By calculating A for the mixture
By calculating ΔA for the mixture, then the postulated Aiso of CPL at λ260 was obtained.
By substituting the absorbance difference ΔA (Aiso − A2) between the two selected wavelengths of the mixture spectrum in Eq. (1), the absorbance Apostulated corresponding to the absorbance of PA only at Aiso is obtained.
Subtracting the postulated absorbance of PA at Aiso from the practically recorded absorbance [ARecorded] at Aiso to get that CDK4 inhibitor corresponding to CPL.equation(2)ARecorded=ACPL+APAARecorded=ACPL+APA
The concentrations of CPL and PA were calculated using the corresponding unified regression equation (obtained by plotting the absorbance of the zero order spectra of CPL or PA at λiso 260 nm against the corresponding concentrations).CRecorded=0.0257CActual-0.011
The absorbance subtraction method has advantage cytoskeleton the two drugs in the mixture can be determined using unified regression equation at λiso in contrary to the previously established isoabsorptive point method ,  and  which determines total concentration of two drugs while one of the drugs is measured by using conventional spectrophotometric method as a complementary method.