Solutions— Prepare a solution of Nicotine in water having a concentration of about 1mg per mL.Transfer 1.0mLof this solution to a 50-mLvolumetric flask,dilute with 0.1Nhydrochloric acid to volume,and mix to obtain the test solution.Transfer an amount of USP Nicotine Bitartrate Dihydrate RS,equivalent to about 50mg of nicotine,to a 25-mLglass-stoppered tube.Add 5mLof 6Nammonium hydroxide,2mLof 1Nsodium hydroxide,and 20mLof n-hexane.Shake for 5minutes,allow the phases to separate,transfer the upper n-hexane layer to a vial,and evaporate with a stream of nitrogen gas.[NOTE—Avoid excessive drying to prevent loss of nicotine.]Dissolve the residue of the nicotine so obtained in water to obtain a solution having a concentration of about 1mg per mL.Dilute 1.0mLof this solution with 0.1Nhydrochloric acid to 50.0mL,and mix to obtain the Standard solution.

Test solution: 20mg per mL,in alcohol.

Test solution— Dissolve about 0.13g of Nicotine,accurately weighed,in dichloromethane,dilute with dichloromethane to 25.0mL,and mix.

Reference solutions— Dilute accurately measured volumes of the Test solutionquantitatively,and stepwise if necessary,with dichloromethane to obtain Reference solution Aand Reference solution Bhaving concentrations of about 26µg per mLand 52µg per mL,respectively.

Chromatographic system(see Chromatography á621ñ)— The gas chromatograph is equipped with a flame-ionization detector maintained at 270and a 0.53-mm ×30-m fused silica column bonded with a 1.5-µm layer of phase G1.Helium is used as the carrier gas at a flow rate of 20mLper minute.The column temperature is maintained at 50for 6seconds,then programmed to rise from 50to 250at 6per minute,and finally held isothermally at 250for 3minutes.

Procedure— Separately inject equal volumes (about 1µL)of theTest solution,Reference solution A,andReference solution Binto the chromatograph,and allow theTest solutionto elute for not less than 2.5times the retention time of nicotine.Record the chromatograms,and measure all of the peak responses.The sum of the peak responses,excluding that of nicotine,from theTest solutionis not more than that of the nicotine response fromReference solution B(1.0%),and no single peak response is greater than that of the nicotine response fromReference solution A(0.5%).