24. Standard stock and working solutions A stock standard solution of Iso and DIF (I mg/ml) were prepared by dissolving separately lso and DIF in methanol s then completing to 100 mL measuring flask with the same sol vent. Aliquot of the prepared stock solution was further r diluted with methanol to get working solution with final con- centration (100 ug mL 1). 2 Procedures For HPLC method Chromatographic conditions: Chromatographic separation was carried out using isocratic mode on a Agilant ODS-C18 (4.6 x 250 mm 1.D.) column with a mobile phase consisting of 80% acetonitrile: 20% methanol. (The phase was filtered using 0.45 m membrane filter mobile and degassed by ultrasonic vibrations for 10 min) with a flow rate of 1 mL min and the eluate was scanned at 230 nm at room temperature. All the injections were run in three repli cates and the injection volume was 20 The run time was th 6 min and the total peak areas were used to quantify the stud- ied components, 2 S. 1. Construction of calibration graph for the determination of the binary mi ture by HPLC method Aliquots of ISO and DIF working standard solution (100 ug mL 1) equivalent to 100-900 ug and 20-180 lug. respectively were accurately transferred into a two series of 10 ml, volumetric flasks, the volume was completed to the mark with the mobile phase. Analyte the prepared samples d using the previously mentioned chromatographic conditions and record the peak area then construct a calibration curve correlating the peak areas of Iso and DIF to the correspond- ing concentrations. Compute the corresponding regression equations d termination of