Identification—
A:
Dissolve a quantity, equivalent to about 12 mg of echothiophate iodide, in 10 mL of water. To 1 mL of this solution add 0.2 mL of 2 N hydrochloric acid and 0.2 mL of 30 percent hydrogen peroxide: the color turns brown. Add a few drops of solvent hexane, and shake: the solvent hexane acquires a pink color.
B:
To 5 mL of the solution prepared for
Identification test
A add 0.5 mL of sodium hydroxide solution (1 in 2), heat at 50
for 2 minutes, cool to room temperature, then add 1 mL of
sodium nitroferricyanide TS: a deep-red color is produced.
Water—
[NOTE—Dry all glassware used in the following procedure at 105
for 4 hours, and store in a desiccator or dry box. Perform as many operations as possible in a dry box.
]
Dry alcohol—
Wash about 150 g of 8- to 12-mesh type 3A molecular sieve with several portions of dehydrated alcohol to remove the fine particles. Place the washed molecular sieve in a shallow borosilicate glass tray, heat in an oven at 350
for 2 hours, and cool in a dry box. Transfer the dry molecular sieve to a 1-liter conical flask, add about 700 mL of dehydrated alcohol, insert the stopper, mix, and allow to stand for not less than 48 hours before using.
Internal standard solution—
[NOTE—Prepare this solution fresh daily.] Place 0.17 mL of methanol in a 100-mL volumetric flask, add Dry alcohol to volume, and mix.
Standard preparations—
[NOTE—Prepare these solutions fresh daily.
] Into three 25-mL volumetric flasks, each containing about 15 mL of
Internal standard solution, transfer 5 µL, 40 µL, and 75 µL of water, respectively. Dilute with
Internal standard solution to volume, and mix.
Test preparation—
Carefully remove the protective retainer and cap from 5 vials of Echothiophate Iodide for Ophthalmic Solution without removing the elastomeric septum closure. Discard the separated parts, and weigh accurately each closed vial and contents. Inject through the septum of each vial 400 µL of
Internal standard solution, accurately measured, using a suitable gas-tight syringe, and allow to stand for 1 hour, swirling occasionally to dissolve the residue. After 1 hour, using a gas-tight syringe, remove 300 µL of solution from each vial, transfer to a dry small-volume sample-collecting vial equipped with a sampling valve system,
* and mix the combined solutions.
Chromatographic system
(see
Chromatography 
621
)—The chromatograph is equipped with a thermal conductivity detector and a 2-mm × 1.8-m silylated glass column packed with 80- to 100-mesh surface-silanized packing S3. The column is maintained at a temperature of about 115
, the injection port and detector block are maintained at temperatures of about 200
and 225
, respectively, and dry helium is used as the carrier gas at a flow rate of about 45 mL per minute. Chromatograph a sufficient number of injections of a
Standard preparation, and record the peak responses as directed for
Procedure: the resolution factor between the water and methanol peaks is not less than 2.0; and the relative standard deviation is not more than 5.0%.
Procedure—
Inject a portion (3 µL to 4 µL) of each
Standard preparation into the chromatograph, record the chromatogram, and measure the responses for the first (water) and second (methanol) major peaks obtained for each. Plot the ratios of the peak responses of water to methanol versus the concentration, in mg per mL, of water in each
Standard preparation.
[NOTE—If the plot is not linear, discard it, and repeat the chromatography on additional portions of the
Standard preparations.
] Similarly inject a portion of the
Test preparation, record the chromatogram, and measure the responses for the two major peaks. By comparison with the linear standard plot, determine the concentration, in mg per mL, of water in the
Test preparation as that corresponding to the ratio of the peak responses of water to methanol from the
Test preparation. Remove the elastomeric septum closure from each test vial, discard the contents, and rinse each vial and closure with several portions of methanol. Dry the vials and the closures in a stream of dry nitrogen, weigh accurately, and subtract this weight from that of the closed vials and contents obtained as directed under
Test preparation. Calculate the water content, in percentage, taken by the formula:
100CV / W,
in which
C is the concentration, in mg per mL, of water in the
Test preparation,
V is the volume, in mL, of
Internal standard solution added to each test specimen vial as directed under
Test preparation, and
W is the average weight, in mg, of the test specimens in the vials: not more than 2.0% is found.
Assay—
[NOTE—In the preparation of all reagents, and throughout this procedure, wherever water is specified, use only water that has been distilled, boiled for 10 minutes, and cooled while protected from the atmosphere.
] Dissolve the contents of a counted number of vials of Echothiophate Iodide for Ophthalmic Solution, equivalent to not less than 30 mg of echothiophate iodide, by adding 5.0 mL of water to each vial. Combine the solutions, and mix. Dilute a portion of the mixture, equivalent to about 12 mg of echothiophate iodide, with water to 40 mL, and proceed as directed for
Procedure in the
Assay under
Echothiophate Iodide, beginning with “Add 10.0 mL of
pH 12 phosphate buffer.”
