.000 claims description 30
- 239000000126 substance Substances 0.000 claims description 29
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 27
- 239000007788 liquid Substances 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 19
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- 239000000523 sample Substances 0.000 claims description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 8
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 7
- 239000012490 blank solution Substances 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 7
- 239000007924 injection Substances 0.000 claims description 7
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 6
- 239000000741 silica gel Substances 0.000 claims description 6
- 229910002027 silica gel Inorganic materials 0.000 claims description 6
- 238000003556 assay Methods 0.000 claims description 4
- 239000012982 microporous membrane Substances 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 3
- 239000012895 dilution Substances 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 239000000047 product Substances 0.000 abstract description 5
- 239000006227 byproduct Substances 0.000 abstract description 4
- 239000001963 growth medium Substances 0.000 abstract description 2
- ZDQSOHOQTUFQEM-NURRSENYSA-N ascomycin Chemical compound C/C([C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@]2(O)O[C@@H]([C@H](C[C@H]2C)OC)[C@@H](OC)C[C@@H](C)C/C(C)=C/[C@H](C(C[C@H](O)[C@H]1C)=O)CC)=C\[C@@H]1CC[C@@H](O)[C@H](OC)C1 ZDQSOHOQTUFQEM-NURRSENYSA-N 0.000 description 95
- 238000000926 separation method Methods 0.000 description 4
- 229960003444 immunosuppressant agent Drugs 0.000 description 3
- 230000001861 immunosuppressant effect Effects 0.000 description 3
- 239000003018 immunosuppressive agent Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000003643 water by type Substances 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 229930105110 Cyclosporin A Natural products 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- 241001647839 Streptomyces tsukubensis Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229940124452 immunizing agent Drugs 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000003120 macrolide antibiotic agent Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Landscapes
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
Abstract
The invention provides an HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor. An HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor is characterized in that the HPLC method is carried out by reversed phase liquid chromatography, a chromatographic column is a chromatographic column with octadecylsilane chemically bonded silica filler, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, inorganic acid and an organic solvent, the mobile phase B comprises an organic phase solvent, isocratic elution is set according to volume fraction, and quantitative analysis is carried out by adopting an external standard method. The method can eliminate the interference of complex components such as a culture medium in the fermentation broth, can simultaneously and rapidly and accurately separate and quantitatively determine the main component tacrolimus and the byproduct ascomycin in the fermentation broth, has short determination time, high accuracy, high precision and simple operation, has high throughput sample detection capability, and can effectively control the quality of products in the production process.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to an HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor.
Background
Tacrolimus (FK 506) is a novel neutral and hydrophobic macrolide powerful immunosuppressant extracted from fermentation liquor of Streptomyces tsukubaensis separated from soil, and the drug effect of the novel powerful immunosuppressant is 10-100 times that of a traditional immunizing agent cyclosporin A, and the novel powerful immunosuppressant is clinically applied to prevention and treatment of rejection of liver or kidney transplantation and is now used as a first-line drug for preventing and treating rejection reaction and autoimmune diseases.
Ascomycin (FK 520) is a byproduct produced in the fermentation process of producing tacrolimus fermentation broth, and is an intermediate of various new drugs. In order to ensure the smooth proceeding of the subsequent process of tacrolimus extraction and purification and control the quality of products, the content of byproduct ascomycin generated in the tacrolimus fermentation process needs to be controlled. Therefore, research on a detection method capable of rapidly and simultaneously measuring the contents of tacrolimus and ascomycin in fermentation broth is very necessary for controlling the intermediate process of the raw material medicine production enterprises.
According to reference documents and patents, the existing separation and measurement methods for tacrolimus, ascomycin and other impurities in tacrolimus fermentation liquor are few, and most of the methods are only concentrated on the detection of tacrolimus main components, and the method for rapidly and simultaneously measuring the contents of tacrolimus and ascomycin in the tacrolimus fermentation liquor is not reported, so that the situation is unfavorable for the control of the product quality of enterprises, and therefore an effective analysis method capable of simultaneously and rapidly measuring the contents of tacrolimus and ascomycin in the fermentation liquor is needed.
