A Novel Hybridization LC-MS/MS Methodology for Quantification of siRNA in Plasma, CSF and Tissue Samples

Abstract

Therapeutic oligonucleotides, such as antisense oligonucleotide (ASO) and small interfering RNA (siRNA), are a new class of therapeutics rapidly growing in drug discovery and development. A sensitive and reliable method to quantify oligonucleotides in biological samples is critical to study their pharmacokinetic and pharmacodynamic properties. Hybridization LC-MS/MS was recently established as a highly sensitive and specific methodology for the quantification of single-stranded oligonucleotides, e.g., ASOs, in various biological matrices. However, there is no report of this methodology for the bioanalysis of double-stranded oligonucleotides (e.g., siRNA). In this work, we investigated hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides in biological samples using an siRNA compound, siRNA-01, as the test compound. The commonly used DNA capture probe and a new peptide nucleic acid (PNA) probe were compared for the hybridization extraction of siRNA-01 under different conditions. The PNA probe achieved better extraction recovery than the DNA probe, especially for high concentration samples, which may be due to its stronger hybridization affinity. The optimized hybridization method using the PNA probe was successfully qualified for the quantitation of siRNA-01 in monkey plasma, cerebrospinal fluid (CSF), and tissue homogenates over the range of 2.00-1000 ng/mL. This work is the first report of the hybridization LC-MS/MS methodology for the quantification of double-stranded oligonucleotides. The developed methodology will be applied to pharmacokinetic and toxicokinetic studies of siRNA-01. This novel methodology can also be used for the quantitative bioanalysis of other double-stranded oligonucleotides.

Keywords: LC-MS/MS; bioanalysis; double-stranded oligonucleotides; hybridization; quantitative; small interfering RNA (siRNA).

Figure 1

Effect of temperature and salt concentration on the recovery of AS1 using DNA probe and PNA probe.

Figure 2

Effect of temperature and salt concentration on the recovery of siRNA-01 using DNA probe and PNA probe.

Figure 3

Optimization of temperature and salt concentration for the recovery of siRNA-01 using DNA probe and PNA probe.

Figure 4

Effect of DNA probe concentration and incubation time on the recovery of siRNA-01.

Figure 5

Comparison of different combination of DNA and PNA probes for recovery of siRNA-01.

Figure 6

Representative MRM chromatograms of siRNA-01 (left) and the internal standard ASO-002 (right) in a blank monkey plasma, a monkey plasma spiked with the analyte at the LLOQ concentration (2.00 ng/mL), and ULOQ (1000 ng/mL) in monkey plasma.

Figure 7

Accuracy and precision of siRNA-01 in monkey liver, kidney, heart, brain, spinal cord, lung, spleen, and colon homogenates (calibration curve prepared in a combined tissue homogenate).