A: Infrared Absorption á197Kñ.

B: The chromatogram obtained from the Assay preparationin the Assayexhibits a major peak for plicamycin the retention time of which corresponds to that exhibited by the Standard preparation,and the chromatogram compares qualitatively to that obtained from the Standard preparation.

Mobile phase— Prepare a suitable filtered and degassed mixture of 650mLof 0.01Mphosphoric acid and 350mLof acetonitrile.

Standard preparations— Dissolve an accurately weighed quantity of USP Plicamycin RSin Mobile phaseto obtain a solution having a concentration of 500µg of plicamycin per mL.Dilute this solution with Mobile phaseto obtain solutions containing 50,100,and 150µg of plicamycin per mL.

Assay preparation— Transfer about 5mg of Plicamycin,accurately weighed,to a 50-mLvolumetric flask.Dissolve in Mobile phase,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 278-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1.3mLper minute.Chromatograph replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationsand the Assay preparationinto the chromatograph by means of a suitable microsyringe or sampling valve,record the chromatograms,and measure the responses for the major peaks.The retention time is about 13minutes for plicamycin.Plot the peak responses of the Standard preparationsversus concentration,in µg per mL,of plicamycin,and draw the straight line best fitting the three plotted points.From the graph so obtained,determine the concentration,in µg per mL,of plicamycin in the Assay preparation.Calculate the potency,in µg of C52H76O24per mg,taken by the formula: in which Cis the concentration,in µg per mL,of plicamycin in the Assay preparation,and Wis the weight,in mg,of Plicamycin taken.