Disclosure of Invention
The invention provides an HPLC method for rapidly and simultaneously measuring the contents of tacrolimus and ascomycin in fermentation liquor, which solves the defect that the prior art lacks a detection method capable of rapidly and simultaneously measuring the contents of tacrolimus and ascomycin in fermentation liquor.
The technical scheme of the invention is realized as follows:
an HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor is characterized in that the HPLC method is carried out by reversed phase liquid chromatography, a chromatographic column is a chromatographic column with octadecylsilane chemically bonded silica filler, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, inorganic acid and an organic solvent, the mobile phase B comprises an organic phase solvent, isocratic elution is set according to volume fraction, and quantitative analysis is carried out by adopting an external standard method.
Further, the method also comprises the preparation of a solution before detection, wherein the solution comprises the following components:
blank solution, including diluent;
a sample solution comprising tacrolimus fermentation broth and a diluent;
the tacrolimus standard curve working solution comprises tacrolimus standard substance and diluent;
an ascomycin standard curve working solution comprising an ascomycin standard and a diluent.
Further, the method comprises the following steps:
a1, preparation of mobile phase A
Weighing a certain amount of inorganic acid, adding water for dilution, uniformly mixing to prepare an inorganic acid solution, adding an organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing a certain amount of tacrolimus fermentation liquor, adding a diluent, oscillating for dissolution, fixing the volume, and filtering to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing a certain amount of tacrolimus standard substance, adding a diluent to prepare tacrolimus standard stock solution, and weighing tacrolimus standard stock solution to prepare a series of tacrolimus standard curve working solutions;
weighing a certain amount of ascomycin standard substance, adding a diluent to prepare ascomycin standard stock solution, and then weighing ascomycin standard stock solution to prepare a series of ascomycin standard curve working solutions;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, the volume ratio of the mobile phase A to the mobile phase B is 58:42 to 75:25, the flow rate of the chromatographic column is 0.7-1.3 mL/min, the column temperature of the chromatographic column is 30-60 ℃, the sample injection amount of the chromatographic column is 5-20 mu L, and the detection wavelength is 205-230 nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the liquid to be detected according to a standard curve.
Further, the method comprises the following steps:
a1, preparation of mobile phase A
Weighing 1.0mL of inorganic acid, adding water to 1000mL, uniformly mixing to prepare an inorganic acid solution with the concentration of 0.1%, adding 50mL of organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing 5.0g of tacrolimus fermentation liquor in a 100mL volumetric flask, adding 50mL of diluent, oscillating for 30 minutes, fixing the volume after dissolution, and filtering the supernatant by an organic microporous membrane of 0.22 mu m to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing 10mg of tacrolimus standard substance, dissolving the tacrolimus standard substance by using a diluent, fixing the volume to 10mL, preparing tacrolimus standard stock solution with the content of 1000mg/L, and preserving the tacrolimus standard stock solution at the temperature of 2-8 ℃; respectively sucking a proper amount of tacrolimus standard stock solution, preparing 5, 10, 50, 150, 300 and 600mg/L tacrolimus standard curve working solution by using a diluent, and preserving at 2-8 ℃ for on-site preparation;
precisely weighing 10mg of ascomycin standard substance, dissolving with a diluent and fixing the volume to 10mL to prepare ascomycin standard stock solution with the content of 1000mg/L, and preserving at the temperature of 2-8 ℃; respectively absorbing a proper amount of ascomycin standard stock solution, preparing 5mg/L ascomycin standard curve working solution, 10mg/L ascomycin standard curve working solution, 50 mg/L ascomycin standard curve working solution, 100 mg/L ascomycin standard curve working solution, and preserving at 2-8 ℃ for preparing at present;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, and a chromatographic column of color 5: YMC-Pack ODS-A,250×4.6mm,5 μm, volume ratio of mobile phase A to mobile phase B of 62:38, flow rate of chromatographic column of 1 m/min, column temperature of 55 deg.C, sample injection amount of chromatographic column of 10 μl, and detection wavelength of 220nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the sample solution according to a standard curve.
