title: 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.
abstract: 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 10mM ammonium formate pH=7: methanol: acetonitrile (5: 50: 45, v/v/v) in isocratic mode of elution at a flow rate of 0.8mL/min on an INERTSIL(®) C18 ODS-3 column (150mm×4.6mm, 3.5μm). 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 3min and the lower limit of quantitation (LLOQ) was (50ng/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 (50-5000ng/ml) for all drugs with good mean regression coefficient for all drugs.
authors.affiliation: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
authors.forename: Sonia T
authors.initials: ST
authors.lastname: Hassib
authors.affiliation: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt.
authors.forename: Ehab F
authors.initials: EF
authors.lastname: Elkady
authors.affiliation: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt. Electronic address: rawda.ahmed@pharma.cu.edu.eg.
authors.forename: Rawda M
authors.initials: RM
authors.lastname: Sayed
mesh_terms: D000818:Animals; D000959:Antihypertensive Agents / Q000032:analysis* / Q000737:chemistry; D001082:Aqueous Humor / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D005260:Female; D005901:Glaucoma / Q000188:drug therapy; D016014:Linear Models; D008297:Male; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013999:Timolol / Q000032:analysis* / Q000737:chemistry
chemical_list: D000959:Antihypertensive Agents; D013999:Timolol
keywords: Anti-glaucoma drugs; Liquid chromatography; Rabbit aqueous humor; Tandem mass spectroscopy; Timolol maleate