Identification—
Concentrate or dilute, if necessary, a suitable volume of Injection to a concentration of about 10 mg per mL. Apply 2 µL of this solution and of a Standard solution of
USP Phenylephrine Hydrochloride RS, containing about 10 mg per mL, at points about 2.5 cm from the bottom edge of a suitable thin-layer chromatographic plate (see
Chromatography 
621
) coated with a 0.25-mm layer of chromatographic silica gel. Dry the spots in a current of warm air, and develop the chromatogram in a suitable chromatographic chamber with a mixture of methanol, water, and ammonium hydroxide (72:25:3) until the solvent front has moved about 12 cm. Dry the plate in warm air, and spray it with
alcoholic potassium hydroxide TS. Dry at 60
for 15 minutes, and spray the plate with
p-nitroaniline TS: the reddish orange spot obtained from the test solution corresponds in color, size, and intensity to that obtained from the Standard solution.
Assay—
Mobile phase—
Prepare and filter a mixture of methanol and water (1:1) containing 1.1 g of sodium 1-octanesulfonate per liter, adjusted with 3
M phosphoric acid to a pH of 3.0. Make adjustments if necessary (see
System Suitability under
Chromatography 621).
Dilution solvent—
Prepare a mixture of methanol and water (1:1), adjusted with 3 M phosphoric acid to a pH of 3.0.
Standard preparation—
Dissolve about 50 mg of
USP Phenylephrine Hydrochloride RS, accurately weighed, in 10 mL of water, dilute with
Dilution solvent to 25.0 mL, and mix. Further dilute 5.0 mL of the resulting solution with
Dilution solvent to 25.0 mL, and mix to obtain a solution having a known concentration of about 0.4 mg per mL.
Assay preparation—
Transfer an accurately measured volume of Injection, equivalent to about 10 mg of phenylephrine hydrochloride, to a 25-mL volumetric flask. Dilute with
Dilution solvent to volume, and mix.
Chromatographic system
(see
Chromatography 621)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm × 25-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the
System suitability solution, and record the responses for the major peaks: the resolution,
R, between epinephrine and phenylephrine is not less than 1.0. Chromatograph replicate injections of the
Standard preparation, and record the peak responses as directed for
Procedure: the relative standard deviation is not more than 2.0%.
Procedure—
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
9H
13NO
2·HCl in each mL of the Injection taken by the formula:
(25C / V)(rU / rS),
in which
C is the concentration, in mg per mL, of
USP Phenylephrine Hydrochloride RS in the
Standard preparation,
V is the volume, in mL, of Injection taken, and
rU and
rS are the peak responses obtained from the
Assay preparation and the
Standard preparation, respectively.
