Quantification of 1,N6-Etheno-Derived Nucleotides

A reverse phased gradient Agilent Technologies 1200 liquid chromatography system equipped with a fluorescence detector (FLD) was used to detect 1,N⁶-etheno-products as described previously (Mutafova-Yambolieva et al., 2007 (link), Durnin et al., 2012 (link), Durnin et al., 2016 (link)). The mobile phase consisted of 0.1 M KH₂PO₄ (pH 6.0) as eluent A. Eluent B consisted of 65% eluent A and 35% methanol. Gradient elution was employed according to the following linear program: time 0, 0% eluent B; 18 min, 100% eluent B. The flow rate was 1 mL/min and the run time was 20 min. Column and autosampler temperatures were maintained at 25 °C and 4 °C, respectively. The fluorescence detector was set to record 1,N⁶-etheno-derivatized nucleotide and nucleoside signals at an excitation wavelength of 230 nm and emission wavelength of 420 nm (Bobalova et al., 2002 (link)).