Agilent 1260 diode array detector

Deoxynucleosides (dN) include dA, dG, dC, and dT. In this study, dG was measured as an indication of how much total dN was in each sample. The ratio of dG to dN was constant. Measurement of dN, expressed as adducts/pg deoxyguanosine (dG), was performed using an Agilent 1290 Infinity series ultra-high performance liquid chromatography system coupled to an Agilent 1260 diode array detector (DAD) (Agilent Technologies, Santa Clara, CA). The DAD was set to 260nm for detection of dG. Chromatographic separation was performed using a Kinetex C18 column (1.7μm; 2.1×100mm) (Phenomenex, Torrance, CA). The mobile phases consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). The following mobile phase gradient was used: 15%B from 0 to 6min, with a linear increase to 95%B between 6 and 8 min, held at 95%B from 8 to 12 min, returned to 15% B between 12 and 12.5 min, and re-equilibration of the column at 15%B from 12.5 to 16 min. The mobile phase flow rate was 0.2 deoxyguanosine/min. A 10 μl sample injection volume was used. A five-point calibration curve, ranging from 0 to 300 ng dG on column, was created and used for quantification of dG in DNA digest samples.