Further, the purity of the tacrolimus standard substance is more than or equal to 99%; the purity of the ascomycin standard substance is more than or equal to 99%; the sample solution is selected from tacrolimus fermentation liquid at different stages of the production process, the diluent is selected from one or more of methanol, ethanol, acetonitrile, acetone and water, and the diluent is an analytical grade.
Further, the inorganic acid of the mobile phase A is selected from one of trifluoroacetic acid, phosphoric acid or acetic acid; the organic solvent of the mobile phase A is selected from one of acetonitrile, ethanol or methanol; the organic phase solvent in the mobile phase B is one or more selected from methanol, acetonitrile, ethanol, isopropanol and water.
Further, the water is ultrapure water, the organic solvent of the mobile phase A is chromatographic purity, and the organic phase solvent of the mobile phase B is chromatographic purity.
Compared with the prior art, the invention has the beneficial effects that:
1. the method can eliminate the interference of complex components such as a culture medium in the fermentation broth, can simultaneously and rapidly and accurately separate and quantitatively determine the main component tacrolimus and the byproduct ascomycin in the fermentation broth, has short determination time (25 minutes/needle), high accuracy, high precision and simple operation, has high throughput sample detection capability, and can effectively control the quality of products in the production process.
2. The separation degree of tacrolimus and ascomycin meets the requirement, the tailing factor is less than 1.5, the peak shape is good, the peak purity is good, the theoretical plate number is more than 3000, the recovery rate is high, the precision is good, the accuracy and the reliability are high, and the reliable and quick technical support can be provided for daily production and inspection work.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram of a blank solution (specifically an ethanol solution);
FIG. 2 is a chromatogram of a standard stock solution of tacrolimus;
FIG. 3 is a chromatogram of an ascomycin standard stock solution;
FIG. 4 is a tacrolimus standard curve;
FIG. 5 is an ascomycin standard curve;
FIG. 6 is a chromatogram of a test solution;
FIG. 7 is a table of peak results of FIG. 6.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
An HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor is characterized in that the HPLC method is carried out by reversed phase liquid chromatography, a chromatographic column is a chromatographic column with octadecylsilane chemically bonded silica filler, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, inorganic acid and an organic solvent, the mobile phase B comprises an organic phase solvent, isocratic elution is set according to volume fraction, and quantitative analysis is carried out by adopting an external standard method.
Further embodiments also include the preparation of a pre-assay solution, the solution comprising:
blank solution, including diluent;
a sample solution comprising tacrolimus fermentation broth and a diluent;
the tacrolimus standard curve working solution comprises tacrolimus standard substance and diluent;
an ascomycin standard curve working solution comprising an ascomycin standard and a diluent.
Further embodiments include the steps of:
a1, preparation of mobile phase A
Weighing a certain amount of inorganic acid, adding water for dilution, uniformly mixing to prepare an inorganic acid solution, adding an organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing a certain amount of tacrolimus fermentation liquor, adding a diluent, oscillating for dissolution, fixing the volume, and filtering to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing a certain amount of tacrolimus standard substance, adding a diluent to prepare tacrolimus standard stock solution, and weighing tacrolimus standard stock solution to prepare a series of tacrolimus standard curve working solutions;
weighing a certain amount of ascomycin standard substance, adding a diluent to prepare ascomycin standard stock solution, and then weighing ascomycin standard stock solution to prepare a series of ascomycin standard curve working solutions;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, the volume ratio of the mobile phase A to the mobile phase B is 58:42 to 75:25, the flow rate of the chromatographic column is 0.7-1.3 mL/min, the column temperature of the chromatographic column is 30-60 ℃, the sample injection amount of the chromatographic column is 5-20 mu L, and the detection wavelength is 205-230 nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the liquid to be detected according to a standard curve.
