Method validation parameters Bimatoprost Timolol maleate D1 HPLC HPLC Linearity Slope Intercept Correlation coefficient(r) 0.0004 0.0007 0.9999 34.59 0.7521 0.9999 77.669 7.5157 0.9999 Linearity range (µg/ml) 100.00-900.00 3.00-3.00 10.00-70.00 Working range (µg/ml) Ringbom 105.00-850.00 5.00-25.00 15.00-65.00 Accuracy 99.52 ± 1.329 100.84 ± 0.639 99.67 ± 0.915 Standard error of slope 0.001 196.98 12.47 Confidence limit of slope 0.336-0.289 335-302 78.385-77.688 Precision Intraday Interday 99.99 ± 0.242 100.44 ± 0.301 101.69 ± 0.247 98.94 ± 0.214 98.08 ± 0.712 100.76 ± 1.060 L.O.D L.O.Q 0.52 1.85 0.62 0.65 1.03 3.15 Intraday (n=3) average of three different concentration repeated three times within a day Interday (n=3) average of three different concentration repeated three times in successive days Ringbom % absorbence against logarithim of concentration L.O.D limit of detection, L.O.Q limit of quantitation

Table 2: Validation results of the proposed method for bimatoprost alone and in presence of timolol maleate.