Determination of methadone and EDDP in oral fluid using the dried saliva spots sampling approach and gas chromatography-tandem mass spectrometry

The present work describes the development and validation of a novel approach to determine methadone (MTD) and its main metabolite (EDDP) in oral fluid samples, using the dried saliva spots (DSS) sampling approach and gas chromatography-tandem mass spectrometry (GC-MS/MS). Oral fluid samples (50 mu L) were applied into Whatman (TM) 903 protein saver filter paper cards and were allowed to dry overnight. The extraction was carried out by immersion of the spot in 1 mL of isopropyl alcohol with agitation for 1min. Afterwards, the extract was centrifuged for 15min at 3500rpm and the supernatant evaporated to dryness and reconstituted with 50 mu L of methanol. The procedure was considered linear in the range of 10 to 250 ng/mL for both compounds, with determination coefficients greater than 0.99. Intra- and inter-day precision and accuracy revealed coefficients of variation (CVs) lower than 15% at the studied concentrations, with mean relative errors within +/- 15% of the nominal concentrations. Recoveries ranged from 45 to 74%. The limits of detection and quantification were 5 and 10 ng/mL respectively for both analytes. All studied parameters complied with the defined criteria and the method enabled the successful determination of MTD and EDDP in oral fluid samples from patients undergoing opiate substitution/maintenance therapy.