Simultaneous determination of timolol maleate in combination with some other anti-glaucoma drugs in rabbit aqueous humor by high performance liquid chromatography-tandem mass spectroscopy

In this work, a sensitive, selective, accurate and precise LC-MS/MS method has been developed for the simultaneous determination of an anti-glaucoma ß-blocker, timolol maleate (TIM) with other co-administered anti-glaucoma drugs of different classes, namely; dorzolamide hydrochloride (DOR), brinzolamide (BRZ) and brimonidine tartrate (BRM) in rabbit aqueous humor (AH) using eslicarbazepine as an internal standard (IS). Liquid-liquid extraction was used for the purification and pre-concentration of analytes from rabbit AH matrix. The chromatographic separation was achieved using a mobile phase consisting of 10 mM ammonium formate pH = 7: methanol: acetonitrile (5: 50: 45, v/v/v) in isocratic mode of elution at a flow rate of 0.8 mL/min on an INERTSIL® C18 ODS-3 column (150 mm × 4.6 mm, 3.5 μm). The method was operated using electrospray ionization source in the positive ionization mode prior to detection by multiple reaction monitoring (MRM) at the following transitions: m/z 317.2 → 261.0 for TIM, m/z 325.1 → 199.0 for DOR, m/z 384.2 → 281.0 for BRZ, m/z 292.1 → 212.0 for BRM and m/z 255.0 → 237.0 for IS. The separation was done in only 3 min and the lower limit of quantitation (LLOQ) was (50 ng/ml) for all cited drugs. A detailed validation of the bio-analytical method was performed as mentioned in US-FDA and EMA guidelines and the standard calibration curves were found to be linear in the range (50-5000 ng/ml) for all drugs with good mean regression coefficient for all drugs.