Four rapid, accurate and very simple derivative spectrophotometric techniques were developed for the quantitative determination of binary mixtures of estradiol (E2) and progesterone (PRG) formulated as a capsule. Method I is the first derivative zero-crossing technique, derivative amplitudes were detected at the zero-crossing wavelength of 239.27 and 292.51 nm for the quantification of estradiol and 249.19 nm for Progesterone. Method II is ratio subtraction, progesterone was determined at λmax 240 nm after subtraction of interference exerted by estradiol. Method III is modified amplitude subtraction, which was established using derivative spectroscopy and mathematical manipulations. Method IIII is the absorbance ratio technique, absorbance of both medicines was measured at two wavelengths λ1= 260, -absorptive point and λ2=240max of progesterone. The Q equations were used to calculate the final concentrations. The calibration curve is linear from 5.0–140 and 2.0–32.0 µg/ml for estradiol and progesterone respectively. The proposed techniques' selectivity was tested using synthetic combinations created in the lab and assessed using the standard addition method. Using one-way ANOVA, the outputs of the proposed ways were compared, and the result showed no significant differences between the proposed techniques.
