Metabolite identification of small interfering RNA duplex by high-resolution accurate mass spectrometry.
On-line liquid chromatography/electrospray ionization high-resolution mass spectrometry (LC/ESI-HRMS) using an LTQ-Orbitrap mass spectrometer was employed to investigate the metabolite profiles of a model siRNA duplex designated HBV263. The HBV263 duplex was incubated in rat and human serum and liver microsomes in vitro. The siRNA drug and its metabolites were then extracted using a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE), and analyzed by LC/ESI-MS. High-resolution accurate mass data enabled differentiation between two possible metabolite sequences with a monoisotopic molecular mass difference of less than 1 Da. ProMass deconvolution software was used to provide semi-automated data processing. In vitro serum and liver microsome incubation samples afforded different metabolite patterns: the antisense strand of the duplex was degraded preferentially in rat and human serum, while the sense strand of the duplex was less stable in rat and human liver microsomes.
Zou
Yan
Y
Department of Drug Metabolism and Pharmacokinetics, Merck Research Laboratories, West Point, PA 19486, USA. yan_zou@merck.com
Tiller
Philip
P
Chen
I-Wu
IW
Beverly
Michael
M
Hochman
Jerome
J
D000818:Animals; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism*; D008970:Molecular Weight; D009862:Online Systems; D016372:RNA, Antisense / Q000032:analysis; D012330:RNA, Double-Stranded / Q000032:analysis*; D034741:RNA, Small Interfering / Q000032:analysis* / Q000097:blood* / Q000737:chemistry; D051381:Rats; D052616:Solid Phase Extraction; D013997:Time Factors
D016372:RNA, Antisense; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering