RESEARCH PAPER Determination of methadone and EDDP in oral fluid using the dried saliva spots sampling approach and gas chromatography-tandem mass spectrometry Andreia Ribeiro 1 & Margarida Prata 1 & Cristiana Vaz 2 & Tiago Rosado 1,3 & José Restolho 1,4 & Mário Barroso 5 & André R. T. S. Araújo 2,6,7 & Eugenia Gallardo 1,3 Received: 11 December 2018 /Revised: 18 January 2019 /Accepted: 29 January 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract 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 μL) were applied into Whatman™ 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 1 min. Afterwards, the extract was centrifuged for 15 min at 3500 rpm and the supernatant evaporated to dryness and reconstituted with 50 μL 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. Keywords Methadone . EDDP . Oral fluid . Dried saliva spots . GC-MS/MS Introduction Methadone (MTD) is the most commonly used drug in opiate substitution therapy [1]. Although heroin still prevails as the most widely consumed illicit opiate, several studies are now pointing to an increase in the consumption of synthetic opioids, such as MTD, buprenorphine, and fentanyl. Despite the fact that these substances are associated with a substantial proportion of overdose deaths, only a few countries, such as Denmark and the Czech Republic, have monitoring systems capable of conducting an analysis of trends in acute intoxica- tions caused by their consumption [2]. Andreia Ribeiro, Margarida Prata, Cristiana Vaz and Tiago Rosado contributed equally to this work. * Mário Barroso mario.j.barroso@inmlcf.mj.pt 1 Centro de Investigação em Ciências da Saúde, Faculdade de Ciências da Saúde da Universidade da Beira Interior (CICS-UBI), Avenida Infante D. Henrique, 6200-506 Covilhã, Portugal 2 Escola Superior de Saúde, Instituto Politécnico da Guarda (IPG), Avenida Rainha D. Amélia, S/N, 6300-749 Guarda, Portugal 3 Laboratório de Fármaco-Toxicologia-UBIMedical, Universidade da Beira Interior, Convento de Sto. António, 6201-001 Covilhã, Portugal 4 Nal von Minden, GmbH, Friedenstr 32, 93053 Regensburg, Germany 5 Serviço de Química e Toxicologia Forenses, Instituto de Medicina Legal e Ciências Forenses - Delegação do Sul, Rua Manuel Bento de Sousa 3, 1169-201 Lisbon, Portugal 6 Unidade de Investigação para o Desenvolvimento do Interior do IPG (UDI/IPG), Av. Dr. Francisco de Sá Carneiro 50, 6300-559 Guarda, Portugal 7 LAQV, Requimte, Departamento de Ciências Químicas, Laboratório de Química Aplicada, Faculdade de Farmácia, Universidade do Porto, 4051-401 Porto, Portugal Analytical and Bioanalytical Chemistry https://doi.org/10.1007/s00216-019-01654-z