CN114216993B - Detection method of prednisolone acetate eye drops related substances - Google Patents
Detection method of prednisolone acetate eye drops related substances Download PDFInfo
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- CN114216993B CN114216993B CN202111620037.0A CN202111620037A CN114216993B CN 114216993 B CN114216993 B CN 114216993B CN 202111620037 A CN202111620037 A CN 202111620037A CN 114216993 B CN114216993 B CN 114216993B
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- LRJOMUJRLNCICJ-JZYPGELDSA-N Prednisolone acetate Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)C[C@@H]2O LRJOMUJRLNCICJ-JZYPGELDSA-N 0.000 title claims abstract description 85
- 229960002800 prednisolone acetate Drugs 0.000 title claims abstract description 85
- 239000003889 eye drop Substances 0.000 title claims abstract description 40
- 229940012356 eye drops Drugs 0.000 title claims abstract description 40
- 239000000126 substance Substances 0.000 title claims abstract description 27
- 238000001514 detection method Methods 0.000 title claims abstract description 26
- 239000012535 impurity Substances 0.000 claims abstract description 98
- XUKUURHRXDUEBC-SXOMAYOGSA-N (3s,5r)-7-[2-(4-fluorophenyl)-3-phenyl-4-(phenylcarbamoyl)-5-propan-2-ylpyrrol-1-yl]-3,5-dihydroxyheptanoic acid Chemical compound C=1C=CC=CC=1C1=C(C=2C=CC(F)=CC=2)N(CC[C@@H](O)C[C@H](O)CC(O)=O)C(C(C)C)=C1C(=O)NC1=CC=CC=C1 XUKUURHRXDUEBC-SXOMAYOGSA-N 0.000 claims abstract description 9
- RTGDFNSFWBGLEC-TVPGTPATSA-N 2-morpholin-4-ylethyl (z)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1h-2-benzofuran-5-yl)-4-methylhex-4-enoate Chemical compound COC1=C(C)C=2COC(=O)C=2C(O)=C1C\C=C(\C)CCC(=O)OCCN1CCOCC1 RTGDFNSFWBGLEC-TVPGTPATSA-N 0.000 claims abstract description 7
- AAEQXEDPVFIFDK-UHFFFAOYSA-N 3-(4-fluorobenzoyl)-2-(2-methylpropanoyl)-n,3-diphenyloxirane-2-carboxamide Chemical compound C=1C=CC=CC=1NC(=O)C1(C(=O)C(C)C)OC1(C=1C=CC=CC=1)C(=O)C1=CC=C(F)C=C1 AAEQXEDPVFIFDK-UHFFFAOYSA-N 0.000 claims abstract description 7
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 79
- 239000003085 diluting agent Substances 0.000 claims description 39
- 239000013558 reference substance Substances 0.000 claims description 34
- 239000000523 sample Substances 0.000 claims description 29
- 239000012488 sample solution Substances 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 20
- 238000012360 testing method Methods 0.000 claims description 14
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims description 8
- 239000007924 injection Substances 0.000 claims description 8
- 238000002347 injection Methods 0.000 claims description 8
- MOVRKLZUVNCBIP-RFZYENFJSA-N cortancyl Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(=O)COC(=O)C)(O)[C@@]1(C)CC2=O MOVRKLZUVNCBIP-RFZYENFJSA-N 0.000 claims description 7
- 230000014759 maintenance of location Effects 0.000 claims description 6
- 238000004090 dissolution Methods 0.000 claims description 5
- 230000004044 response Effects 0.000 claims description 5
- 240000001008 Dimocarpus longan Species 0.000 abstract description 6
- 235000000235 Euphoria longan Nutrition 0.000 abstract description 6
- 238000003908 quality control method Methods 0.000 abstract description 6
- 239000000725 suspension Substances 0.000 abstract description 6
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 54
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 27
- 238000002156 mixing Methods 0.000 description 24
- 239000011550 stock solution Substances 0.000 description 20
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 230000000903 blocking effect Effects 0.000 description 14
- 230000006378 damage Effects 0.000 description 14
- 239000012490 blank solution Substances 0.000 description 11
- 238000005303 weighing Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 8
- 238000010790 dilution Methods 0.000 description 8
- 239000012895 dilution Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 239000002253 acid Substances 0.000 description 7
- 239000011259 mixed solution Substances 0.000 description 7
- 239000002585 base Substances 0.000 description 6
- 230000001590 oxidative effect Effects 0.000 description 6
- 229940068196 placebo Drugs 0.000 description 6
- 239000000902 placebo Substances 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 239000003513 alkali Substances 0.000 description 5
