Mobile phase— Prepare a filtered and degassed mixture containing 4volumes of ammonium acetate solution (1in 100)and 6volumes of a mixture of methanol,acetonitrile,and glacial acetic acid (400:200:0.6).Adjust this solution to a pHof 6.6±0.1by the dropwise addition of glacial acetic acid,and make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ),to obtain a retention time of about 5minutes for the fentanyl peak.

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

Assay preparation— If necessary,dilute the Injection with water so that each mLcontains the equivalent of about 50µg of fentanyl.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak response as directed for Procedure:the tailing factor for the fentanyl peak is not more than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in µg,of fentanyl (C22H28N3O)in each mLof the Injection taken by the formula: in which 336.48and 528.59are the molecular weights of fentanyl and fentanyl citrate,respectively;Cis the concentration,in µg per mL,of USP Fentanyl Citrate RSin the Standard preparation;Dis the dilution factor used to obtain the Assay preparation;and rUand rSare the peak responses for the fentanyl peak obtained from the Assay preparationand the Standard preparation,respectively.