Journal of Chromatography A, 1226 (2012) 71–76 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rn al h om epage: www.elsevier.com/locat e/chroma An automated method for the analysis of phenolic acids in plasma based on ion-pairing micro-extraction coupled on-line to gas chromatography/mass spectrometry with in-liner derivatisation Sonja Peters a,b,∗ , Erwin Kaal c , Iwan Horsting d , Hans-Gerd Janssen a,b a Unilever Research and Development, Advanced Measurement and Data Modelling, P.O. Box 114, 3130 AC Vlaardingen, The Netherlands b Analytical-Chemistry Group, University of Amsterdam, Postbus 94157, 1090 GD Amsterdam, The Netherlands c DSM Biotechnology Center, DSM, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands d ATAS GL International, P.O. Box 17, Olivier van Noortlaan 130, 5500 AA Veldhoven, The Netherlands a r t i c l e i n f o Article history: Available online 28 October 2011 Keywords: Automation Gas chromatography Micro-extraction in packed sorbent (MEPS) Ion-pairing extraction In-liner derivatisation Plasma Phenolic acids a b s t r a c t A new method is presented for the analysis of phenolic acids in plasma based on ion-pairing ‘Micro- extraction in packed sorbent’ (MEPS) coupled on-line to in-liner derivatisation-gas chromatography– mass spectrometry (GC–MS). The ion-pairing reagent served a dual purpose. It was used both to improve extraction yields of the more polar analytes and as the methyl donor in the automated in-liner derivati- sation method. In this way, a fully automated procedure for the extraction, derivatisation and injection of a wide range of phenolic acids in plasma samples has been obtained. An extensive optimisation of the extraction and derivatisation procedure has been performed. The entire method showed excellent repeatabilities of under 10% and linearities of 0.99 or better for all phenolic acids. The limits of detection of the optimised method for the majority of phenolic acids were 10 ng/mL or lower with three phenolic acids having less-favourable detection limits of around 100 ng/mL. Finally, the newly developed method has been applied in a human intervention trial in which the bioavailability of polyphenols from wine and tea was studied. Forty plasma samples could be analysed within 24 h in a fully automated method including sample extraction, derivatisation and gas chromatographic analysis. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Metabolic profiling and metabolomics are rapidly gaining importance in pharmaceutical and nutritional intervention studies. Metabolomics is the comprehensive study of the metabolome, i.e. it involves the comprehensive identification and quantification of all metabolites present in biological systems such as plants, animals or humans. When gas chromatography (GC) is used as the analytical method, the metabolic fingerprint includes small molecules only. These molecules are usually analysed in complex matrices such as plasma, urine or faeces. GC fingerprints offer an unsurpassed peak capacity and sensitivity allowing the analysis of thousands of compounds at good detection limits. Although GC systems are very robust, sample preparation is essential. When body fluids are to be analysed by GC, large-molecular weight compounds need to be removed before analysis and many compounds of interest require a prior derivatisation step. These steps are often offline, ∗ Corresponding author at: Unilever Research and Development, Advanced Mea- surement and Data Modelling, P.O. Box 114, Olivier van Noortlaan 130, 3130 AC Vlaardingen, The Netherlands. Tel.: +31 010 4606397; fax: +31 010 4605310. E-mail address: sonja.peters@unilever.com (S. Peters). labour-intensive and require the use of high amounts of solvents. Key aspects bringing sample preparation forward are therefore automation and miniaturisation. Automation of sample preparation has been progressed sub- stantially with the introduction of solid-phase microextraction (SPME) [1] and robotic solid-phase extraction (SPE) systems. SPME and GC are nowadays routinely coupled with or without (prior) derivatisation. While SPME possesses some advantages over tra- ditional sample preparation methods such as low or no solvent consumption and the relative ease of online coupling to chromato- graphic systems, it also has some major disadvantages, mainly related to the lack of coatings that allow the adsorption of polar compounds [2]. Coupling robotic SPE systems with GC is more complicated. The amount and nature of the extraction effluent is usually not compatible with GC and most analytes require a derivatisation step in order to make them amendable for GC anal- ysis. ‘Micro-extraction in packed sorbent’ (MEPS) [3] is a relatively new miniaturised SPE method that has been shown to be an excel- lent tool to automate sample preparation protocols. MEPS does not require (expensive) robotic systems as it utilises the ‘normal’ syringe of the auto-sampler of the chromatographic system. The sorbent material is inserted into the syringe needle and the sam- ple extraction is performed by pulling and pushing the plunger up 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.10.055