- 238000007865 diluting Methods 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 239000012088 reference solution Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000006196 drop Substances 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000012494 forced degradation Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 206010022941 Iridocyclitis Diseases 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 201000004612 anterior uveitis Diseases 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000013065 commercial product Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000013643 reference control Substances 0.000 description 1
- 238000007430 reference method Methods 0.000 description 1
- 239000013074 reference sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000006467 substitution reaction Methods 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
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/30—Control of physical parameters of the fluid carrier of temperature
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Steroid Compounds (AREA)
Abstract
The invention discloses a detection method of prednisolone acetate eye drops related substances, which adopts high performance liquid chromatography for detection. The detection method can simultaneously and accurately detect the EP impurity A, EP impurity B, EP impurity C, the EP impurity D and the EP impurity E in the prednisolone acetate eye drops, thereby solving the quality control problem of the suspension for the prednisolone acetate longan and being beneficial to quality control of the suspension for the prednisolone acetate longan.
Description
Technical Field
The invention relates to the field of medicine detection, in particular to a detection method of prednisolone acetate eye drops related substances.
Background
The prednisolone acetate eye drops are anti-inflammatory drugs, belong to hormone eye drops, are mainly used for patients with ocular inflammation, such as iridocyclitis, and also can be used for treating ocular uveitis, and have good effect on controlling ocular inflammation.
The number of degradation impurities determined according to the EP (European pharmacopoeia) and the research on the short-term stability of the finished product process is 3, namely an EP impurity A, EP impurity B, EP impurity C, and the number of process impurities is two, namely an EP impurity D, EP impurity E.
In EP (european pharmacopoeia) and USP (united states pharmacopoeia), only the detection method of the related substances of API (crude drug) is given, the method is complex, the auxiliary materials in the finished product have a certain interference, the peak purity is insufficient, and the detection of the related substances is quite difficult. The RP-HPLC method for measuring the content of the prednisolone acetate and related substances thereof in the palibuted, pharmaceutical service and research, 2003,3 (2) provides a method for detecting impurities in the preparation for the prednisolone acetate, and experiments show that the peak purity angle (purity angle) of the EP impurity A in the chromatogram obtained by the method is larger than the purity threshold (purity threshold), namely the peak purity is insufficient, so that the content of the related substances of the prednisolone acetate eye drops cannot be accurately measured.
Disclosure of Invention
In order to solve the problems, the invention provides a detection method of prednisolone acetate eye drops related substances, which adopts high performance liquid chromatography for detection, and comprises the following specific steps:
a. sample solution preparation: taking prednisolone acetate eye drops to be detected, and adding a diluent for dissolution to obtain the prednisolone acetate eye drops;
b. preparing a reference substance solution: taking prednisolone acetate as a reference substance, and adding a diluent for dissolution to obtain the prednisolone acetate;
b. respectively taking reference substance solution and test sample solution, and injecting into chromatograph under the following chromatographic conditions:
chromatographic column: octadecylsilane chemically bonded silica of 3.5 μm is used as a filler;
mobile phase: volume ratio 70:30 water-acetonitrile;
flow rate: 0.8ml/min;
the diluent is 30% v/v acetonitrile in water.
Further, the volume ratio of the prednisolone acetate eye drops to the diluent in the step a) is 0.5-2.5: 10ml.
Further, the concentration of the prednisolone acetate in the reference substance solution in the step b) is 1-5 mug/mL.
Further, the column was XTERRAMS C18, 150×4.6mm,3.5 μm in size.
