Liquid chromatography–mass spectrometry method for determination of amphotericin B in rabbit tears and its application to ocular pharmacokinetic study

Abstract

A rapid and sensitive LC/MS/MS method was developed for the quantification of amphotericin B in rabbit tears using natamycin as internal standard. The analyte and internal standard were extracted from tear sample by solid phase extraction method using Oasis HLB cartridges. Cchromatographic separation was achieved on a cyano column using methanol–ammonium acetate (3.5 mM, adjusted to pH 4 with acetic acid) (90:10, v/v) as mobile phase. Quantitation was performed on a triple quadrupole mass spectrometer employing electrospray ionization technique, operating in multiple reaction monitoring (MRM) in negative ion mode. The assay was validated with linear range of 100–3200.00 ng/mL for amphotericin B using 10ul of tear sample. The intra- and inter-day assay precision ranged from 1.67 to 7.75% and 2.49 to 5.56%, respectively, and intra and inter-day assay accuracy was between from -3.96 to 4.63%and 2.49 to 5.56%, respectively. Amphotericin-B was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days storage in a freezer at -80 ◦ C. The method was successfully applied to the pharmacokinetic studies of amphotericin B eye drops in rabbit tears