A: The IRabsorption spectrum of a potassium bromide dispersion of it exhibits maxima only at the same wavelengths as that of a similar preparation of USP Phenobarbital RS.If a difference appears,dissolve portions of both the test specimen and the USP Reference Standard in a suitable solvent,evaporate the solutions to dryness,and repeat the test on the residues.

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

Solvent— Use dimethyl sulfoxide.

pH4.5Buffer solution— Dissolve about 6.6g of sodium acetate trihydrate and 3.0mLof glacial acetic acid in 1000mLof water,and adjust,if necessary,with glacial acetic acid to a pHof 4.5±0.1.

Mobile phase— Prepare a filtered and degassed mixture of pH4.5Buffer solutionand methanol (3:2),making adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard solution— Dissolve a sufficient quantity of caffeine in a mixture of methanol and pH4.5Buffer solution(1:1)to obtain a solution having a concentration of about 125µg per mL.

Standard preparation— Dissolve about 20mg of USP Phenobarbital RS,accurately weighed,in 15.0mLof Internal standard solution.Sonicate if necessary.

Assay preparation— Transfer about 20mg of Phenobarbital,accurately weighed,to a conical flask,add 15.0mLof Internal standard solution,mix,and sonicate for 15minutes.Filter through a membrane filter (0.5µm or finer porosity)before use.

Chromatographic system (see Chromatography á621ñ)— The liquid chromatograph is equipped with a 254-nm detector and a 4-mm ×25-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 resolution,R,between the analyte and the internal standard peaks is not less than 1.2,the tailing factor for the analyte and the internal standard peaks is not greater than 2.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.6for caffeine and 1.0for phenobarbital.Calculate the quantity,in mg,of C12H12N2O3in the portion of Phenobarbital taken by the formula: in which Wis the weight,in mg,of USP Phenobarbital RStaken for the Standard preparation,and RUand RSare the peak response ratios obtained from the Assay preparationand the Standard preparation,respectively.