Further, in the chromatographic condition in the step c), the sample injection amount is 10 mu L, the wavelength is 254nm, the column temperature is 35 ℃, and the sample temperature is as follows: 5 ℃.
Further, the relative retention time of the relevant substance chromatographic peak and prednisone acetate Long Sepu peak in the chromatogram of the test solution is EP impurity A1.08, EP impurity B0.35, EP impurity C2.03, EP impurity D0.66 and EP impurity E2.11 respectively.
The invention also provides a content determination method of the prednisolone acetate eye drops related substances, which comprises the following specific operation steps:
1) Taking a sample to be detected, and detecting according to the method;
2) The content of the related substances is calculated according to the following formula:
wherein Aspl is Long Feng area of prednisone acetate in the sample solution; astd is the average peak area of prednisolone acetate in the six-needle control solution; wstd is the weighed amount of prednisolone acetate in the reference substance solution; dstd is the dilution factor of the reference solution; l is the marked amount of prednisolone acetate in the sample to be detected; dspl is the dilution factor of the sample to be tested; p is the purity of prednisolone acetate reference substance; RRF is the relative response factor of impurities.
Further, the relative response factors of the impurities are respectively: EP impurity a1.02, EP impurity B1.12, EP impurity C0.87, EP impurity D1.17, EP impurity E0.88, the remainder of impurity 1.
The detection method provided by the invention adopts a specific chromatographic column and chromatographic conditions, and can accurately detect the EP impurity A, EP impurity B, EP impurity C, the EP impurity D and the EP impurity E in the prednisolone acetate eye drops at the same time, thereby solving the quality control problem of the suspension for the prednisolone acetate longan and being beneficial to quality control of the suspension for the prednisolone acetate longan.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Reference of FIG. 1, reference control and mixed solution of impurities
Hollow white solution in the reference of FIG. 2
FIG. 3 reference sample solution
FIG. 4 reference acid breaking solution
FIG. 5 reference acid blank
FIG. 6 reference base disruption solution-1
FIG. 7 reference base disruption solution-2
FIG. 8 reference base blank
FIG. 9 reference oxidative destruction solution
FIG. 10 reference oxidation blank
FIG. 11 reference high temperature destruction solution-1
FIG. 12 reference high temperature destruction solution-2
FIG. 13 Linear investigation result
FIG. 14 blank solution
FIG. 15 control solution
FIG. 16 control and impurity mixed solution
FIG. 17Placebo solution
FIG. 18LOQ signal to noise ratio solution
FIG. 19 Normal sample map
FIG. 20 acid damage profile
FIG. 21 alkali blank map
FIG. 22 base destruction profile-1
FIG. 23 base destruction profile-2
FIG. 24 oxidation blank
FIG. 25 oxidative destruction profile
FIG. 26 high temperature destruction graph
Detailed Description
The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.
Example 1 detection of prednisolone acetate eye drops
a. Sample solution preparation: precisely transferring prednisolone acetate eye drops, diluting with diluent by 10 times v/v, and mixing well;
b. preparing a reference substance solution:
preparation of control stock solution: weighing prednisolone acetate as reference, adding diluent, dissolving and diluting to obtain prednisolone acetate concentration of 2 μg/mL;
wherein the diluent is 30% acetonitrile water solution;
c. respectively sucking the sample solution and the mixed reference solution, and injecting into a chromatograph under the following chromatographic conditions:
mobile phase: purified water: acetonitrile=70:30 (v: v);
detection wavelength: 254nm;
chromatographic column: XTERRAMS C18,3.5 μm,150x 4.6mm or equivalent;
flow rate: 0.8ml/min;
sample injection amount: 10 mu L
Column temperature: 35 DEG C
Sample temperature: 5 ℃;
run time: 40min
d. The relative retention time of the chromatogram of the test solution and the prednisone acetate Long Sepu peak is 1.08, 0.35, 2.03, 0.66 and 2.11 respectively, and the EP impurity A, EP impurity B, EP, C, EP and D, EP impurity E are judged to exist in the prednisolone acetate eye drops.
