ascending technique to a distance of about 8 cm, dried at room temperature. The spots were detected under UV-lamp and scanned at 215 nm and 237 nm for ISO and DIF, respectively. The peak areas were recorded for the drug. The calibration curves were constructed by plotting the area under the peak versus the corresponding concentrations. The corresponding regression equations were computed.
2.5.3. Determination of the binary mixture in pharmaceutical dosage form by the proposed methods
One gram of cream was extracted by 35 mL methanol, heated to 50 C till it melted and sonicated for ten minutes. The extracted sample was cooled for 2 h till base coalescence. The extract was filtered into a 50 mL volumetric flask and
the residual mass was re-extracted by 10 mL methanol, cooled and filtered then the volume was completed to 50 mL with the same solvent.
The proposed methods were applied for the analysis of the pharmaceutical preparation solutions using the procedures mentioned under linearity and construction of calibration curve for each method and the concentrations of the cited drugs were calculated from the corresponding regression equations.
3. Results and discussion
This work is concerned with the simultaneous determination of ISO and DIF using two different chromatographic techniques. First method is HPLC which represents an advantage over the previously published HPLC method7,8 in sensitivity. Different mobile phases were tried for the chromatographic separation of the components and the best resolution was achieved using a mobile phase composed of 80% acetonitrile–20% methanol. Using the specific chromatographic conditions retention times were found to be 4.7 and 3.6 for ISO and DIF, respectively Fig. 2. Statistical analysis of the parameters required for sys- tem suitability test of HPLC method indicates a good resolu- tion of the two components as in Table 1.
HPLC method allows selective determination of ISO and DIF in the range of 10–90 lg mL 1 and 2–18 lg mL 1, respec- tively. The regression equations were calculated and found to be:
A 1⁄4 34:703C þ 36:745 r 1⁄4 0:9999 for ISO at 230 nm A1⁄442:689Cþ20:1 r1⁄40:9999 for DIF at 230nm
where, ‘‘A” is the peak area. ‘‘C” is the concentration in lg mL 1 and ‘‘r” is the regression coefficient. The mean per- centage recoveries and standard deviations of the pure drug were calculated as shown in Table 2.
The second method is a TLC-densitometric procedure, dif- ferent systems were tried, where complete separation of ISO and DIF was achieved using ethyl acetate: chloroform: toluene (60:10:10 by volume) as the mobile phase. The Rf values were 0.18 for ISO and 0.84 for DIF as in Figs. 3 and 4.
TLC-Densitometric technique allows selective determina- tion of ISO and DIF in the range of 0.1–4 lg spot 1 for ISO and 0.1–1.4 lg spot 1 for DIF. The regression equations were calculated and found to be: