Abstract

A reliable, repeatable and a sensitive analytical method was developed and validated for the quantification of ofloxacin (a polar molecule) in human Aqueous Humour (AH) of the eye. The chromatography analysis was performed in tandem mass spectrometry (LC- ESI-MS/MS). Using Synergy Hydro PR column polar endcapping (mainly via H-bonding) interacts with polar compounds resulting in high retention. The method was validated over a linear concentration range of 0.1-100.0 ng/mL with the R2 value being higher than 0.999. The Lower Limit of Quantification (LLOQ) for ofloxacin was quantified at 0.05 ng/mL with sufficient specificity, accuracy, and precision. Both the precision (Coefficient of Variation (CV); 4.28%) and accuracy (Relative Error (RE); 4.46%) were within acceptable criteria of<15%. The chromatographic run time was short within 7 minutes, while the recovery was found ≥100%. The analytical method was successfully validated for the stability of ofloxacin in human AH at various storage conditions (room temperature, fridge, freezer, and freeze-thaw). Furthermore, the developed method was applied to quantify the level of ofloxacin in twenty-one human AH samples as collected intraoperatively from the anterior chamber of patients undergoing cataract surgery following topical application. The mean concentration of ofloxacin was measured at 644.33 ± 1.16 ng/mL.