Adsorbent: chromatographic silica gel.

Developing solvent: mixture of chloroform and diethylamine (9:1).

Test preparation— Use undiluted.Apply 15µL.

Detection reagent: potassium iodoplatinate TS.

Procedure— Proceed as directed for Procedureunder Thin-Layer Chromatographic Identification Test á201ñ,the spots on the plate located by spraying with Detection reagent.

Acetate buffer— Prepare a solution in water containing in each L0.05mole of sodium acetate and 2.9mLof glacial acetic acid.

Mobile phase— Transfer 5.1g of tetrabutylammonium hydrogen sulfate to a 1-Lvolumetric flask,add 50mLof acetonitrile,and dilute with Acetate bufferto volume.Adjust with 5Nsodium hydroxide to a pHof 5.5±0.1.

Standard preparation— Dissolve an accurately weighed quantity of USP Atropine Sulfate RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 80µg per mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 2mg of atropine sulfate,to a 25-mLvolumetric flask,dilute with water to volume,and mix.

Resolution solution— Prepare a solution in water containing about 2.5µg of p-hydroxybenzoic acid per mL.Dilute one volume of this solution with four volumes of the Standard preparation.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and 30-cm ×3.9-mm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 1.5%.In a similar manner,chromatograph the Resolution solution:the retention time of p-hydroxybenzoic acid is about 1.6relative to that of atropine,and the resolution,R,between the p-hydroxybenzoic acid and atropine peaks is not less than 2.2.

Procedure— Separately inject equal volumes (about 100µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of (C17H23NO3)2·H2SO4·H2Oin each mLof the Injection taken by the formula: in which 694.85and 676.83are the molecular weights of atropine sulfate monohydrate and anhydrous atropine sulfate,respectively;Cis the concentration,in mg per mL,of USP Atropine Sulfate RSin the Standard preparation;Vis the volume,in mL,of Injection taken;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.