Quantitative analysis of dicer substrate oligonucleotides in mouse liver by ultra-high-performance liquid chromatography-electrospray ionization tandem mass spectrometry.

Abstract A method using multi-mode solid-phase extraction and ultra-high-performance liquid chromatography (UHPLC)–electrospray mass spectrometry was developed to quantify Dicer-substrate small interfering RNA (DsiRNA) directed against the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene transcript in mouse liver tissue. The oligonucleotides were separated into sense and antisense strands using a UHPLC C18 column with mobile phases containing 1,1,1,3,3,3-hexafluoro-2-propanol in both water (mobile phase A) and methanol (mobile phase B) with triethylamine as the ion pairing agent at a column temperature of 65°C. The lower limits of detection for the sense and antisense strands were ∼1 ng/mg. The dynamic ranges for the sense and antisense strands were 5 ng/mg–1,000 ng/mg and 1 ng/mg–1,000 ng/mg, respectively. The lower limits of quantification for the sense and antisense strands were 5 ng/mg and 1 ng/mg, respectively, each with a relative standard deviation <15% over the range of calibration curve with...