(M1230-09-51) Improving Sensitivity and Separation of Multiple Nucleotides Utilizing a Metal Free UHPLC System

Purpose: Chromatographic surfaces can significantly influence the detection and accurate quantification of metal sensitive analytes, such as phosphorylated molecules. This can negatively affect chromatographic separation due to adhesion resulting in poor assay sensitivity. By utilizing an inert flow path, we can reduce the interaction between analytes and the metal surfaces thus improving separation. This study investigated the use of a metal free system, the Nexera XS Inert system, to display how it improved sensitivity and quantification of NTs. Nucleotides (NTs) cytidine 5’-diphosphate (CDP), and cytidine 5’ monophosphate (CMP) were analyzed on a Shimadzu Nexera XR, with a metal flow path and the Shimadzu Nexera XS inert, metal free system.

Methods: The NTs cytidine 5’-diphosphate (CDP) and cytidine 5’ monophosphate (CMP) were analyzed on a regular HPLC system, the Nexera XR, with a metal flow path (metal system) and the metal free system, the Shimadzu Nexera XS inert. Detection was achieved using a PDA detector at 254nm in both instruments, only the inert system was equipped with an inert flow cell. Columns used were the metal and metal free Shim-pack Scepter C18-120 columns (3µm 2.1x100mm). The mobile phase comprised of 10mM ammonium formate and acetonitrile with a ratio of 99.5:0.5, respectively. Individual NT standards were diluted in water and the peak intensity and linearity of each compound was evaluated in a range of 1-100 µg/mL. The peak intensity, tailing factor (Tf), linearity and statistical analysis of each compound was evaluated.

Results: The results revealed a significant difference in the peak intensity for CDP, and CMP between the metal and metal free system. When analyzed on the metal free system, all samples (CDP, and CMP), had improved sensitivity yielding a peak height of approximately double the height observed on the metal system and a strong linearity with an R2 value of 0.9997 and 0.9999, respectively. Tailing factor (Tf) for NTs on the metal free system was less than 1.4 indicating improved separation. The difference in column material had a large impact on quantitative reliability. Polymer-lined stainless body with polymer frit inside the column maintains a metal free flow path reducing tailing effects and adsorption of compounds to metal surfaces observed in a standard metal column. RSD (%) of samples collected on the metal free system demonstrates greater precision and accuracy when compared to the metal system even at the lowest concentration. The limit of detection and limit of quantitation for all samples (CDP and CMP) were 0.08 and 0.25 and 0.04 and 0.15, respectively.

Conclusion: This study demonstrated increased sensitivity and accuracy of quantitation for phosphorylated molecules when using a fully inert flow path and is recommended for use with biomolecules.

CMP Calibration Curves and Chromatograms collected on metal and metal free UHPLC system



CDP Calibration Curves and Chromatograms collected on metal and metal free UHPLC system



Statistical data and LOD/LOQ from nucleotide analysis on metal and metal free UHPLC