4). At experimental pH, Amlodipine besylate form strong 1:1 complexes with Ca2+ ion. Absorbance differences at pH 1.2, 2.2, 6.4 and 7.4 were (Fig. 5, Fig. 6, Fig. 7 and Fig. 8) IWR-1 chemical structure indicated as “ˆ” shaped curves

and the break points were found at absorbance difference of 0.15, 0.16, 0.17 and 0.18 at pH 1.2, 2.2, 6.4 and 7.4 respectively. It confirmed the formation of 1:1 complexes of Amlodipine besylate with Ca (II) ion. Ardon’s plot confirmed the formation of 1:1 complex of Amlodipine besylate with Ca (II) ion at pH 1.2, 2.2, 6.4 and 7.4, since the method is valid for only 1:1 complexes. The Ardon’s plots gave straight lines intercept which are presented in Fig. 9, Fig. 10, Fig. 11 and Fig. 12 indicate the formation of 1:1 complexes at experimental pH. The value of stability constant check details for the complexation of Amlodipine besylate with Ca (II) ion at pH 1.2, 2.2, 6.4 and 7.4 were obtained from the spectral data using Ardon’s plot. The values of stability constant were given as [(Intercept)/(slope)] by using Ardon’s equation. The values of stability constants for the drug–metal system at pH 1.2, 2.2, 6.4 and 7.4 presented in Table 1 The in vitro determination of percentage of protein binding of Amlodipine besylate and their 1:1 mixture with Ca (II) ion was done by equilibrium dialysis method at physiological temperature (37 ± 0.5)°C and at pH 7.4. The observed values of protein

binding for drug alone and with metal are given in Fig. 13. The spectra of drug molecules alone and (1:1) mixture of drug and metal showed significant change in their absorption intensities. This may be due to interaction of Ca2+ with drug that may alter the absorption intensities but the position of the compound does not shift. Job’s plots showed, for a constant total concentration of drug and metal, the complex was at its greatest concentration at a point where the species of drug and metal are combined in the ratio in which they occur in complex. The straight lines which cross each other showed a break at nearly 5 mol fractions indicating the 1:1 complexes for all the systems. At experimental pH, Amlodipine besylate forms

strong 1:1 complexes with Ca2+ indicated as ‘ˆ’ shaped curves. These curves may indicate strong kinetics of complexation between Amlodipine besylate with else Ca2+. The stability constants obtained from the Ardon’s plot for Amlodipine–Ca2+ system was remain quite close at all pH systems except at pH 7.4. At pH 7.4 the stability constant was 0.11, higher than all other systems. So, we can conclude that a stable complex was formed at pH 7.4 i.e. in blood. In protein binding studies it was found that at a low drug concentration the percentage of protein binding attains a steady state plateau condition (84%). This indicated the saturation of the sites of protein by the drugs or its complexes as observed by other investigators.