Identification—
Pipet a volume of Injection, equivalent to 10 mg of dexamethasone phosphate, into a 100-mL volumetric flask, add water to volume, and mix. Pipet 5 mL of this solution into a 125-mL separator, and wash with two 10-mL portions of water-washed methylene chloride, discarding the washings. Transfer the solution into a glass-stoppered, 50-mL tube, and add 5 mL of alkaline phosphatase solution, prepared by dissolving 50 mg of alkaline phosphatase enzyme in 50 mL of
pH 9 Buffer with magnesium (prepared as directed in
Identification test
A under
Dexamethasone Sodium Phosphate). Allow to stand at 37
for 45 minutes, and extract with 25 mL of methylene chloride. Evaporate 15 mL of the methylene chloride extract on a steam bath to dryness, and dissolve the residue in 1 mL of methylene chloride. Apply 5 µL of this solution and 5 µL of a solution of
USP Dexamethasone RS in methylene chloride containing 300 µg per mL to a 20- × 20-cm, thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel. Allow the spots to dry, and develop the chromatogram in a tank completely lined with a strip of filter paper, using a solvent system consisting of a mixture of 50 parts of chloroform, 50 parts of acetone, and 1 part of water, until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing tank, mark the solvent front, and allow the spots to dry. Spray the plate with dilute sulfuric acid (1 in 2), and heat at 105
until brown or black spots appear: the
RF value of the principal spot obtained from the test specimen corresponds to that obtained from the Reference Standard.
Assay—
Mobile phase—
Prepare a suitable degassed solution of 0.01 M monobasic potassium phosphate in a mixture of methanol and water (1:1) which, at ambient temperature and at a flow rate of about 1.6 mL per minute, gives a retention time of about 5 minutes for dexamethasone phosphate.
Standard preparation—
[NOTE—Prepare this solution at the time of use.
] Dissolve an accurately weighed quantity of
USP Dexamethasone Phosphate RS in
Mobile phase to obtain a solution having a known concentration of about 80 µg per mL.
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 8 mg of dexamethasone phosphate, to a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm × 30-cm column that contains packing L1. Chromatograph five replicate injections of the
Standard preparation, and record the peak responses as directed under
Procedure: the relative standard deviation is not more than 1.5%.
Procedure—
By means of a suitable sampling valve, separately inject equal volumes (about 20 µL) of the
Standard preparation and the
Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C
22H
30FO
8P in each mL of the Injection taken by the formula:
0.1(C / V)(rU / rS),
in which
C is the concentration, in µg per mL, of
USP Dexamethasone Phosphate RS in the
Standard preparation,
V is the volume, in mL, of Injection taken, and
rU and
rS are the peak responses at equivalent retention times obtained from the
Assay preparation and the
Standard preparation, respectively.
