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A new, simple and selective HPLC method was implemented for the simultaneous estimation of tafluprost (TFL) and timolol (TIM) in their new anti-glaucoma combination in the challengeable ratio of 3 and 1000 for TFL and TIM, respectively. Separation was achieved using a BDS Hypersil phenyl column and a mobile phase made up of acetonitrile: 0.015 M phosphate buffer (50:50 v/v, pH 3.5) delivered at 1 mL min −1  and the separation was completed in less than 6 min. UV detection was time programmed at 220 nm for the first 4.5 min and later at 254 nm. Mebeverine (MEB) was used as an internal standard (I.S.). The linearity was observed in the ranges of 0.6–45 and 50–2000 µg mL −1  with limits of detection (LOD) of 0.18, 16.48 µg mL −1  and limits of quantification (LOQ) of 0.55, 49.94 µg mL −1  for TFL and TIM, respectively. The method satisfied International Council for Harmonization (ICH) validation guidelines. The study was extended to the estimation of the studied drugs in their co-formulated eye drops as well as in their single dosage forms with acceptable percentage recoveries. Moreover, Green Analytical Procedure Index (GAPI) and analytical Eco-scale were investigated to confirm the greenness of the proposed HPLC method.

Green HPLC method with time programming for the determination of the co-formulated eye drops of tafluprost and timolol in their challengeable ratio