Ion exchange liquid chromatography method for the direct determination of small ribonucleic acids

• We developed and validated a novel IEC method to quantitate siRNA.
• Method can simultaneously quantitate metabolites of the siRNA.
• Develop a novel ion-pair LC–MS method to determine the metabolites.
• Develop a novel lysis buffer which provides greater than 95% recovery of siRNA.
• Applied the method to determine the uptake and metabolism of an siRNA in prostate cancer cells.

Bioanalysis of siRNAs is challenging due to their size (5–14 kDa) and negative charge across the backbone, which complicates both sample preparation and chromatography. We present here a one step sample preparation combined with non-denaturing anion exchange chromatography with UV detection for the quantitation of siRNA and its chain shortened metabolites. The sample preparation uses a novel lysis buffer with proteinase K to effectively isolate siRNA from cells and formulated media with greater than 95% recovery. The ion exchange chromatography allows for a lower limit of quantitation of 6 ng mL−1 in cells and media equivalent to 6 ng/200,000 cells. This method is applied to study the uptake of siRNA in prostate cancer cells and the disappearance in the media and siRNA metabolism. siRNA metabolites are identified by matching the retention time of standards to metabolite peaks. Identification is further confirmed by mass spectrometry. To our knowledge this is the first ion exchange method reported for the quantitation of siRNA from a biological matrix. It is also the first non-denaturing chromatographic method reported for siRNA quantitation.

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