EXAMPLE 2 determination of the content of substances related to prednisolone acetate eye drops
a. Sample solution preparation: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, adding a diluent to dilute to a scale, and uniformly mixing;
b. preparing a reference substance solution:
preparation of control stock solution: weighing 10.0mg of prednisolone acetate as a reference substance, precisely weighing, adding a diluent into a 50mL volumetric flask, shaking to dissolve, fixing the volume to a scale, and shaking uniformly; and then taking the solution from 4mL to 20mL of volumetric flask, fixing the volume by using a diluent, and uniformly mixing. (control stock solution concentration: 40. Mu.g/mL);
preparing a reference substance solution: precisely transferring the control stock solution into a volumetric flask with volume of 0.5mL to 10mL, adding a diluent to dilute to a scale, shaking, uniformly mixing, and finally obtaining the concentration of the control solution: 2 μg/mL;
wherein the diluent is 30% acetonitrile water solution;
c. respectively sucking the sample solution and the reference substance solution, and injecting into chromatograph under the following chromatographic conditions:
mobile phase: purified water: acetonitrile=70:30 (v: v);
detection wavelength: 254nm;
chromatographic column: XTERRA MS C18,3.5 μm,150x 4.6mm or equivalent;
flow rate: 0.8ml/min;
sample injection amount: 10 mu L
Column temperature: 35 DEG C
Sample temperature: 5 ℃;
run time: 40min
d. The content of the related substances is calculated according to the following formula:
wherein Aspl is Long Feng area of prednisone acetate in the sample solution; astd is the average peak area of prednisolone acetate in the six-needle control solution; wstd is the weighed amount of prednisolone acetate in the reference substance solution, and mg; dstd is the dilution factor of the reference solution; l is the marked quantity of prednisolone acetate in the sample to be detected, and is 10.0mg/mL; dspl is the dilution factor of the sample to be tested; p is the purity of prednisolone acetate reference substance, calculated in mg/mg; RRF is the relative response factor of the impurity, nonspecific impurity rrf=1; specific impurities RRF are respectively: EP impurity A1.02, EP impurity B1.12, EP impurity C0.87, EP impurity D1.17, EP impurity E0.88,
the beneficial effects of the invention are further illustrated by way of test examples:
test example 1 study of prednisolone acetate eye drops
Method for detecting prednisolone acetate eye drops related substances by referring to RP-HPLC method for determining content of prednisolone acetate and related substances thereof in palibuted
1. Method of
1.1 preparation of solutions
A diluent: acetonitrile
Control and mixed solution of various impurities: weighing prednisolone acetate standard substance, adding diluent, dissolving and diluting to obtain reference substance solution with concentration of 0.4 μg/mL for use; weighing EP impurity A, B, C, D, E respectively, adding acetonitrile for dissolution, then absorbing each impurity single solution, mixing, and then adding a diluent for dilution to obtain impurity mixed solutions with the impurity concentration of 0.04 mg/mL; finally, respectively sucking 0.5mL of reference substance solution and impurity mixed solution, mixing, and then fixing the volume by using a diluent to obtain the reference substance with the concentration of 2 mug/mL of each substance and each impurity mixed solution.
Sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 15mL centrifuge tube, adding 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling;
blank solution: diluent agent
Alkaline destruction sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 15mL centrifuge tube, adding 0.1mL of 1N sodium hydroxide solution, blocking, uniformly mixing, standing at room temperature for 16h, adding 0.1mL of 1N hydrochloric acid solution and 2.0mL of acetonitrile, blocking, shaking, centrifuging, and taking supernatant for sample injection;
alkali blank solution: precisely transferring 0.1mL of 1N sodium hydroxide solution into a 15mL centrifuge tube, blocking, uniformly mixing, standing at room temperature for 16h, adding 0.1mL of 1N hydrochloric acid solution and 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling;
acid-destroyed sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 15mL centrifuge tube, adding 0.1mL of 1N hydrochloric acid solution, blocking, uniformly mixing, standing at room temperature for 16h, adding 0.1mL of 1N sodium hydroxide solution and 2.0mL of acetonitrile, blocking, shaking, centrifuging, and taking supernatant for sample injection;
acid blank solution: precisely transferring 0.1mL of 1N hydrochloric acid solution into a 15mL centrifuge tube, blocking, uniformly mixing, standing at room temperature for 16h, adding 0.1mL of 1N sodium hydroxide solution and 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling;
oxidative destruction of the sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 15mL centrifuge tube, adding 2 drops of 30% hydrogen peroxide, blocking, uniformly mixing, standing at room temperature for 16h, adding 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling;
oxidizing a blank solution: taking two drops of 30% hydrogen peroxide into a 15mL centrifuge tube, blocking, uniformly mixing, standing at room temperature for 16h, adding 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling;
high temperature destruction of the sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 15mL centrifuge tube, placing in a water bath at 80 ℃ for 5h, adding 2.0mL of acetonitrile, blocking, shaking, centrifuging, taking supernatant and sampling.
