Mobile phase— Dissolve 1.4 g of monobasic ammonium phosphate and 2.0 g of phosphoric acid in 500 mL of water in a 1000-mL volumetric flask. Dilute with water to volume, mix, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Internal standard solution— Transfer about 75 mg of hypoxanthine to a 500-mL volumetric flask, dissolve in and dilute with water to volume, and mix.

Standard stock preparation— Dissolve an accurately weighed amount of USP Ganciclovir RS in water to obtain a solution having a known concentration of about 250 µg per mL.

Standard preparation— Transfer 20.0 mL of the Standard stock preparation and 10.0 mL of the Internal standard solution to a 100-mL volumetric flask. Dilute with Mobile phase to volume, and mix.

Assay stock preparation— Constitute Ganciclovir for Injection in a portion of water, quantitatively transfer with water to a suitable volumetric flask, and dilute with water to volume to obtain a solution having a concentration of about 1 mg per mL.

Assay preparation— Transfer 5.0 mL of the Assay stock preparation and 10.0 mL of the Internal standard solution 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 4.6-mm × 10-cm column that contains packing L1. The flow rate is about 1.2 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for hypoxanthine and 1.0 for ganciclovir; the resolution, R, between hypoxanthine and ganciclovir is not less than 3.0; the column efficiency is not less than 1000 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 peak response ratios for the major peaks. Calculate the quantity, in mg, of ganciclovir (C9H13N5O4) in the container of Ganciclovir for Injection taken by the formula: in which C is the concentration, in mg per mL, of USP Ganciclovir RS in the Standard preparation; D is the dilution factor, in mL, used to prepare the Assay preparation; and RU and RS are the peak response ratios of ganciclovir to the internal standard obtained from the Assay preparation and the Standard preparation, respectively.