Determination of natamycin in rabbit cornea by high-performance liquid chromatography–tandem mass spectrometry with protective soaking extraction technology

• A new LC–MS/MS analysis of natamycin in cornea was developed and validated.
• Cornea samples were processed with simple, effective and protective soaking extraction technology.
• The LC–MS/MS analysis was successfully applied for evaluating the ocular pharmacokinetics of natamycin in rabbit cornea.

A new selective and sensitive high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) method was developed for the quantification of natamycin (NAT) in rabbit corneas with amphotericin B as the internal standard (IS). The cornea samples were processed by a simple and protective methanol soaking extraction technology. The NAT could be extracted completely from rabbit cornea after 24 h of soaking with methanol under a mild condition. Chromatographic separation was performed on a C18 column (2.1 mm × 50 mm, 3.5 μm) using mobile phase with ammonium acetate buffer (pH 4.5; 4.0 mM):acetonitrile (40:60, v/v) at a flow rate of 0.25 ml/min. Quantification was performed using the transitions 666.2 → 503.2 m/z for NAT and 924.5 → 906.6 m/z for IS by positive ion electrospray ionization in multiple reaction monitoring mode. The assay was validated over a concentration range of 8.64 ng/ml to 843 ng/ml with lower limit of detection of 4.32 ng/ml. The method was validated with respect to linearity, accuracy, precision, recovery, stability and extracting efficiency. The extraction recovery of NAT from cornea samples was approximately 100% with the new methanol soaking extraction procedure. The method has been successfully applied to the ocular pharmacokinetic studies of NAT eye drops in the cornea of Japanese white rabbit.