A: Developing solvent—Prepare a mixture of chloroform,acetone,and diethylamine (40:5:5).

Test solution— Cut several pieces of Gum into small pieces with scissors,and weigh.Transfer a portion of the Gum,equivalent to about 4mg of nicotine,to a centrifuge tube.Add 5mLof chloroform,sonicate for about 30minutes to dissolve the nicotine,and centrifuge for about 10minutes.Cool to about 15,and add two 3-mLportions of 0.5Nhydrochloric acid with gentle mixing,and release excess pressure if necessary.Mix the contents of the tube by shaking,and centrifuge the mixture for about 10minutes.Transfer 5mLof the upper aqueous layer to a separatory funnel,and adjust with 0.5Nsodium hydroxide solution to a pHgreater than 10.0.Add 3mLof chloroform,and shake gently.Use the chloroform layer as the Test solution.

Standard solution— Transfer 10mg of USP Nicotine Bitartrate Dihydrate RSto a separatory funnel,add 10mLof water,and mix to dissolve.Adjust with 0.5Nsodium hydroxide to a pHgreater than 10.0.Add 3mLof chloroform,with gentle shaking,and use the chloroform layer as the Standard solution.

Procedure— Separately apply 10µLeach of the Test solutionand the Standard solutionabout 1.5cm from the lower edge of a thin-layer chromatographic plate (see Chromatography á621ñ).Air-dry,place the plate in a chromatographic tank that has been saturated with Developing solvent,and develop the chromatogram until the solvent front has moved about 7cm.Remove the plate from the chamber,and allow to air-dry.Examine the plate under short-wavelength UVlight:the RFvalue of the principal spot obtained from the Test solutioncorresponds to that obtained from the Standard solution(presence of nicotine).

B: Infrared Absorption á197Kñ—Use USP Polacrilex Resin RSand a test specimen prepared as follows.Cut a piece of Gum into small pieces with scissors.Place the pieces in a 50-mLcentrifuge tube,add about 20mLof n-hexane,and place in an ultrasonic bath for about 30minutes.Centrifuge at about 2500rpm for about 5minutes.Decant the hexane phase,and add 10mLof 2Nhydrochloric acid.Shake the tube carefully,and open the stopper slightly to relieve any excess pressure.Add 10mLof alcohol,and shake the tube carefully with the stopper slightly open.Centrifuge again as described above,and decant the liquid,taking care to avoid contamination of the precipitate with the gum material.Add 1mLto 3mLof water,mix gently to resuspend the precipitate,and filter.Wash the residue on the filter with water and then with alcohol.Dry the filter and residue at about 105for 1hour (presence of polacrilex).

Acetate buffer— Prepare a mixture containing 13.6g of sodium acetate and 57.2mLof glacial acetic acid in 1000mLof water.

Solvent— Prepare a mixture of water,acetonitrile,0.25Msodium 1-decanesulfonate,and Acetate buffer(785:150:40:25).

Mobile phase— Prepare a mixture containing water,acetonitrile,Acetate buffer,and 0.25Msodium 1-decanesulfonate (685:200:75:40).

Standard preparation— Dissolve an accurately weighed quantity of USP Nicotine Bitartrate Dihydrate RSin Solventto obtain a Standard stock solutionhaving a known concentration of about 1.25mg per mL.Dilute a volume of this solution quantitatively with Solventto obtain a Standard preparationhaving a known concentration of about 125µg per mL(40µg of nicotine per mL).

Assay preparation— Place one piece of Gum,accurately weighed,in a stoppered flask,add about 50mLof n-hexane,and transfer 50.0mLof Solvent.Add a stirring bar,insert the stopper in the flask,and stir vigorously for about 30minutes or until the test specimen has been dispersed.Remove from the stirring mechanism,and allow to stand for about 30minutes or until the phases have separated.Remove an aliquot of the lower layer,taking care not to remove a large quantity of the insoluble excipients,and filter,discarding the first few mLof the filtrate.Use the clear filtrate as the Assay preparation.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×30-cm stainless steel column containing packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,record the chromatograms,and measure the peak responses as directed for Procedure:the column efficiency is not less than 2500theoretical plates,the tailing factor is not more than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— [NOTE—Perform the following procedure on 10individual pieces of Gum,and use the average of the calculated values as the assay value.Use peak areas where peak responses are indicated.]Separately inject equal volumes (about 50µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak responses.Calculate the quantity,in mg,of nicotine (C10H14N2)in the Gum taken by the formula: in which Cis the concentration,in mg per mL,of USP Nicotine Bitartrate Dihydrate RSon the anhydrous basis in the Standard preparation,162.23and 462.41are the molecular weights of nicotine and anhydrous nicotine bitartrate,respectively,and rUand rSare peak responses obtained from the Assay preparationand the Standard preparation,respectively.