In this method, absorbance was measured at pH 1.2, 2.2, 6.4 and 7.4 at various concentrations (1 × 10−5 to 8 × 10−5 M) of Amlodipine besylate with Ca2+ (2 × 10−5 to 9 × 10−5) at 365 nm. The observed absorbance of the mixtures at various mole fractions was subtracted from the sum of the values for free drug and free metal. The absorbance differences (D) were then plotted against the mole fractions of drugs in the mixtures. This method

was conducted according to Ardon.10 In this method, concentrations of drug were varied while keeping the concentration of the metal fixed (2 × 10−5 M). All the experiments were performed in buffer at pH 1.2, 2.2, 6.4 and pH 7.4. The absorbance was measured at 365 nm by using UV–VIS spectrophotometer. From Ardon’s plot, the BMS-354825 cost value of stability constants of the drug–metal complex was calculated. For calculation, the Ardon’s equation was used. This equation is given below: 1(D−∈AC)=1KC(∈com−∈A)[B]n+1C(∈com−∈A)Here D = Absorbance of the mixture; B = Molar concentration of the drug; C = Molar concentration of the metal; ∈com = Molar Epigenetic inhibitors extinction co-efficient of the complex and ∈A = Molar extinction co-efficient of the drug. Equilibrium dialysis is one of the methods used for the determination of the protein binding of any compound developed by Singlass.11 Before conducting this method the dialysis membrane are activated.12

The membrane pieces were filled with BSA solution with different concentrations of drug and their (1:1) drug–metal mixture, keeping the total volume 4 ml. The membrane bags were immersed in 60 ml of solution having pH 7.4 and were shaken gently at (37 ± 0.5)°C for about 6 h in metabolic shaker. The absorbance of buffer (outside the membrane bags) was measured at 365 nm using the UV–VIS spectrophotometer and the concentrations of the bound and unbound drugs were calculated using a standard curve. The percentage of protein binding (F) mafosfamide was determined by the formula: F=[B]−[A][Totaldrug]×100where, A and B was the Molar concentration of free drug in buffer compartment and Molar concentration of total drug in protein compartment respectively. The Scatchard method13 and 14 was used for this purpose

and a curve was produced by plotting ‘r/[A]’ versus ‘r’ using the equation: r=[B]−[A][Protein]where, r = the ratio between the molar concentration of the bound drug and the molar concentration of protein. The results were expressed as Mean ± SEM values for each experiment. Differences in mean values between experimental groups were analyzed by unpaired t-test. A probability values less than 0.05 (p < 0.05) was defined to be significant. 15 It was seen that Amlodipine besylate gives a sharp peak at 365 nm. But when (Ca2+) mixed with Amlodipine besylate in 1:1 ratio, the intensity of the peak of Amlodipine besylate changes remarkably (absorbance decreases) i.e., absorption characteristics are altered due to interaction but the position of the compound do not shift (Fig. 1, Fig. 2, Fig.