To provide some case studies demonstrating the use of a selective/specific, sensitive, robust and high throughput LC/MS-HRAM method to quantify siRNA compounds and to evaluate the in vivo metabolite profiles in plasma and tissues.
The biological samples were extracted by solid phase extraction (SPE) before injection onto the LC/MS-HRAM for analysis. A gradient of an aqueous mobile phase and organic mobile phase of acetonitrile in the presence of ion-pairing reagents was utilized in the liquid chromatography. The siRNA compounds and their metabolites were quantified with parallel reaction monitoring (PRM) on the mass spectrometer. Using full scan mode, metabolites were identified by high-resolution mass spectrometry using deconvolution software.
Isolation of siRNA oligonucleotides from biological matrices using the SPE allows for a rapid and high throughput method. The metabolic profiling data showed that the siRNA parent compound is quickly metabolized to one major metabolite and one minor metabolite in plasma and tissues. Calibration curves of parent compound and the two metabolites range from an LLOQ (lower limit of quantification) of 10 to 5000 ng/mL in plasma, and 100 to 50000 ng/g in liver and kidney tissues. The rat liver assay was reproducible with an intra-day precision of below 18.2% CV (coefficient of variance) and inter-day precision of below 19.2% CV for parent and its two metabolites. The assay showed good accuracy, with intra-assay concentrations within 80.5% and 118.0% of the expected value, and inter-assay concentrations within 96.0% to 106.4 % of the expected value for 3 analytes in rat liver lysate. The data showed similar amounts of siRNA parent and major metabolite detected in plasma samples, while the major metabolite was dominant in both liver and kidney. The concentration-time profiles of parent and metabolites in liver and kidney tissues showed the metabolites had longer half-lives than the parent compound in liver and kidney. The quantification of siRNA drugs in plasma and tissues by this LC/MS-HRAM method can support preclinical and clinical studies.
The LC/MS-HRAM mass spectrometry method provides highly sensitive and selective quantitation of siRNA and its metabolites and accurate identification of siRNA metabolites in biological matrices.
Chris Tran– Alnylam Pharmaceuticals Inc.
Krishna Aluri– Alnylam Pharmaceuticals Inc.
Jing-Tao Wu– Alnylam Pharmaceuticals Inc.
Valerie Clausen– Associate Director, Alnylam Pharmaceuticals Inc., Cambridge, Massachusetts
Yuanxin Xu– Alnylam Pharmaceuticals Inc.
Jing Li– Alnylam Pharmaceuticals Inc.
Ju Liu– Alnylam Pharmaceuticals Inc.