Developing solvent— Shake equal volumes of isobutyl alcohol and water in a separator,and allow the layers to separate.Use the upper layer as the Mobile phaseand the lower layer as the Stationary phase.

p-Dimethylaminobenzaldehyde solution,1%— Dissolve 1.0g of p-dimethylaminobenzaldehyde in 50mLof alcohol,add 2mLof hydrochloric acid,and dilute with alcohol to 100.0mL.

pH6.5Buffer solution— Mix 700mLof 0.2Mdibasic sodium phosphate and 300mLof 0.1Mcitric acid.

Standard preparation— Prepare a solution of urea in water,containing 0.1mg per mL.

Test preparation— Dissolve 10.0mg of Hydroxyurea in 1.0mLof water.

Procedure— Treat a suitable chromatographic paper strip (Whatman No.1or equivalent)by dipping it in pH6.5Buffer solution.Dry the paper strip,and apply 100µLof the Test preparationand 50µLof the Standard preparation.Place the strip in a chromatographic chamber for descending chromatography containing the Stationary phasein the bottom of the chamber and the Mobile phasein the trough.Develop for 24hours,remove the strip from the chamber,air-dry,and develop again for 24hours.Remove the strip,air-dry,spray with p-Dimethylaminobenzaldehyde solution,1%,and heat at 90for 1to 2minutes.Not more than two spots,other than the major component,are present in the Test preparation,and their intensities are not greater than the intensity of the spot from the Standard preparation(0.5%of each impurity).The Rrvalues relative to hydroxyurea,the principal spot,are 0.65and 1.26(urea).

Solution A— Dissolve 1.7g of tetrabutylammonium hydrogen sulfate and 1.74g of dibasic potassium phosphate,anhydrous,in 1000mLof water,and adjust with 1Nsodium hydroxide or 85%phosphoric acid to a pHof 5.0.

Solution B: methanol.

Mobile phase— Prepare a solution of filtered,degassed Solution Aand Solution B(8.5:1.5).Make adjustments if necessary (see System Suitability under Chromatography á621ñ).

Resolution solution— Dissolve accurately weighed quantities of USP Hydroxyurea RSand hydroxylamine hydrochloride in Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phase to obtain a solution having a known concentration of about 0.4mg per mLof each.

Standard preparation— Dissolve an accurately weighed quantity of USP Hydroxyurea RSin Mobile phase,and dilute quantitatively,and stepwise if necessary,with Mobile phase to obtain a solution having a known concentration of about 0.4mg per mL.

Assay preparation— Transfer about 200mg of Hydroxyurea,accurately weighed,to a 500-mLvolumetric flask,dissolve in and dilute with Mobile phase to volume,and mix.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 214-nm detector and a 4.6-mm ×25-cm column that contains 5-µm packing L1.The flow rate is about 0.5mLper minute.Chromatograph the Resolution solution,and record the peak responses as directed for Procedure:the resolution,R,between the hydroxylamine and hydroxyurea peaks is not less than 1.5;for the hydroxyurea peak,the column efficiency is not less than 5000;and the tailing factor is not more than 1.5.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)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 CH4N2O2in the portion of Hydroxyurea taken by the formula: in which Cis the concentration,in mg per mL,of USP Hydroxyurea RSin the Standard preparation;and rUand rSare the peak responses of the hydroxyurea peaks obtained from the Assay preparation and the Standard preparation,respectively.