Separation and quantification of RNA molecules using size‐exclusion chromatography hyphenated with inductively coupled plasma‐mass spectrometry

Abstract

The hyphenation of SEC with ICP-MS was successfully applied to RNA quantification. The developed method combines the separation technique for large biomolecules and element selective detection of ICP-MS. The separation of RNA molecules was performed under the SEC condition without additive reagents such as salts to prevent the adhesion of RNA molecules on the column resin. Fragments of RNA, which were commercially available as a ladder marker solution and certified reference materials, were successfully separated and analyzed by measuring ³¹P⁺ with this method. RNA was quantified with good repeatability (RSD of peak area; 2.7%, n = 3) and linearity (R² = 0.999) using a P standard solution as a calibrant. LOD and absolute detection limit of RNA were 6.7 μg/kg and 67 pg, respectively, which were equal to the values obtained by the analysis of a P standard solution. The accuracy of the proposed measurement was evaluated by measuring certified reference materials of RNA solutions for quantitative analysis (NMIJ CRM 6204-a). The results obtained by this method agreed with the certified values within uncertainty. The proposed analysis method, which demonstrates good accuracy and high precision and is free from interference by nucleotide analogues, qualifies as a method of quality control for the RNA synthesis and extraction process.