Thin-layer chromatographic identification test
201
Test solution
Transfer a quantity of Oral Solution, equivalent to about 5 mg of dexamethasone, to a 50-mL separator, add 10 mL of water, and extract with two 20-mL portions of chloroform. Filter the lower layers through chloroform-saturated cotton into a 50-mL conical flask, and evaporate to dryness. Dissolve the residue in 10 mL of chloroform.
Developing solvent system:
a mixture of methylene chloride and methanol (180:16).
Procedure
Visualize the spots, using a 1 in 5 solution of p-toluenesulfonic acid in a mixture of alcohol and propylene glycol (9:1) followed by heat.
Assay
Mobile phase
Prepare a filtered and degassed mixture of methanol, water, and glacial acetic acid (55:43:2). Make adjustments if necessary (see
System Suitability under
Chromatography
621![](images/chars/rang.gif)
).
Diluting solution
Prepare a mixture of methanol and water (1:1).
Standard preparation
Dissolve an accurately weighed quantity of
USP Dexamethasone RS in
Diluting solution, and dilute quantitatively, and stepwise if necessary, with
Diluting solution to obtain a solution having a known concentration of about 0.04 mg per mL.
Assay preparation
Transfer an accurately measured volume of Oral Solution, equivalent to about 4 mg of dexamethasone, to a 100-mL volumetric flask, dilute with Diluting solution to volume, and mix.
Chromatographic system
(see
Chromatography
621![](images/chars/rang.gif)
)The liquid chromatograph is equipped with a 254-nm detector and a 3.9-mm × 30-cm column that contains packing L1. The flow rate is about 1.5 mL per minute. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the column efficiency is not less than 2000 theoretical plates, the tailing factor is not more than 2.0, and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure
Separately inject equal volumes (about 10 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 per mL, of dexamethasone (C
22H
29FO
5) in the portion of Oral Solution taken by the formula:
LC / D(rU / rS),
in which
L is the labeled quantity, in mg per mL, of dexamethasone in the Oral Solution;
D is the concentration, in mg per mL, of dexamethasone in the
Assay preparation on the basis of the labeled quantity and the extent of dilution;
C is the concentration, in mg per mL, of
USP Dexamethasone RS in the
Standard preparation; and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.