Example 2
An HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor is characterized in that the HPLC method is carried out by reversed phase liquid chromatography, a chromatographic column is a chromatographic column with octadecylsilane chemically bonded silica filler, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, inorganic acid and an organic solvent, the mobile phase B comprises an organic phase solvent, isocratic elution is set according to volume fraction, and quantitative analysis is carried out by adopting an external standard method.
Further embodiments also include the preparation of a pre-assay solution, the solution comprising:
blank solution, including diluent;
a sample solution comprising tacrolimus fermentation broth and a diluent;
the tacrolimus standard curve working solution comprises tacrolimus standard substance and diluent;
an ascomycin standard curve working solution comprising an ascomycin standard and a diluent.
Further embodiments include the steps of:
a1, preparation of mobile phase A
Weighing 1.0mL of inorganic acid, adding water to 1000mL, uniformly mixing to prepare an inorganic acid solution with the concentration of 0.1%, adding 50mL of organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing 5.0g of tacrolimus fermentation liquor in a 100mL volumetric flask, adding 50mL of diluent, oscillating for 30 minutes, fixing the volume after dissolution, and filtering the supernatant by an organic microporous membrane of 0.22 mu m to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing 10mg of tacrolimus standard substance, dissolving the tacrolimus standard substance by using a diluent, fixing the volume to 10mL, preparing tacrolimus standard stock solution with the content of 1000mg/L, and preserving the tacrolimus standard stock solution at the temperature of 2-8 ℃; respectively sucking a proper amount of tacrolimus standard stock solution, preparing 5, 10, 50, 150, 300 and 600mg/L tacrolimus standard curve working solution by using a diluent, and preserving at 2-8 ℃ for on-site preparation;
precisely weighing 10mg of ascomycin standard substance, dissolving with a diluent and fixing the volume to 10mL to prepare ascomycin standard stock solution with the content of 1000mg/L, and preserving at the temperature of 2-8 ℃; respectively absorbing a proper amount of ascomycin standard stock solution, preparing 5mg/L ascomycin standard curve working solution, 10mg/L ascomycin standard curve working solution, 50 mg/L ascomycin standard curve working solution, 100 mg/L ascomycin standard curve working solution, and preserving at 2-8 ℃ for preparing at present;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, and a chromatographic column of color 5: YMC-Pack ODS-A,250×4.6mm,5 μm, volume ratio of mobile phase A to mobile phase B of 62:38, flow rate of chromatographic column of 1 m/min, column temperature of 55 deg.C, sample injection amount of chromatographic column of 10 μl, and detection wavelength of 220nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the sample solution according to a standard curve.
Example 3
This embodiment differs from embodiment 2 in that: the purity of the tacrolimus standard substance is more than or equal to 99%; the purity of the ascomycin standard substance is more than or equal to 99%; the sample solution is selected from tacrolimus fermentation liquid at different stages of the production process, the diluent is selected from one or more of methanol, ethanol, acetonitrile, acetone and water, and the diluent is an analytical grade; the inorganic acid of the mobile phase A is selected from one of trifluoroacetic acid, phosphoric acid or acetic acid; the organic solvent of the mobile phase A is selected from one of acetonitrile, ethanol or methanol; the organic phase solvent in the mobile phase B is one or more selected from methanol, acetonitrile, ethanol, isopropanol and water; the water is ultrapure water, the organic solvent of the mobile phase A is chromatographic purity, and the organic phase solvent of the mobile phase B is chromatographic purity.
Example 4
An HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation liquor is characterized in that the HPLC method is carried out by reversed phase liquid chromatography, a chromatographic column is a chromatographic column with octadecylsilane chemically bonded silica filler, a mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, phosphoric acid and acetonitrile, the mobile phase B comprises isopropanol, isocratic elution is carried out according to volume fraction, and quantitative analysis is carried out by adopting an external standard method.
