Development and validation of a new stability indicating reversed phase liquid chromatographic method for the determination of prednisolone acetate and impurities in an ophthalmic suspension.

A new stability indicating reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated under current International Conference of Harmonisation (ICH) guidance for the determination of prednisolone acetate (PAC) and impurities in an ophthalmic suspension. The developed method is presented as an alternative to a modified version of the current RP-HPLC method described in the USP monograph for the assay of PAC in an ophthalmic suspension. Along with the assay of PAC, the new method is also capable of identifying and quantifying eight selected PAC impurities and degradation products in an ophthalmic suspension. Using an Agilent Poroshell 120 EC-C18 100 mm × 4.6mm (dp: 2.7 µm) column set to 60°C with step gradient elution generated using mobile phase A: acetonitrile/water (10:90) (v/v) and mobile phase B: acetonitrile delivered at 1.2 mL min(-1), all peaks of interest are eluted in 33 min with resolution of 1.5 between the critical pairs. The developed method was validated for PAC and impurities to ICH recommendations for accuracy, linearity, precision (repeatability), limit of detection, limit of quantitation, robustness and specificity.

Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method.

An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5µm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin(-1) resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin(-1)), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.