1.2 chromatographic conditions
Mobile phase: purified water: acetonitrile=69:31 (v: v)
Detection wavelength: 254nm
Chromatographic column:100RP-185μm,250x4mm or equivalent
flow rate: 1.0mL/min
Sample injection amount: 20 mu L
Column temperature: 30 DEG C
Sample temperature: 25 DEG C
Dilution solvent: acetonitrile
Run time: 60min
2. Results
2.1 control and peak-out results of the mixed solution of each impurity are shown in fig. 1 and table 1.
Table 1 peak time of each impurity
| Names of Compounds | Retention time min | Relative retention time |
| Name | RT | RRT |
| EP impurity B | 6.88 | 0.32 |
| EP impurity D | 13.91 | 0.65 |
| Prednisolone Acetate | 21.54 | - |
| EP impurity A | 23.44 | 1.09 |
| EP impurity C | 42.68 | 1.98 |
| EP impurity E | 47.14 | 2.19 |
2.2 forced degradation chromatogram results
TABLE 2 Strong degradation experiment results
From the results, it can be seen that: the peak purity angle (purity angle) of the EP impurity a in the chromatograms of the sample in the normal state and the acid, base, oxidized and high-temperature destroyed sample in the reference method is greater than the purity threshold (purity threshold), that is, the peak purity is insufficient, so that the method is insufficient to accurately and quantitatively detect all the impurities.
Through further large-scale screening of sample solvents, chromatographic columns, mobile phase elution programs and flow rates, the fact that the chromatographic peaks of prednisolone acetate and EP impurity A in a liquid chromatogram are closely adjacent is found, the separation degree is difficult to reach pharmacopoeia regulations, and even if the separation degree accords with expectations, the tail of the prednisolone acetate Long Sepu peak still has influence on the starting position of the chromatographic peak of the EP impurity A, so that accurate quantitative determination of the EP impurity A is interfered.
Through continuous trial and error tests, the chromatographic column and the mobile phase can obviously influence the chromatographic behavior of the prednisolone acetate and the EP impurity A, when a sample is diluted by a specific solvent, the accurate quantification of the EP impurity A can be realized under the action of the specific chromatographic column and the flow rate, and the determination method and the calculation formula of the relevant substances of the prednisolone acetate eye drops are determined through repeated experiments, and are specifically as follows:
1) Sample solution preparation: precisely transferring prednisolone acetate eye drops, diluting with diluent for 10 times, and mixing well;
2) Control solution preparation
Preparing a reference substance solution:
preparation of control stock solution: weighing prednisolone acetate as reference substance, precisely weighing, adding 30% acetonitrile water solution, dissolving and diluting to obtain reference substance stock solution with concentration of 40 μg/mL;
preparation of a control solution: precisely transferring the reference substance stock solution, and adding 30% acetonitrile water solution to dilute the reference substance stock solution into a reference substance solution with the final concentration of 2 mug/mL;
3) Respectively sucking the sample solution and the reference substance solution, and injecting into chromatograph under the following chromatographic conditions:
mobile phase: purified water: acetonitrile=70:30 (v: v);
detection wavelength: 254nm;
chromatographic column: XTERRA MS C18,3.5 μm,150x 4.6mm or equivalent;
flow rate: 0.8ml/min;
sample injection amount: 10 mu L
Column temperature: 35 DEG C
Sample temperature: 5 ℃;
run time: 40min
4) The relative retention time of the chromatogram of the test solution and the prednisone acetate Long Sepu peak is 1.08, 0.35, 2.03, 0.66 and 2.11 respectively, and the EP impurity A, EP impurity B, EP, C, EP and D, EP impurity E are judged to exist in the prednisolone acetate eye drops.
And the content of the related substances is calculated according to the following formula:
wherein Aspl is Long Feng area of prednisone acetate in the sample solution; astd is the average peak area of prednisolone acetate in the six-needle control solution; wstd is the weighed amount of prednisolone acetate in the reference substance solution, and mg; dstd is the dilution factor of the reference solution; l is the marked quantity of prednisolone acetate in the sample to be detected, and is 10.0mg/mL; dspl is the dilution factor of the sample to be tested; p is the purity of prednisolone acetate reference substance, calculated in mg/mg; RRF is the relative response factor of the impurity, nonspecific impurity rrf=1; specific impurities RRF are respectively: EP impurity a1.02, EP impurity B1.12, EP impurity C0.87, EP impurity D1.17, EP impurity E0.88.
Test example 2 methodology study
1. Linear investigation
1.1 preparation of solution:
CHK stock solution: weighing a prednisolone acetate standard 10.36 mg-50 mL volumetric flask, and dissolving with a diluent to fix the volume; and taking a volumetric flask with the volume of 4mL to 20mL of the solution, fixing the volume by using a diluent, and uniformly mixing.
Impurity mixed stock solution: dissolving 2mg of EP impurity A, B, C, D, E to five 5mL volumetric flasks with acetonitrile to fix volume; and then respectively taking 1mL of each impurity single solution into the same 10mL volumetric flask, fixing the volume by using a diluent, and uniformly mixing.
Sucking CHK stock solution, impurity mixed stock solution and diluent according to the proportion of the table 3,
preparing a series of solutions to be tested.
TABLE 3 Linear solution formulation Table
| Level | L1(LOQ) | L2 | L3 | L4 | L5 | L6 |
| CHK stock solution (mL) | 0.1 | 0.3 | 0.5 | 1 | 2.5 | 3 |
| Impurity mixed stock solution (mL) | 0.1 | 0.3 | 0.5 | 1 | 2.5 | 3 |
| Final constant volume (mL) | 10 | 10 | 10 | 10 | 10 | 10 |
| Concentration (μg/mL) | 0.4 | 1.2 | 2 | 4 | 10 | 12 |
Simultaneously preparing a blank solution and a prednisolone acetate reference substance solution, and taking the blank solution and the prednisolone acetate reference substance solution as references:
blank solution: diluent agent
Prednisolone acetate control solution: precisely weighing prednisolone acetate as reference substance, and adding 30% acetonitrile water solution to dissolve and dilute into 2 μg/mL reference substance solution;
1.2 detection results
Taking the solution to be detected, blank solution and prednisolone acetate reference substance solution with the series of concentrations, and detecting according to the chromatographic conditions finally determined in test example 1, wherein specific results are shown in table 4 and fig. 13-16, and the results can be seen as follows: the separation degree of the prednisolone acetate and the specific impurities is good, mutual interference does not exist, and when the concentration of the prednisolone acetate and the specific impurities is in the concentration range from 0.4 mug/mL (LOQ) to 12 mug/mL, the R of each component is more than 0.99, the linear relation is good, and the acceptable standard is met.
Table 4 results of linear range investigation
2. Accuracy and repeatability investigation
2.1 preparation of solutions
CHK stock solution: weighing a prednisolone acetate standard 10.36 mg-50 mL volumetric flask, and dissolving with a diluent to fix the volume; and then removing the solution from a 4mL volumetric flask to a 20mL volumetric flask, fixing the volume by using a diluent, and uniformly mixing.
Impurity mixed stock solution: dissolving 2mg of EP impurity A, B, C, D, E to five 5mL volumetric flasks with acetonitrile to fix volume; and then respectively taking 1mL of each impurity single solution into the same 10mL volumetric flask, fixing the volume by using a diluent, and uniformly mixing.
