Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (65:35)that contains 1mLof phosphoric acid in each 1000mL.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

0.01M Methanolic sodium hydroxide— Dissolve 400mg of sodium hydroxide in 500mLof water.Cool,add 500mLof methanol,and mix.

Standard preparation— Dissolve an accurately weighed quantity of USP Levothyroxine RSin 0.01M Methanolic sodium hydroxide,and dilute quantitatively and stepwise with 0.01M Methanolic sodium hydroxideto obtain a solution having a known concentration of about 4µg per mL.

Assay preparation— Transfer an accurately weighed portion of Oral Powder,equivalent to about 5mg of levothyroxine sodium,to a 250-mLvolumetric flask.Dilute with 0.01M Methanolic sodium hydroxideto volume,mix,and allow to stand for 4hours,with occasional mixing.Pass a portion of this mixture through a filter that does not absorb levothyroxine.Transfer 10.0mLof the filtrate to a 50-mLvolumetric flask,dilute with 0.01M Methanolic sodium hydroxideto volume,and mix.

Chromatographic system (seeChromatography á621ñ)— The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×25-cm column that contains packing L10.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.8;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of levothyroxine sodium (C15H10I4NNaO4)in the portion of Oral Powder taken by the formula: in which 798.85and 776.87are the molecular weights of levothyroxine sodium and levothyroxine,respectively;Cis the concentration,in µg per mL,of USP Levothyroxine RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.