High temporal resolution coupling of low-flow push-pull perfusion to capillary electrophoresis for ascorbate analysis at the rat vitreoretinal interface

Abstract

A system is presented demonstrating the high-temporal resolution coupling of low-flow push-pull perfusion sampling (LFPS) to capillary electrophoresis for the absorbance measurement of ascorbate at the rat vitreoretinal interface. This system holds all separation components at a low pressure as the means for withdrawing sample during LFPS. The system uses a flow-gated interface to directly couple the withdrawal capillary from the LFPS probe to a separation capillary and eliminates the need for any offline sample handling. The temporal resolution of the system was limited by injection time and is less than 16 s. This high temporal resolution was applied to the monitoring of in vivoascorbate levels at the rat vitreoretinal interface. Baseline concentrations of ascorbate were found to be 86 µM ± 18 µM at the vitreoretinal interface. Baseline concentrations matched well with those obtained for the postmortem bulk vitreous analysis. Upon stimulation with 145 mM K⁺, a maximum increase in baseline values between 32–107% for n = 3 was observed. This system demonstrates the first in vivo temporal study of ascorbate at the rat vitreoretinal interface.