Research Article QuEChERS-based extraction procedure for multifamily analysis of phytohormones in vegetables by UHPLC-MS/MS A new method has been developed and validated for the simultaneous analysis of different phytohormones (auxins, cytokinins and gibberellins) in vegetables. The compounds were extracted using a QuEChERS-based method (acronym of quick, easy, cheap, effective, rugged and safe). The separation and determination of the selected phytohormones were carried out by ultra high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), using electrospray ionization source (ESI) in positive and negative ion modes. The method was validated and mean recoveries were evaluated at three concentration levels (50, 100 and 250 mg/kg), ranging from 75 to 110% at the three levels assayed. Intra- and interday precisions, expressed as relative standard deviations (RSDs), were lower than 20 and 25%, respectively. Limits of quantification (LOQs) were equal or lower than 10 mg/kg. The developed procedure was applied to seven courgette samples, and naphthylacetic acid, naphthylacetamide and benzyladenine were found in some of the analysed samples. Keywords: Phytohormones / QuEChERS / UHPLC-MS/MS / Vegetables DOI 10.1002/jssc.201100093 1 Introduction Phytohormones are important regulators produced by plants, and they are usually present at low concentrations [1]. They regulate several processes such as growth, differentiation, metabolism, morphogenesis [2], and also the protective response to biotic and abiotic stresses such as pathogen challenge, herbivore insects, drought, cold and heat [3, 4]. Phytohormones are classified into four groups: auxins, gibberellins, cytokinins and inhibitors, and they may act in a different way to achieve the regulation of a range of physiological processes [5, 6]. Auxins are a group of compounds that regulate plant development, affecting cell division and root formation [7]. Gibberellins are a group of compounds that play a crucial role in promoting seed germination [8], while cytokinins could affect chloroplast formation and chloroplast structure, as well as photosynth- esis rate and pathogen resistance [9]. Nowadays, they are widely used in agriculture, because in the last few years there is a growing interest in ecological agriculture, replacing the massive use of pesticides, fertili- zers and synthetic plant growth regulators (analogous to the phytohormones) by natural products [10, 11]. As a conse- quence, the European Union has established maximum residue limits (MRLs) for some phytohormones, i.e. naph- thylacetemide, naphthylacetic acid, 2,4-D (0.05 mg/kg) (http://ec.europa.eu/sanco_pesticides/public/index.cfm). Therefore, it is necessary to develop sensitive analytical methods to comply with the current legislation, allowing for the simultaneous determination of several classes of phytohormones in vegetables, and thus ensuring the safety of the supplied products. Common extraction procedures such as solid-liquid extraction (SLE) [12], applying dichloromethane as extrac- tant solvent in tomato samples, solid-phase extraction (SPE) using C 18 [13], solid-phase microextraction (SPME) [14] and vapour-phase extraction [15] have been employed for the extraction of phytohormones from plants. However, most of the extraction procedures are time consuming and they involve the use of toxic organic solvents. Furthermore, some methods involve the freezing or lyophilisation of the samples before the extraction [16], avoiding decomposition of the target compounds. In the last few years, QuEChERS (acronym of quick, easy, cheap, effective, rugged and safe) method has been used for the extraction of a wide variety of compounds Marı´a Isabel Alarco ´ n Flores Roberto Romero-Gonza ´ lez Antonia Garrido Frenich Jose ´ Luis Martı´nez Vidal Group ‘‘Analytical Chemistry of Contaminants’’, Department of Analytical Chemistry, Almeria University, Almeria, Spain Received February 2, 2011 Revised March 21, 2011 Accepted April 2, 2011 Abbreviations: BA, N 6 -benzyladenine; 2,4-D, 2,4- dichlorophenoxyacetic acid; GA, gibberelin A 3 ; IAA, indole- 3-acetic acid; MRM, multiple reaction monitoring; NA, naphthylacetic acid; NAA, naphthylacetamide; QuEChERS, quick, easy, cheap, effective, rugged and safe; RTW, retention time window Correspondence: Professor Antonia Garrido Frenich, Group ‘‘Analytical Chemistry of Contaminants’’, Department of Analy- tical Chemistry, Almeria University, E-04071 Almeria, Spain E-mail: agarrido@ual.es Fax: 134950015483 & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com J. Sep. Sci. 2011, 34, 1517–1524 1517