Placebo: the traditional Chinese medicine composition is prepared from other components except raw materials according to the formula components of the commercial product.
Placebo, CHK stock solution and impurity mixed stock solution were respectively aspirated according to the ratios in Table 5, and the diluent was formulated into three concentration levels of the solution to be tested.
TABLE 5 accuracy solution formulation table
| Level | A1(LOQ) | A-0.2％ | A-1.2％ |
| Placebo(mL) | 1 | 1 | 0.5 |
| CHK stock solution (mL) | 0.1 | 0.5 | 1.5 |
| Impurity mixed stock solution (mL) | 0.1 | 0.5 | 1.5 |
| Final constant volume (mL) | 10 | 10 | 5 |
| Concentration (μg/mL) | 0.4 | 2 | 12 |
2.2 detection results
The above test solution and Placebo solution were taken and tested according to the chromatographic conditions finally determined in test example 1, and the recovery rates of the contents of the three concentration levels prepared in parallel were calculated, and specific results are shown in tables 6 to 11, FIG. 17.
From the results, it can be seen that: no specific impurity was detected in placebo, and the results all met acceptable criteria (L-0.04 recovery at 70% -130%; recovery at 80% -120% for the remaining two levels); repeatability in% RSD at 9-needle recovery at three concentration levels for each impurity all met acceptable criteria (% RSD +.15%).
TABLE 6 EP impurity B accuracy results
TABLE 7 EP impurity D accuracy results
TABLE 8 EP impurity A accuracy results
TABLE 9 EP impurity C accuracy results
Table 10 EP impurity E accuracy results
TABLE 11 unspecific impurity accuracy results
3. Limit of quantitation (0.04%)
The solution was prepared by continuously feeding 6 needles with reference to the linear solution L1 (LOQ), and the results were shown in Table 12 and FIG. 18, according to the chromatographic conditions finally determined in test example 1.
From the results, it can be seen that: the peak area RSD% of the 6-needle solution is smaller than 10% of the acceptable standard, and the signal to noise ratio is larger than 10, so that the method meets the requirements.
Table 12 LOQ results table
4. Solution stability
4.1 Standard solution
The standard solution (prepared by using the control solution preparation method finally determined in test example 1) was stored at room temperature in a dark place, sampled at 0h, 47h and 67h, and tested according to the chromatographic conditions finally determined in test example 1, and the results are shown in Table 13.
TABLE 13 stability of standard solutions
From the results, it can be seen that: the standard solution was stable for 67 hours at room temperature in the dark.
4.2 sample solution
The sample solutions (prepared by the method for preparing the sample solutions finally determined in test example 1) were stored at room temperature in the dark, during which time samples were taken at 0h, 7h, 25h and 45h, and were tested according to the chromatographic conditions finally determined in test example 1, and the results are shown in Table 14.
TABLE 14 sample solution stability
From the results, it can be seen that: the sample solution was stable at room temperature for 7 hours in the dark.
5. Forced degradation
5.1 solution preparation
Normal sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, adding a diluent to dilute to a scale, and uniformly mixing;
alkaline destruction sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, adding 0.5mL of 1N sodium hydroxide solution, gently shaking, standing at room temperature for 0.5h, adding 0.5mL of 1N hydrochloric acid solution, fixing the volume with a diluent, and uniformly mixing.
Alkali blank solution: precisely transferring 0.5mL of 1N sodium hydroxide solution into a 10mL volumetric flask, adding, gently shaking, standing at room temperature for 0.5h, adding 0.5mL of 1N hydrochloric acid solution, fixing volume with diluent, and mixing well.
Acid-destroyed sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, adding 0.5mL of 1N hydrochloric acid solution, gently shaking, standing at room temperature for 1h, adding 0.5mL of 1N sodium hydroxide solution, fixing the volume with a diluent, and uniformly mixing.
Oxidative destruction of the sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, adding 0.5mL of 30% hydrogen peroxide, gently shaking, standing in water bath at 70 ℃ for 1h, fixing the volume with a diluent, and uniformly mixing.
