RESUMO
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 C(18) 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 sufficient precision and accuracy. Oligonucleotides were quantified at different time intervals after intravenous administration of living mice with lipid nanoparticle formulated HPRT1 DsiRNA and were detected as early as 15 min after administration, but not detected beyond the 24 h time point.
