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

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 C 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