A Simple Emulsification-Assisted Extraction Method for the GC–MS/SIM Analysis of Wine Markers of Aging and Oxidation: Application for Studying Micro-Oxygenation in Madeira Wine Ana I. Freitas 1 & Vanda Pereira 1,2 & João M. Leça 1,2 & Ana C. Pereira 1,3 & Francisco Albuquerque 4 & José C. Marques 1,2 Received: 21 April 2017 /Accepted: 25 January 2018 /Published online: 15 February 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Sotolon and the heterocyclic acetals of glycerol are known as potential aging and oxidation markers in fortified wines such as Madeira, Port, and Sherry. Thus, determining the evolution of these compounds under different oxidative aging conditions is important for fortified wine quality purposes. This study proposes a new methodology based on a miniaturized emulsification extraction followed by GC–MS/SIM, which was developed and optimized to follow the formation of sotolon and heterocyclic acetals in fortified wines that were submitted to traditional accelerated aging and micro-oxygenation. The optimization was achieved by means of a mixed-level factorial design, considering 3 factors: sample volume, extractant volume, and concentrated extract volume, by performing 19 experiments in duplicate. The extraction was optimized using 8 mL of wine sample, 5 mL of dichloromethane, concentrating the extract up to 10-fold. The method performance was evaluated for sotolon, using a matrix- matched calibration between 10 and 2000 μg/L. The selectivity was confirmed through the analysis of real samples. The methodology showed good linearity (R 2 = 0.999), high sensitivity (LOQ = 6.8 μg/L), recovery about 105%, and good precision (less than 8 and 9%, evaluated by the variation of intra- and inter-day measurements, respectively). This is the first methodology that revealed to be an excellent tool to simultaneously follow the formation of sotolon and heterocyclic acetals in Madeira wines, using an inexpensive, simple, efficient, and effective experimental layout. Indeed, it was shown that traditional accelerated aging and micro-oxygenation have impact on the formation of such molecules. Keywords Alcoholic beverages . Sotolon . Heterocyclic acetals . Liquid–liquid extraction . Design of experiments Introduction Madeira wines are recognized as one of the world’ s great classic fortified wines and are characterized by a peculiar winemaking process. Their fermentation is stopped, by the addition of neutral spirit of vine origin up to 17–22% (v/v: mL ethanol/100 mL wine), according to the desired sweetness (dry to sweet wines are produced, with total sugars usually higher than 49.1 g/L) and desired marketing characteristics (Carvalho et al. 2015; Pereira et al. 2011). Traditionally, their aging process usually includes an artificial heating at about 45 °C for at least 3 months (estufagem), followed by aging in wood casks placed in sunshine-heated lodges (canteiro). More information about its processing can be found elsewhere (Pereira et al. 2016; Pereira et al. 2011). These strong oxida- tive conditions contribute to their specific organoleptic char- acteristics, quality, and longevity, as exceptional 100-year-old Madeira wines are easily found. Indeed, it is their longevity that has made them unique in the world of wine. Thus, the economic value of these wines is strongly associated with their age. Several aging-derived compounds have been point- ed out as playing an important role on the aroma of these wines (Câmara et al. 2006; Campo et al. 2006; Pereira et al. 2014; Perestrelo et al. 2011); however, sotolon (3-hydroxy- 4,5-dimethyl-2(5H)-furanone) is recognized as being the key * Vanda Pereira vpereira@uma.pt 1 Faculty of Exact Sciences and Engineering, University of Madeira, Campus da Penteada, 9020-105 Funchal, Portugal 2 Institute of Nanostructures, Nanomodelling and Nanofabrication (I3N), University of Aveiro, Aveiro, Portugal 3 CIEPQPF, Department of Chemical Engineering, University of Coimbra, Pólo II - Rua Sílvio Lima, 3030-790 Coimbra, Portugal 4 Madeira Wine Company, SA, Plataforma 3, Pavilhão T, Zona Franca Industrial, 9200-047 Caniçal, Machico, Portugal Food Analytical Methods (2018) 11:2056–2065 https://doi.org/10.1007/s12161-018-1176-3