A validated capillary microsampling liquid chromatography-tandem mass spectrometry method for quantification of antisense oligonucleotides in mouse serum.

Background: Capillary microsampling (CMS) has been used for quantitative bioanalysis of small molecules; however, there is no report of applying this technique in the bioanalysis of antisense oligonucleotides (ASOs). Results: A CMS liquid chromatography-tandem mass spectrometry method was successfully developed and validated for the quantification of ASO1 in mouse serum. The validated method was applied in a safety study in juvenile mice. Equivalent performance between CMS samples and conventional samples was demonstrated in the mouse study. Conclusion: This work is the first to report using CMS for liquid chromatography-tandem mass spectrometry quantitative bioanalysis of ASOs. The validated CMS method was successfully applied to support good laboratory practice safety studies in mice and the CMS strategy has subsequently been applied to other ASOs.

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

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.

Validation and application of hybridization liquid chromatography-tandem mass spectrometry methods for quantitative bioanalysis of antisense oligonucleotides.

Background: Antisense oligonucleotide (ASO), an emerging modality in drug research and development, demands accurate and sensitive bioanalysis to understand its pharmacokinetic and pharmacodynamic properties. Results: By combining the advantages of both ligand binding and liquid chromatography-mass spectrometry/tandem mass (LC-MS/MS), hybridization LC-MS/MS methods were successfully developed and validated/qualified in a good lab practice (GLP) environment for the quantitation of an ASO drug candidate in monkey serum, cerebrospinal fluid (CSF) and tissues in the range of 0.5-500 ng/ml. Special treatment of CSF samples was employed to mitigate nonspecific binding, improve long-term storage stability and enable the usage of artificial CSF as a more accessible surrogate matrix. The method was also qualified and applied to ASO quantitation in various monkey tissue samples using a cocktail tissue homogenate as a surrogate matrix. Conclusion: This work was the first reported GLP validation and application of ASO bioanalysis using the hybridization LC-MS/MS platform.