Spe discovery dsc 18 columns

When ready to analyze, the aliquots were thawed and centrifuged again at 2000× g for 10 min at room temperature to break down mucins and to obtain a clear fluid. The supernatants were collected and poured into freshly labeled tubes. The extraction of saliva samples was carried out using the solid phase extraction (SPE) technique modified from Elio et al. [27 (link)]. SPE Discovery DSC-18 columns (1 mL, Supelco, Bellefonte, PA, USA) were used for saliva sample clean-up and enrichment. Prior to extraction, 500 µL of centrifuged saliva sample or cortisol standard (10, 20, 40, 60, 80, and 100 nmol/L) were spiked with 5 µL of IS (final concentration: 53 nmol/L). Before adding the sample, the SPE columns were equilibrated with 3 mL of methanol followed by 1.5 mL of deionized water. After sample introduction, the columns were washed sequentially with 0.25 mL of deionized water, 0.5 mL of acetone:water mixture (1:4, v/v), and 1 mL of hexane. Finally, 1.5 mL of diethyl ether was used to elute the sample. Each eluted sample was dried in a vacuum chamber and consecutively resuspended with 100 µL of HPLC mobile phase prior to injection.