A: It responds to the Thin-layer Chromatographic Identification Test á201ñ,the test solution and the Standard solution of USP Isoetharine Hydrochloride RSbeing prepared at a concentration of 2.5mg per mLin methanol,the solvent mixture being n-butanol,water,and formic acid (64:25:11),and the spots being located by spraying with sodium hydroxide solution (1in 10).

B: Mix about 50mg with about 200mg of powdered sodium hydroxide,transfer the mixture to a small test tube,heat in a small flame to fusion,and continue the heating for a few minutes longer.Cool,add about 0.5mLof water,then add a moderate excess of hydrochloric acid,and warm:starch iodate paper placed over the mouth of the test tube turns blue.

Mobile phase— Prepare a filtered and degassed mixture of 0.1Msodium sulfate in 0.8%acetic acid.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 60mg of USP Isoetharine Hydrochloride RS,accurately weighed,to a 25-mLvolumetric flask,add 4.0mLof alcohol,and mix.Add 3drops of 1Nhydrochloric acid,dilute with water to volume,and mix.

Assay preparation— Transfer about 75mg of Isoetharine Mesylate,accurately weighed,to a 25-mLvolumetric flask,add 4.0mLof alcohol,and mix.Add 3drops of 1Nhydrochloric acid,dilute with water to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L9.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed under Procedure:the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in mg,of C13H21NO3·CH4O3Sin the portion of Isoetharine Mesylate taken by the formula: in which Cis the concentration,in µg per mL,of USP Isoetharine Hydrochloride RSin the Standard preparation,335.42and 275.77are the molecular weights of isoetharine mesylate and isoetharine hydrochloride,respectively,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.