Rapid, sensitive and selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of topically applied azithromycin in rabbit conjunctiva tissues

A simple, rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of azithromycin in rabbit conjunctiva tissues using roxithromycin as internal standard. Following a deproteinization procedure, the samples were eluted isocratically at a flow rate of 0.3 mL/min utilizing a mobile phase containing of 10 mM ammonium acetate (adjusted pH to 5.2 with 0.1% acetic acid)–methanol (18:82, v/v) and a SHISEIDO CAPCELL PAK C18 (3.0 mm × 75 mm, 3 μm). Azithromycin and its internal standard were measured by a triple-quadrupole mass spectrometer in the selected reaction monitoring (SRM) mode with precursor-to-product qualifier transition m/z 375 [M+2H]2+ → 591 and m/z 837 [M+H]+ → 679 respectively. The method demonstrated that good linearity ranged from 10 to 2000 ng/mL with r = 0.9998. The lower limit of quantification for azithromycin in conjunctiva tissues was 10 ng/mL with good accuracy and precision. The intra- and inter-day precision (RSD) values were below 15% and accuracy (%) ranged from 90% to 110% at all QC levels. The method was applicable to ocular pharmacokinetic studies of azithromycin.