A: Transfer to a separator a volume of Injection,equivalent to about 50mg of phenobarbital sodium,add 15mLof water,add 2mLof hydrochloric acid,shake,and extract the liberated phenobarbital with four 25-mLportions of chloroform.Filter the combined extracts through a pledget of cotton or other suitable filter into a beaker,and wash the separator and the filter with several small portions of chloroform.Evaporate a 50-mLportion of the chloroform solution of phenobarbital on a steam bath with the aid of a current of air.Add 10mLof ether,again evaporate,and dry the residue at 105for 2hours:the IRabsorption spectrum of a potassium bromide dispersion of the residue so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Phenobarbital RS.

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.

pH4.5Buffer solution,Mobile phase,Internal standard solution,and Chromatographic system— Prepare as directed in the Assayunder Phenobarbital.

Standard preparation— Transfer about 15mg of USP Phenobarbital RS,accurately weighed,to a 50-mLvolumetric flask,add 25mLof Mobile phase,and sonicate if necessary to dissolve.Add 15.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.3mg of USP Phenobarbital RSper mL.

Assay preparation— Transfer an accurately measured volume of Injection,equivalent to about 65mg of phenobarbital sodium,to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Transfer 25.0mLof this solution to a 50-mLvolumetric flask,add 15.0mLof Internal standard solution,dilute with Mobile phaseto volume,and mix.

Procedure— Proceed as directed for Procedurein the Assayunder Phenobarbital.Calculate the quantity,in mg,of C12H11N2NaO3in each mLof the Injection taken by the formula: in which 254.22and 232.24are the molecular weights of phenobarbital sodium and phenobarbital,respectively,Vis the volume,in mL,of Injection taken,and the other terms are as defined therein.