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Sample preparation is often the bottleneck in the analysis of phytohormones in plant samples. In the present study, sample pretreatment involving extraction by acidified and non-acidified organic solvents and extract purification using solid-phase extraction (SPE) and/or liquid-liquid extraction (LIE) has been optimized and compared for the determination of multiple phytohormones, including auxins, cytokinins (CKs), abscisic acid (ABA), gibberellins (GAs), jasmonic acid (JA) and salicylic acid (SA) in biological samples by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) under multiple-reaction monitoring (MRM). A novel and efficient sample pretreatment method for multiple phytohormones in acidified acetonitrile extracts of plant samples using polymer-based mixed-mode cation-exchange solid phase extraction (SPE) with Oasis MIX cartridges combined with ethyl acetate LLE has been developed. The method detection limits (MDLs) for the target phytohormones ranged from 0.0013 to 0.021 ng.mL(-1), and the recoveries were obtained by spiking the target phytohormones in the two-month-old leaves of oilseed rape (Brassica napus L) at concentrations of 020, 2.0 and 10 ng.g(-1), which ranged from 75.1 to 111%, 79.6 to 113% and 892 to 111%, respectively. The intra-day precisions were in the range of 1.15 to 10.2%, and the inter-day precisions ranged from 2.92 to 12.4%. Additionally, matrix effects were substantially decreased. The proposed sample pretreatment method has been successfully applied to the analysis of multiple phytohormones in the leaf samples of oilseed rape (B. rtapus L). (C) 2015 Elsevier B.V. All rights reserved.