High-throughput determination of oxidative stress biomarkers in saliva by solvent-assisted dispersive solid-phase extraction for clinical analysis

A reliable analytical method for the simultaneous determination of two oxidative stress biomarkers (i.e., 8‑hydroxy-2′-deoxyguanosine (8-oxodG) and 8‑hydroxy-2′-deoxyadenosine (8-oxodA)) in saliva samples is presented. These biomarkers are produced by an oxidative DNA damage and have gained prominence in the field of medicine as early diagnostic and disease control tools. The method is based on solvent-assisted dispersive solid-phase extraction (SA-DSPE) as a clean-up step, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For this purpose, a commercial polymer with a hydrophilic-hydrophobic balance has been used as extraction phase. This balance makes the material suitable for extracting compounds from polar matrices such as saliva. Those variables involved in the extraction were optimized by a Box-Behnken design, whereas those variables affecting the desorption were optimized by a Doehlert design, except the desorption solvent that was optimized by using a Simplex-Centroid design. The method was successfully validated, showing a good linearity at least up to 20 ng mL⁻¹, limits of detection and quantification at the low ng mL⁻¹ level, and good precision values (< 15%). Standard addition calibration was employed to correct the observed matrix effects. Finally, this new approach was successfully applied to saliva samples from nine volunteers, three of them with type II diabetes, obtaining notable differences in the concentration values between both groups. The proposed methodology overcomes some of the drawbacks of the only previous work with the same purpose, such as the time-consuming procedure and the consumption of large volumes of organic solvents. To increase the sample throughput and reduce the analysis time, a thermostatic stirrer that allows the extraction of several samples simultaneously was used.