METHOD FOR CONTROLLING IMPURITY OF CYCLOSPORIN A EYE GEL

Abstract

A method of controlling the impurities in a cyclosporin A eye gel. A high performance liquid chromatography is performed, and chromatographic conditions are as follows: the detection wavelength is 210-230 nm; the column temperature is 60-68? C.; the flow rate is 0.8-1 ml/min; and the mobile phase A is: THF-water-phosphoric acid. The method of controlling impurities solves the problem of excipients interference and separation of many impurities at the same time, it also provides an effective method for the formulation of quality standard of impurities in this kind of preparation.

Claims

1. A method of controlling the impurities in a cyclosporin A eye gel, comprising: performing high performance liquid chromatography, wherein, chromatographic conditions are as follows: a detection wavelength is 210-230 nm; a column temperature is 60-68? C.; a flow rate is 0.8-1 ml/min; and a mobile phase is THF-water-phosphoric acid.

2. The method of controlling impurities according to claim 1, wherein a volume ratio of THF, water, and phosphoric acid in the mobile phase is 400:600:1.5-1.8.

3. The method of controlling impurities according to claim 1, wherein the column temperature is 65? C.

4. The method of controlling impurities according to claim 1, wherein the chromatographic column has octadecylsilane-bonded silica gel as a filler.

5. The method of controlling impurities according to claim 1, wherein the detection wavelength is 220 nm.

6. The method of controlling impurities according to claim 1, wherein the column is a waters column, a Thermo column, a Pheromone column or a YMC column.

7. The method of controlling impurities according to claim 1, wherein the volume ratio of THF, water, and phosphoric acid in the mobile phase is 400:600:1.58.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is an adaptive mixed standard chromatogram of the impurity system in Chinese Pharmacopoeia.

[0028] FIG. 2 is an adaptive mixed standard chromatogram of the impurity system of example 12;

[0029] FIG. 3 is an adaptive mixed standard chromatogram of the impurity system of example 1;

[0030] FIG. 4 is an adaptive mixed standard chromatogram of the impurity system of example 2;

[0031] FIG. 5 is an adaptive mixed standard chromatogram of the impurity system of example 3;

[0032] FIG. 6 is an adaptive mixed standard chromatogram of the impurity system of example 4;

[0033] FIG. 7 is an adaptive mixed standard chromatogram of the impurity system of example 5;

[0034] FIG. 8 is an adaptive mixed standard chromatogram of the impurity system of example 6;

[0035] FIG. 9 is an adaptive mixed standard chromatogram of the impurity system of example 7;

[0036] FIG. 10 is an adaptive mixed standard chromatogram of an impurity system in example 8;

[0037] FIG. 11 is an adaptive mixed standard chromatogram of the impurity system of example 9;

[0038] FIG. 12 is an adaptive mixed standard chromatogram of the impurity system in example 10; and

[0039] FIG. 13 is an adaptive mixed standard chromatogram of the impurity system in example 11.

DETAILED DESCRIPTION

[0040] The present invention discloses a method for controlling impurities of cyclosporin A eye gel, and those skilled in the art can learn from the contents of the disclosure and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are apparent to those skilled in the art and are considered to be included in the present invention. The method of the present invention has been described in terms of preferred embodiments, and it is apparent that the method and application described herein may be changed or modified and combined to implement and apply the present invention without departing from the content, spirit, and scope of the invention.

[0041] In order to make those skilled in the art better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific examples.

EXAMPLE 1

Chromatographic Conditions

[0042] Mobile phase: Acetonitrile-water-Methyl-t-butyl Ether-phosphoric acid (430:520:50:1) [0043] Wavelength: 210 nm; [0044] Flow rate: 1.0 ml/min; [0045] Column temperature: 70? C.; [0046] Sample volume: 80 ul; [0047] Chromatographic column: watersC18 (150mm*4.6mm, 5 um) filler

[0048] The solution chromatogram for the systematic application is shown in FIG. 3. The results showed that there are many interference peaks interfering with the effective detection of impurities, and the separation degree of the main peak from the latter impurity (retention time 51.9 min) is poor, the last two impurities do not achieve baseline separation, therefore this method can not be used to detect the related substances of cyclosporine A eye gel.

EXAMPLE 2

Chromatographic Conditions

[0049] Reference is made to article Assay of Cyclosporin and its degradation products in cyclosporine capsules by HPLC method of the Chinese Journal of antibiotics, Vol. 27, No. 4, April, 2002. The flow rate is 1 ml/min, HPLC cloume is HypersilBDSC18250 mm*4.6 mm,5 um, Mobile phase: water-THF-0.4 mol/L N-propylamine phosphate solution (0.4 mol/L N-propylamine solution, adjusting pH value to 2.6 with phosphoric acid)=590: 400: 10, column temperature is 70? C., and the wavelength is 220 nm.

[0050] The chromatogram is shown in FIG. 4. The results showed that the separation degree of impurity was poor when the retention time was 34.0 min or 35.3 min, baseline separation was not achieved.

EXAMPLE 3

Chromatographic Conditions

[0051] On the basis of example 2, the proportion of organic phases was reduced. The specific conditions are as follows: the flow rate is 1 ml/min, the HPLC column is Hypersil BDS C18 250 mm*4.6 mm, 5 u, Mobile phase: [Water-0.4 mol/L N-propylamine phosphate solution(0.4 mol/L N-propylamine solution, adjusting pH value to 2.6 with phosphoric acid)=(885:15)]?THF=650:350, column temperature is 70? C., and the wavelength is 220 nm.

[0052] The chromatogram is shown in FIG. 5. The results show that when the retention time was 69.4 min and 71.0 min, baseline separation of impurities was not achieved.

EXAMPLE 4

Chromatographic Conditions

[0053] On the basis of example 3, the chromatographic column was changed. The specific conditions are as follows: the flow rate is lml/min, the HPLC column is Waters Nova-Pak C18 350 mm*3.9 mm, 4 um, the mobile phase: [Water-0.4 mol/L N-propylamine phosphate solution(0.4 mol/L N-propylamine solution, adjusting pH value to 2.6 with phosphoric acid)=(885:15)]?THF=650: 350, the column temperature is 70? C., and the wavelength is 220 nm.

[0054] The chromatogram is shown in FIG. 6. The result showed that the collecting time was too long , which was 130 minutes.

EXAMPLE 5

Chromatographic Conditions

[0055] On the basis of example 4, the isometric condition is changed to a gradient condition. Considering that the baseline noise of the mixed mobile phase through the instrument proportional valve is high, a mixed mobile phase was prepare. The specific conditions are as follows: the flow rate is lml/min, the HPLC column is Waters Nova-Pak C18 350 mm*3.9 mm, 4 um, the mobile phase A: [Water-0.4 mol/L N-propylamine phosphate solution(0.4 mol/LN propylamine solution, adjusting pH value to 2.6 with phosphoric acid)=(885: 15)]?THF=650:370, the mobile phase B: [Water-0.4 mol/L N-propylamine phosphate solution (0.4 mol/LN propylamine solution, adjusting pH value to 2.6 with phosphoric acid)=(885: 15)]?THF=600:400, the column temperature is 70? C., and the wavelength is 220 nm.

Gradient Srocedure:

[0056]

TABLE-US-00001 Time (min) A % B % 0 100 0 40 100 0 50 0 100 80 0 100 81 100 0 90 100 0,

[0057] The chromatogram is shown in FIG. 7. The results show that the baseline noise is large and impurity detection is interfered.

