An UPLC-MS/MS Method for Simultaneous Determination of Tropicamide and Phenylephrine in Rabbit Ocular Tissues and...

Mixed eye drops containing 0.5% tropicamide and 0.5% phenylephrine are commercially available for cycloplegic refraction. Determining the pharmacokinetics (PK) and distribution of tropicamide and phenylephrine simultaneously in ocular tissues is an important but challenging issue. Herein, we developed a sensitive and selective ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for simultaneous determination of tropicamide and phenylephrine concentrations in rabbit ocular tissues and plasma. The two analytes were extracted with ethyl acetate using etofesalamide as an internal standard and separated using a chromatographic C₈ column with isocratic elution. Mass spectrometry analysis was performed with positive electrospray ionization and data were acquired in a multiple reaction monitoring mode. We validated this method over a concentration range of 5-1,600 ng/mL for tropicamide and 1-320 ng/mL for phenylephrine in ocular tissues, as well as 0.5-64 ng/mL for both compounds in plasma. Inter- and intraday precisions in all samples were both <12.9% and the accuracy was within 92.1%-108.4%. The highest concentration of tropicamide was found in aqueous humor (C_max: 29430 ng/g), while was in cornea for phenylephrine (C_max: 3465 ng/g). All the ocular tissues concentrations were much higher than those of blood. This UPLC-MS/MS method allowed us to determine the PK and distribution of tropicamide and phenylephrine in rabbit ocular tissue, which may be helpful in the future development and application of mydriatic agents.