Agilent 1200 series

The 8-oxodG level was determined by HPLC (Agilent 1200 series) coupled to electrochemical (Coulochem III, ESA) and UV (Agilent 1200 series) detection. The limit of detection is about 0.01 pmol of 8-oxodG (one 8-oxodG lesion per 10⁶ dG). Separation of 8-oxodG and 2’-deoxyribosides was carried out by using an Ultrasphere pre-column (5C18, Interchim) and an Uptisphere column (5ODB, Interchim). Elution was performed in isocratic mode using a mobile phase composed of 10% (v/v) methanol and 100 mM sodium acetate pH 5.2. The guard and the measure cells were respectively set to an oxidation potential of 460, 150 and 380 mV. The quantification of 8-oxodG was performed in accordance with a calibration curve previously obtained with known picomole amounts of authentic 8-oxodG. The standard expression of the number of 8-oxodG residues per 10⁶ dG, deoxyguanosine were quantified by UV detection (254 nm) of the output of the HPLC column. For the conditions described, the retention times of 8-oxodG and dG were 11.5 and 8.5 min respectively at 35°C. For each condition (stationary and exponential growth phases), the average of three measurements (n = 3) from two biological replicates was used for statistical analyses. To enumerate viable cells, most-probable-number (MPN) assays were performed as previously published (Blodgett, 2006 ).

HPLC analysis of CC-1065 and 7 were performed by reversed-phase column (Grace Alltima, C18, 5 μm, 100 Å, 10 × 250 mm) on Agilent 1200 series. The column was equilibrated with 85% solvent A (H₂O)/15% solvent B (CH₃CN) for 3 min. The gradient of elution was as follow: 0–3 min: a linear gradient increase from 85% A/15% B to 60% A/40% B, 3–10 min: a linear gradient increase to 45% A/55% B, 10–12 min: a linear gradient increase to 30% A/70% B, 12–21 min: a linear gradient increase to 15% A/85% B, 21–22 min: a linear gradient decrease to 85% A/15% B, 22–25 min: 85% A/15% B. Flow rate was 1 mL•min^-1 and detection wavelength was at 374 nm, the HPLC peak at retention time 13.7 min and 14.8 min correspond to CC-1065 and 7, respectively.
HPLC analysis of SAH and 5′-dA was performed by reversed-phase column (Diamonsil, C18, 5 μm, 4.6 × 250 mm) on Agilent 1200 series. The column was equilibrated with 95% solvent A (H₂O + 1‰ TFA)/5% solvent B (CH₃CN + 1‰ TFA) for 3 min. The gradient of elution was as follow: 0–5 min: 95% A/5% B, 5–20 min: a linear gradient increase to 80% A/20% B, 20–24 min: a linear gradient increase to 10% A/90% B, 24–27 min: 10% A/90% B, 27–29 min: a linear gradient decrease to 95% A/5% B, 29–30 min: 95% A/5% B. Flow rate was 1 mL•min^-1 and detection wavelength was at 260 nm, the HPLC peak at retention time 9.0 min and 12.2 min correspond to SAH and 5′-dA, respectively.

DNA extraction was performed using 20 mg of digestive gland tissue according to the chaotropic NaI method derived from Helbock et al. [56 (link)], slightly modified by Akcha et al. [57 (link)]. In addition, 8-oxodGuo levels were determined by HPLC (Agilent 1200 series, Les Ulis, France) coupled to electrochemical (Coulochem III, ESA, Illkirch, France) and UV (Agilent 1200 series) detection as described in [58 (link)].

The HPLC analysis was performed on an Agilent Series 1200 chromatographic system (Agilent Technologies, Santa Clara, CA, USA) coupled with a photodiode array detector (PAD) (Agilent Technologies, Santa Clara, CA, USA) and using a Zorbax Extend C18 (4.6 × 250 mm, 5 μm i.d.) chromatographic column (Agilent Technologies, USA). The elution was performed in a gradient with a mobile phase consisting of (A) 1% (v/v) formic acid solution in water and (B) 1% (v/v) formic acid solution in acetonitrile. The regimen was as follows: 0 min—7% B; 5 min—7% B; 45 min—40% B; 47 min—70%; 52 min—70% B. The flow was 1 mL min⁻¹, the injection volume 5 μL and the column temperature 25 °C. The chromatograms were recorded at 278 and 320 nm. The analysis for all samples was performed in duplicate. All samples were filtered through a 0.45 μm membrane filter (Nylon Membranes, Supelco, Bellefonte, PA, USA) prior to the analysis. Identification of individual phenolic compounds and caffeine was performed by comparing the retention times and the characteristic absorption spectrum (190–400 nm) with commercially available standards, while quantification was enabled by establishing calibration curves (20–100 µg mL⁻¹).