Further embodiments also include the preparation of a pre-assay solution, the solution comprising:
blank solution, including ethanol;
a sample solution comprising tacrolimus fermentation broth and ethanol;
the tacrolimus standard curve working solution comprises tacrolimus standard substance and ethanol;
ascomycin standard curve working solution comprising ascomycin standard and ethanol.
Further embodiments include the steps of:
a1, preparation of mobile phase A
Weighing 1.0mL of phosphoric acid, adding water to 1000mL of phosphoric acid, uniformly mixing to prepare a phosphoric acid solution with the concentration of 0.1%, adding 50mL of acetonitrile, and uniformly mixing;
a2, preparation of sample solution
Weighing 5.0g of tacrolimus fermentation liquor in a 100mL volumetric flask, adding 50mL of ethanol, oscillating for 30 minutes, dissolving, fixing the volume, and filtering the supernatant through an organic microporous membrane of 0.22 mu m to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing 10mg of tacrolimus standard substance, dissolving the tacrolimus standard substance by using ethanol, fixing the volume to 10mL, preparing tacrolimus standard stock solution with the content of 1000mg/L, and preserving at the temperature of 2-8 ℃; respectively absorbing a proper amount of tacrolimus standard stock solution, preparing 5, 10, 50, 150, 300 and 600mg/L tacrolimus standard curve working solution by using ethanol, and preserving at 2-8 ℃ for preparation at present;
precisely weighing 10mg of ascomycin standard substance, dissolving with ethanol and fixing the volume to 10mL, preparing ascomycin standard stock solution with the content of 1000mg/L, and preserving at 2-8 ℃; respectively absorbing a proper amount of ascomycin standard stock solution, preparing 5mg/L ascomycin standard curve working solution, 10mg/L ascomycin standard stock solution, 50 mg/L ascomycin standard curve working solution, 100 mg/L ascomycin standard curve working solution, and preserving at 2-8 ℃ for preparing at present;
a4, detection and analysis
Waters e2695 high performance liquid chromatograph with ultraviolet detector, external standard method quantification;
the Waters e2695 high performance liquid chromatograph selects octadecylsilane chemically bonded silica filler chromatographic column, color 5 chromatographic column: YMC-Pack ODS-A,250×4.6mm,5 μm, volume ratio of mobile phase A to mobile phase B of 62:38, flow rate of chromatographic column of 1 m/min, column temperature of 55 deg.C, sample injection amount of chromatographic column of 10 μl, and detection wavelength of 220nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the sample solution according to a standard curve.
Wherein, the chromatogram of the blank solution (specifically, ethanol solution) is shown in fig. 1, the chromatogram of the tacrolimus standard stock solution is shown in fig. 2, the chromatogram of the ascomycin standard stock solution is shown in fig. 3, the tacrolimus standard curve is shown in fig. 4, the ascomycin standard curve is shown in fig. 5, the chromatogram of the test solution is shown in fig. 6, and fig. 7 is a table of peak results of fig. 6.
The tacrolimus or ascomycin content in the tacrolimus fermentation broth is calculated according to formula (1):
wherein:
x, the content of tacrolimus or ascomycin in the test sample is expressed in milligrams per kilogram (mg/kg);
c, determining the concentration of tacrolimus or ascomycin in the test solution according to a standard curve, wherein the unit is milligrams per liter (mg/L);
v. final volume of the sample solution, in milliliters (mL);
w is the mass of the test piece, and the unit is gram (g);
theoretical plate number=16 (retention time/peak width) 2 Or theoretical plate number=5.54× (retention time/half-width) 2
In the method, in the standard solution, the retention time of tacrolimus and ascomycin is 15.040 minutes and 13.403 minutes respectively, and the specific data of the linear range, the linear equation, the correlation coefficient and the quantitative limit of tacrolimus and ascomycin are as shown in the following table 1:
TABLE 1 Linear Range, linear equation, correlation coefficient, detection Limit, quantitative Limit for Tacrolimus and ascomycin
| Compounds of formula (I) | Linear range | Linear equation | Correlation coefficient R 2 | Detection limit | Quantitative limit |
| Tacrolimus | 5.08～609.6mg/L | Y＝3249X+6674.26 | 0.999734 | 0.08mg/L | 0.20mg/L |
| Ascomycin | 5.05～404.0mg/L | Y＝3104X-3618.25 | 0.999868 | 0.10mg/L | 0.25mg/L |
In the sensitivity, a sample without a target object is taken as a matrix in an experiment, the concentration of the target object is marked according to the detection limit of an instrument, and the signal to noise ratio of the target object is measured. Determining the qualitative detection limit of the method by using a signal-to-noise ratio (S/N) of 3 times, wherein tacrolimus is 0.08mg/L and ascomycin is 0.10mg/L; the limit of quantification of the method was determined at a 10-fold signal to noise ratio, wherein tacrolimus was 0.20mg/L and ascomycin was 0.25mg/L.
Precision: the recovery rate of tacrolimus in the added concentration level of tacrolimus fermentation liquor is 98.0-98.6%, ascomycin is 97.5-98.1%, and the specific data are shown in the following table 2:
TABLE 2 standard recovery and relative standard deviation of Tacrolimus and ascomycin
By the detection method, the contents of tacrolimus and ascomycin in the samples are measured by sampling fermentation liquor from different culture stages of production, and the specific data are shown in the following table 3:
TABLE 3 detection results of Tacrolimus and ascomycin in fermentation broth
| Sample numbering | 1 | 2 | 3 | 4 | 5 |
| Tacrolimus content (mg/kg) | 0.12 | 0.55 | 1.13 | 1.65 | 2.01 |
| Ascomycin content (mg/kg) | 0.05 | 0.12 | 0.24 | 0.30 | 0.35 |
The results show that the blank solvent does not interfere with the content measurement of tacrolimus and ascomycin in the fermentation liquor, the separation degree of tacrolimus and ascomycin in the test sample solution is at least 1.75, the separation degree of ascomycin and adjacent impurities is at least 1.95, both are larger than 1.5, the peak shape is sharp, the peak purity is good, the theoretical plate numbers are tacrolimus 3969 respectively, ascomycin 3831 is larger than 3000 respectively, the tailing factors are tacrolimus 1.01 respectively, and ascomycin 1.02 both accord with the standard.
In summary, the HPLC method for rapidly and simultaneously determining the tacrolimus and ascomycin contents in the fermentation broth is suitable for detecting and analyzing the tacrolimus and ascomycin contents in the fermentation broth, and has the advantages of rapidness (25 minutes per needle), high accuracy, high precision, simple operation, high throughput sample detection capability and capability of effectively controlling the quality of products in the production process.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (7)
1. An HPLC method for rapidly and simultaneously determining the contents of tacrolimus and ascomycin in fermentation broth is characterized in that: the method comprises the steps of measuring by reversed-phase liquid chromatography, wherein the chromatographic column adopts octadecylsilane chemically bonded silica filler, the mobile phase comprises a mobile phase A and a mobile phase B, the mobile phase A comprises water, inorganic acid and an organic solvent, the mobile phase B comprises the organic phase solvent, isocratic elution is set according to volume fraction, and quantitative analysis is carried out by adopting an external standard method.
2. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in fermentation broth according to claim 1, characterized in that: also included is the preparation of a pre-assay solution comprising:
blank solution, including diluent;
a sample solution comprising tacrolimus fermentation broth and a diluent;
the tacrolimus standard curve working solution comprises tacrolimus standard substance and diluent;
an ascomycin standard curve working solution comprising an ascomycin standard and a diluent.
3. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in fermentation broth according to claim 2, characterized in that: the method comprises the following steps:
a1, preparation of mobile phase A
Weighing a certain amount of inorganic acid, adding water for dilution, uniformly mixing to prepare an inorganic acid solution, adding an organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing a certain amount of tacrolimus fermentation liquor, adding a diluent, oscillating for dissolution, fixing the volume, and filtering to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing a certain amount of tacrolimus standard substance, adding a diluent to prepare tacrolimus standard stock solution, and weighing tacrolimus standard stock solution to prepare a series of tacrolimus standard curve working solutions;
weighing a certain amount of ascomycin standard substance, adding a diluent to prepare ascomycin standard stock solution, and then weighing ascomycin standard stock solution to prepare a series of ascomycin standard curve working solutions;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, the volume ratio of the mobile phase A to the mobile phase B is 58:42 to 75:25, the flow rate of the chromatographic column is 0.7-1.3 mL/min, the column temperature of the chromatographic column is 30-60 ℃, the sample injection amount of the chromatographic column is 5-20 mu L, and the detection wavelength is 205-230 nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the liquid to be detected according to a standard curve.
4. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in fermentation broth according to claim 3, characterized in that: the method comprises the following steps:
a1, preparation of mobile phase A
Weighing 1.0mL of inorganic acid, adding water to 1000mL, uniformly mixing to prepare an inorganic acid solution with the concentration of 0.1%, adding 50mL of organic solvent, and uniformly mixing;
a2, preparation of sample solution
Weighing 5.0g of tacrolimus fermentation liquor in a 100mL volumetric flask, adding 50mL of diluent, oscillating for 30 minutes, fixing the volume after dissolution, and filtering the supernatant by an organic microporous membrane of 0.22 mu m to obtain a sample solution;
a3, preparation of tacrolimus standard curve working solution and ascomycin standard curve working solution
Weighing 10mg of tacrolimus standard substance, dissolving the tacrolimus standard substance by using a diluent, fixing the volume to 10mL, preparing tacrolimus standard stock solution with the content of 1000mg/L, and preserving the tacrolimus standard stock solution at the temperature of 2-8 ℃; respectively sucking a proper amount of tacrolimus standard stock solution, preparing 5, 10, 50, 150, 300 and 600mg/L tacrolimus standard curve working solution by using a diluent, and preserving at 2-8 ℃ for on-site preparation;
precisely weighing 10mg of ascomycin standard substance, dissolving with a diluent and fixing the volume to 10mL to prepare ascomycin standard stock solution with the content of 1000mg/L, and preserving at the temperature of 2-8 ℃; respectively absorbing a proper amount of ascomycin standard stock solution, preparing 5mg/L ascomycin standard curve working solution, 10mg/L ascomycin standard curve working solution, 50 mg/L ascomycin standard curve working solution, 100 mg/L ascomycin standard curve working solution, and preserving at 2-8 ℃ for preparing at present;
a4, detection and analysis
Quantitative analysis by an external standard method by adopting a high performance liquid chromatograph with an ultraviolet detector;
the high performance liquid chromatograph with the ultraviolet detector selects a chromatographic column filled with octadecylsilane chemically bonded silica gel, and a chromatographic column of color 5: YMC-Pack ODS-A,250×4.6mm,5 μm, volume ratio of mobile phase A to mobile phase B of 62:38, flow rate of chromatographic column of 1 m/min, column temperature of 55 deg.C, sample injection amount of chromatographic column of 10 μl, and detection wavelength of 220nm;
a5, calculating results
The prepared tacrolimus standard curve working solution and ascomycin standard curve working solution are respectively detected according to the sequence from low concentration to high concentration, the peak area is taken as the ordinate, the concentration is taken as the abscissa, the standard curve is drawn, and the linear correlation coefficient R of the curve is drawn 2 ≥0.999；
And (3) injecting the filtered sample solution into a high performance liquid chromatograph with an ultraviolet detector to obtain corresponding peak areas, and obtaining the concentrations of tacrolimus and ascomycin in the sample solution according to a standard curve.
5. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in fermentation broth according to any of claims 2-4, characterized in that: the purity of the tacrolimus standard substance is more than or equal to 99%; the purity of the ascomycin standard substance is more than or equal to 99%; the sample solution is selected from tacrolimus fermentation liquid at different stages of the production process, the diluent is selected from one or more of methanol, ethanol, acetonitrile, acetone and water, and the diluent is an analytical grade.
6. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in a fermentation broth according to any of claims 1-4, characterized in that: the inorganic acid of the mobile phase A is selected from one of trifluoroacetic acid, phosphoric acid or acetic acid; the organic solvent of the mobile phase A is selected from one of acetonitrile, ethanol or methanol; the organic phase solvent in the mobile phase B is one or more selected from methanol, acetonitrile, ethanol, isopropanol and water.
7. The HPLC method for rapid simultaneous determination of tacrolimus and ascomycin content in fermentation broth according to claim 6, characterized in that: the water is ultrapure water, the organic solvent of the mobile phase A is chromatographic purity, and the organic phase solvent of the mobile phase B is chromatographic purity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311182678.1A CN117214368A (en) | 2023-09-14 | 2023-09-14 | HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311182678.1A CN117214368A (en) | 2023-09-14 | 2023-09-14 | HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117214368A true CN117214368A (en) | 2023-12-12 |
Family
ID=89036562
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311182678.1A Pending CN117214368A (en) | 2023-09-14 | 2023-09-14 | HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117214368A (en) |
-
2023
- 2023-09-14 CN CN202311182678.1A patent/CN117214368A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103543224B (en) | Detection method for residues of abamectin and ivermectin | |
| CN108152418B (en) | HPLC detection method for ketorolac tromethamine or/and impurities in preparation of ketorolac tromethamine | |
| CN108152425B (en) | Method for detecting lignanoids in sesame oil by high performance liquid chromatography | |
| CN108802243B (en) | Method for simultaneously detecting bentazone, 2,4-D, 2, 4-dichlorophen, 2,4, 6-trichlorophenol and pentachlorophenol in water | |
| CN117214368A (en) | HPLC method for rapidly and simultaneously determining tacrolimus and ascomycin content in fermentation liquor | |
| CN107907616B (en) | Method for detecting erythromycin residue in mushroom dregs | |
| CN111220730A (en) | Analysis method of related substances in irbesartan and hydrochlorothiazide compound preparation | |
| CN113866318A (en) | Method for detecting (6-aminopyridine-2-yl) (1-methylpiperidine-4-yl) methanone dihydrochloride | |
| CN114609295A (en) | High performance liquid chromatography analysis method for quinic acid content in tala enzymolysis waste liquid | |
| CN111220722B (en) | Method for simultaneously determining 8 p-hydroxybenzoate compounds in soil | |
| CN109061009B (en) | Method for measuring content of itaconic acid in fermentation liquor | |
| CN114354810A (en) | Method for detecting impurity N in clindamycin phosphate and method for separating impurity | |
| CN110208419B (en) | Method for detecting impurities in bivalirudin | |
| CN114034799A (en) | Method for detecting fluxapyroxad residual quantity in grapes | |
| Yao et al. | Ultra-sensitive speciation analysis of inorganic platinum-chloride complexes in platinum-based drugs by HPLC-ICP-MS | |
| CN113219089A (en) | Method for detecting urea by post-column derivatization-liquid chromatography | |
| Mahmoudi et al. | Novel liquid chromatographic method for the simultaneous evaluation of erythromycin and azithromycin in human urine | |
| CN111751466A (en) | Method for simultaneously determining DON toxin and NIV toxin | |
| CN116858978B (en) | Method for simultaneously detecting insulin aspart and insulin deglutition and plasma sample processing method thereof | |
| CN114113416B (en) | Method for determining veterinary drug chlorprostaenol in wastewater by liquid chromatography | |
| CN114544850B (en) | Dapagliflozin intermediate and impurity detection method | |
| CN115541755B (en) | Quality control method of nifuratel tablet | |
| CN114216990B (en) | Method for detecting dibromohydantoin in bromotrityl tetrazole biphenyl | |
| CN113945658B (en) | Method for simultaneously measuring contents of 7 antibiotics in water sample | |
| CN108760961B (en) | Method for detecting purity of solid potassium fomesate by liquid chromatography |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |