BP 2025 (Ph. Eur. 11.6 update)
Action and use
Detection of corneal lesions, retinal angiography and pancreatic function testing.
DEFINITION
Fluorescein Eye Drops are a sterile solution of Fluorescein Sodium in Purified Water.
The eye drops comply with the requirements stated under Eye Preparations and with the following requirements.
Content of fluorescein sodium, C20H10Na2O5
90.0 to 110.0% of the stated amount.
IDENTIFICATION
- Evaporate a volume containing 20 mg of fluorescein sodium and dry at 105° for 30 minutes. The infrared absorption spectrum of the residue, Appendix II A, is concordant with the reference spectrum of fluorescein sodium (RS 151).
- The eye drops are strongly fluorescent, even in extreme dilution; the fluorescence disappears when the solution is made acidic and reappears when it is made alkaline.
- Dilute with water to produce a solution containing 0.05% w/v of fluorescein sodium. One drop of the solution, absorbed by a piece of filter paper, colours the paper yellow. On exposing the moist paper to bromine vapour for 1 minute and then to ammonia vapour the yellow colour becomes deep pink.
TESTS
Acidity or alkalinity
pH, 7.0 to 9.0, Appendix V L.
Chloroform-soluble matter
To a volume containing 0.1 g of fluorescein sodium add 1 mL of 2M sodium hydroxide, extract with 10 mL of chloroform and dry the chloroform layer with anhydrous sodium sulfate. The absorbance of the resulting solution at 480 nm is not more than 0.05, Appendix II B, using chloroform in the reference cell.
Related substances and resorcinol
Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
- Dilute the eye drops with 1M methanolic hydrochloric acid to contain 0.5% w/v of fluorescein sodium.
- Add to a volume of the eye drops containing 25 mg of fluorescein sodium 2 mL of phosphate buffer pH 8.0, 3 mL of water and 2.5 g of sodium chloride, shake to dissolve the sodium chloride and extract with two 25-mL quantities of peroxide-free ether. Dry the combined extracts over anhydrous sodium sulfate, evaporate to dryness under reduced pressure and dissolve the residue in 1 mL of 1M methanolic hydrochloric acid.
- Dilute 1 volume of solution (1) to 100 volumes with 0.1M methanolic hydrochloric acid.
- Dilute 2 volumes of solution (3) to 5 volumes with 0.1M methanolic hydrochloric acid.
- 0.0125% w/v of resorcinol in 0.1m methanolic hydrochloric acid.
- Mix 5 volumes of a 0.025% w/v solution of resorcinol in 0.1M methanolic hydrochloric acid with 2 volumes of solution (1) and add sufficient methanol to produce 10 mL.
CHROMATOGRAPHIC CONDITIONS
- Use a silica gel 60 F254 precoated plate (Merck plates are suitable).
- Use the mobile phase as described below.
- Apply 10 μL of solution (1) and 5 pL of solutions (2) to (6).
- Develop the plate to 15 cm.
- After removal of the plate, dry in air, expose the plate to iodine vapour for 30 minutes and examine in daylight and under ultraviolet light (254 nm).
MOBILE PHASE
10 volumes of methanol and 90 volumes of dichloromethane.
SYSTEM SUITABILITY
The test is not valid unless the chromatogram obtained with solution (6) shows two clearly separated spots in daylight.
LIMITS
In the chromatogram obtained with solution (1), under ultraviolet light (254 nm):
any secondary spot, other than any spot corresponding to resorcinol, is not more intense than the spot in the chromatogram obtained with solution (3) (0.5%);
not more than one such spot is more intense than the spot in the chromatogram obtained with solution (4) (0.2%).
In the chromatogram obtained with solution (2), in daylight:
any spot corresponding to resorcinol in the chromatogram obtained with solution (2) is not more intense than the spot in the chromatogram obtained with solution (5) (0.5%).
ASSAY
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
- Dilute the eye drops with the mobile phase to produce a solution containing 0.005% w/v of fluorescein sodium.
- Dissolve 55 mg of diacetylfluorescein BPCRS in a mixture of 5 mL of ethanol (96%) and 1 mL of 5m sodium hydroxide, heat on a water bath for 20 minutes, mixing frequently, cool and add sufficient water to produce 50 mL. Dilute 5 volumes of this solution to 100 volumes with the mobile phase.
CHROMATOGRAPHIC CONDITIONS
- Use a stainless steel column (25 cm x 4.6 mm) packed with end-capped octadecylsilyl silica gel for chromatography (5 μm) (Spherisorb ODS 2 is suitable).
- Use isocratic elution and the mobile phase described below.
- Use a flow rate of 1.5 ml per minute.
- Use an ambient column temperature.
- Use a detection wavelength of 254 nm.
- Inject 20 μL of each solution.
MOBILE PHASE
5 volumes of triethylamine, 400 volumes of acetonitrile and 595 volumes of water, adjust the pH to 3.0 with orthophosphoric acid.
DETERMINATION OF CONTENT
Calculate the content of C20H10Na2O5 in the eye drops using the declared content of anhydrous diacetylfluorescein in diacetylfluorescein BPCRS. Each mg of anhydrous diacetylfluorescein is equivalent to 0.9037 mg of C20H10Na2O5.
STORAGE
Fluorescein Eye Drops should be protected from light.