Oxidizing a blank solution: taking two drops of 30% hydrogen peroxide into a 10mL volumetric flask, gently shaking, standing in a water bath at 70 ℃ for 1h, fixing the volume by using a diluent, and uniformly mixing.
High temperature destruction of the sample solution: precisely transferring 1.0mL of prednisolone acetate eye drops into a 10mL volumetric flask, placing in a 70 ℃ water bath for 2h, fixing the volume with a diluent, and uniformly mixing.
5.2 detection results
The forced degradation solution was measured under the chromatographic conditions finally defined in test example 1, and the results are shown in Table 15.
TABLE 15 conditions and results for Strong degradation experiments
| Strength of | Time | Temperature (temperature) | Link map numbering | |
| Normal sample | NA | NA | Room temperature | FIG. 19 |
| Acid(s) | 1N HCl(0.5mL) | 1h | Room temperature | FIG. 20 |
| Alkali blank | 1N NaOH(0.5mL) | 0.5h | Room temperature | FIG. 21 |
| Alkali | 1N NaOH(0.5mL) | 0.5h | Room temperature | Fig. 22 and 23 |
| Oxidation blank | 30％H 2 O 2 (0.5mL) | 1h | 70℃ | FIG. 24 |
| Oxidation | 30％H 2 O 2 (0.5mL) | 1h | 70℃ | FIG. 25 |
| High temperature | NA | 2h | 70℃ | FIG. 26 |
From the profile of each experimental condition, it can be seen that: the diluent and the placebo in the sample have no interference at the peak positions of the prednisolone acetate and the specific impurities; the sample is stable to acid, oxidation and high temperature, does not generate interference peaks, is unstable under the alkaline condition, but the degraded impurities have no interference to the determination of the main component and other specific impurities.
In conclusion, the detection method of the related substances of the prednisolone acetate eye drops can simultaneously and accurately detect the EP impurity A, EP impurity B, EP impurity C, the EP impurity D and the EP impurity E in the prednisolone acetate eye drops, so that the quality control problem of the suspension for the prednisolone acetate longan is solved, the quality control of the suspension for the prednisolone acetate longan is facilitated, and the detection method has practical application and popularization values.
Claims (3)
1. A detection method of prednisolone acetate eye drops related substances is characterized in that: the method adopts high performance liquid chromatography for detection, and comprises the following specific steps:
a) Preparing a sample solution: taking prednisolone acetate eye drops to be detected, and adding a diluent for dissolution to obtain the prednisolone acetate eye drops;
b) Preparing a reference substance solution: taking prednisolone acetate as a reference substance, and adding a diluent for dissolution to obtain the prednisolone acetate;
c) Respectively taking a reference substance solution and a test substance solution, and injecting into a chromatograph under the following chromatographic conditions:
chromatographic column: XTERRA MS C18, 150×4.6mm,3.5 μm;
mobile phase: volume ratio 70:30 water-acetonitrile;
flow rate: 0.8ml/min;
sample injection amount 10 μl, wavelength 254nm, column temperature 35 ℃, sample temperature: 5 ℃;
the volume ratio of the prednisolone acetate eye drops to the diluent is 0.5-2.5: 10;
the diluent is 30% v/v acetonitrile water solution;
the related substances are as follows: EP impurity A, EP impurity B, EP impurity C, EP impurity D and EP impurity E, the chemical structural formulas of the respective related substances are shown in the following table:
2. the method of detection according to claim 1, wherein: the concentration of the prednisolone acetate in the reference substance solution in the step b) is 1-5 mug/mL.
3. The method of detection according to claim 1, wherein: the relative retention time of the chromatographic peak of related substances and the prednisone acetate Long Sepu peak in the chromatogram of the sample solution is EP impurity A1.08, EP impurity B0.35, EP impurity C2.03, EP impurity D0.66 and EP impurity E2.11, and the relative response factors of the impurities are respectively: EP impurity a1.02, EP impurity B1.12, EP impurity C0.87, EP impurity D1.17, EP impurity E0.88.
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