Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex

The ocular metabolism of an siRNA duplex, SIRNA-027, was examined by ion-pair reversed-phase liquid chromatography (IP-RP-LC) coupled to electrospray ionization mass spectrometry (ESI–MS). The RNA duplex was injected intraocularly into the eyes of New Zealand white rabbits. Rabbits were sacrificed at different timepoints and the vitreous and retina/choroid tissue analyzed for siRNA by IP-RP-LC-MS. The method used a hexafluoroisopropanol (HFIP)/triethylamine (TEA) ion-pairing buffer with a methanol gradient. Using electrospray ionization, the duplex was preserved in the gas phase for analysis by a triple quadrupole mass spectrometer. With this methodology metabolites from rabbit ocular vitreous humor and retina/choroid tissue were identified and a pattern of siRNA degradation was established. Results showed that the duplex was metabolized predominantly from one end. This end of the siRNA duplex was calculated to have the weakest binding energy of the two ends indicating that the ability of the siRNA to split into single strands is a factor in its degradation.