EXAMPLE 6

[0058] chromatographic conditions: It plans to remove N-propylamine from the mobile phase. The specific chromatographic conditions are as follows:

[0059] The flow rate is 0.7 ml/min, the HPLC column is Waters Nova-Pak C18 350 mm*3.9 mm, 4 um, the mobile phase: THF-water-85% phosphoric acid=400:600:1.58 ml, the column temperature is 70? C., and the wavelength is 220 nm.

[0060] The chromatogram is shown in FIG. 8. The results showed that the acquisition time was 120 min and the acquisition time was too long.

EXAMPLE 7

Chromatographic Conditions

[0061] It plans to change the amount of phosphoric acid in the mobile phase. The specific chromatographic conditions are as follows: the flow rate is 0.8 ml/min, the HPLC column is Waters Nova-Pak C18 350 mm*3.9 mm, 4 um, the mobile phase: THF-water-85% phosphoric acid=400:600:1.8, the column temperature is 65? C., and the wavelength is 220 nm.

[0062] The chromatogram is shown in FIG. 9. The results show that the detection of impurity at 39.3 min was interfered by excipient peak at 37.7 min.

EXAMPLE 8

Chromatographic Conditions

[0063] It plan to reduce the proportion of organic phase in the mobile phase. The specific chromatographic conditions are as follows: the flow rate is 0.8 ml/min, the HPLC column is Waters Nova-Pak C18 350 mm*3.9 mm, 4 um, the mobile phase: THF-water-85% phosphoric acid=380:620:1.58, the column temperature is 65? C., and the wavelength is 220 nm.

[0064] The chromatogram is shown in FIG. 10. The results showed that the peak of excipient at 140 min was not finished and the collecting time was too long.

EXAMPLE 9

Chromatographic Conditions

[0065] Mobile phase: Water-tetrahydrofuran-85% phosphoric acid (600:400:1.58) [0066] Wavelength: 220 nm; [0067] Flow rate: 0.7 ml/min; [0068] Column temperature: 70? C.; [0069] Sample volume: 80 ul; [0070] Chromatographic column: C18, 300 mm*3.9 mm, 4 um

[0071] The chromatogram of the solution for systematic application is shown in FIG. 11. What to be improved is: there is excipient peak at 90min and collecting time 120 minutes is too long.

EXAMPLE 10

Chromatographic Conditions

[0072] Mobile phase: Water-tetrahydrofuran-85% phosphoric acid (600:400:1.80); [0073] Wavelength: 220 nm; [0074] Flow rate: 0.8 ml/min; [0075] Column temperature: 65? C.; [0076] Chromatographic column: C18, 300 mm*3.9 mm, 4 um [0077] Sample volume: 100 ul;

[0078] The chromatogram of the solution for systematic application is shown in FIG. 12.

[0079] What to be improved is: the excipient peak 37 is not completely separated from the latter impurity and interferes with the impurity detection.

EXAMPLE 11

Chromatographic Conditions

[0080] Mobile phase: Water-tetrahydrofuran-85% phosphoric acid (600:400:1.58); [0081] Wavelength: 220 nm; [0082] Flow rate: 0.8 ml/min; [0083] Column temperature: 65? C.; [0084] hromatographic column: C18, 300 mm*3.9 mm, 4 um [0085] Sample volume: 100 ul;

[0086] The chromatogram of the solution for systematic application is shown in FIG. 13.

[0087] What to be improved is: the peak retention time of excipient was 140 minutes and the collecting time was too long.

EXAMPLE 12

Chromatographic Conditions

[0088] Mobile phase: Water-tetrahydrofuran-85% phosphoric acid (600:400:1.58); [0089] Wavelength: 220 nm; [0090] Flow rate: 0.8 ml/min; [0091] Column temperature: 65? C.; [0092] Chromatographic column: C18, 300 mm*3.9 mm, 4 um [0093] Sample volume: 100 ul;

[0094] The chromatogram of the solution for systematic application is shown in FIG. 2.

[0095] The above description only shows preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make a number of improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should be considered falling within the scope of protection of the present invention.