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 a...

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Bibliographic Details
Published in:Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2006-05, Vol.835 (1), p.62-70
Main Authors: Beverly, Michael, Hartsough, Kim, Machemer, Lynn, Pavco, Pam, Lockridge, Jennifer
Format: Article
Language:English
Subjects:
Analysis
Analytical, structural and metabolic biochemistry
Animals
Biological and medical sciences
Chromatography, High Pressure Liquid - methods
Electrospray
ESI
Eye - metabolism
Female
Fundamental and applied biological sciences. Psychology
General pharmacology
HPLC
LC–MS
Mass spectrometry
Medical sciences
Metabolites
Oligonucleotides
Pharmacology. Drug treatments
Rabbits
RNA, Antisense - analysis
RNA, Double-Stranded - analysis
RNA, Small Interfering - analysis
RNAi
siRNA
Spectrometry, Mass, Electrospray Ionization - methods
Spectrophotometry, Ultraviolet
VEGF
Vitreous Body - chemistry
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Summary: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.
ISSN:1570-0232
1873-376X
DOI:10.1016/j.jchromb.2006.03.008