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

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-06, Vol.1022, p.109-117
• Main Authors: Hassib, Sonia T., Elkady, Ehab F., Sayed, Rawda M.
• Format: Article
• Language: English
• Subjects: Animals; Anti-glaucoma drugs; Antihypertensive Agents - analysis; Antihypertensive Agents - chemistry; Aqueous Humor - chemistry; Chromatography; Chromatography, High Pressure Liquid - methods; Drug Stability; Drugs; Female; Glaucoma - drug therapy; Ionization; Linear Models; Liquid chromatography; Male; Maleates; Methyl alcohol; Rabbit aqueous humor; Rabbits; Reproducibility of Results; Sensitivity and Specificity; Separation; Tandem Mass Spectrometry - methods; Tandem mass spectroscopy; Timolol - analysis; Timolol - chemistry; Timolol maleate
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.04.012

•Validated LC–MSMS method for the simultaneous determination of anti-glaucoma drugs.•Separation and quantitation of the four drugs in rabbit aqueous humor.•The method is validated according to US-FDA and EMA guidelines.•The method has high sensitivity, high selectivity and fast analysis. 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.