A: Pipet 1mLof Injection into a 125-mLseparator containing 10mLof water and 1mLof 1Nsodium hydroxide,mix,extract with two 10-mLportions of methylene chloride,and evaporate the extracts on a steam bath just to dryness.Dissolve the residue in methanol,add 1mLof 1.2Nhydrochloric acid,then add methanol to make 100mL.Dilute 10mLof this solution with methanol to 100mL:the UVabsorption spectrum of this solution exhibits a maximum at the same wavelength as that of a similar solution of USP Amitriptyline Hydrochloride RS,concomitantly measured.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

C: Pipet a volume of Injection,equivalent to 50mg of amitriptyline hydrochloride,into a separator containing 25mLof water.Proceed as directed in the test for Identification—Organic Nitrogenous Bases á181ñ,beginning with “In a second separator,”and using water in place of 0.01Nhydrochloric acid in the Reference Standard solution:the solution of the test specimen so obtained meets the requirements of the test for Identification—Organic Nitrogenous Bases á181ñ.

Phosphate buffer— Dissolve 11.04g of monobasic sodium phosphate in 900mLof water,adjust with phosphoric acid to a pHof 2.5±0.5,dilute with water to make 1000mL,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Phosphate bufferand acetonitrile (58:42).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Amitriptyline Hydrochloride RSin water,and dilute quantitatively with water to obtain a solution having a known concentration of about 0.2mg per mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 50mg of amitriptyline hydrochloride,to a 250-mLvolumetric flask,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-mm ×30-cm 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 tailing factor is not more than 2.0,the column efficiency is not less than 800theoretical plates,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)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 amitriptyline hydrochloride (C20H23N·HCl)in each mLof the Injection taken by the formula: in which Cis the concentration,in mg per mL,of USP Amitriptyline Hydrochloride 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.