Simultaneous quantification of siRNA antisense and sense strands by hybrid liquid chromatography-mass spectrometry.

BACKGROUND

Most oligonucleotide bioanalytical assays currently only quantify the pharmacologically-active antisense strand, though there have been recent efforts to simultaneously quantify the sense strand using hybridization ELISA or solid phase extraction LC-MS. Hybrid LC-MS, which offers both high sensitivity and specificity unlike the currently used platforms, has not been applied to quantify both siRNA strands simultaneously.

MATERIALS & METHODS: A hybrid LC-MS assay utilizing LNA capture probes was developed and applied to quantify both strands of a 21-mer lipid-conjugated siRNA (SIR-3) using tandem mass spectrometry (MS/MS). A similar approach using high-resolution mass spectrometry (HRMS) was also evaluated.

RESULTS

The final LC-MS/MS method was capable of quantifying both strands of SIR-3 at concentrations between 0.600 and 1000 ng/mL in cynomolgus monkey tissue homogenates with acceptable accuracy and precision. The LC-HRMS assay demonstrated similar sensitivity and assay performance as the LC-MS/MS assay.

CONCLUSIONS

Overall, this manuscript presents orthogonal methods to existing siRNA bioanalytical workflows that with high sensitivity and specificity can provide greater information about the concentration and biotransformation of an siRNA analyte.