Journal of Chromatography B, 988 (2015) 116–120 Contents lists available at ScienceDirect Journal of Chromatography B jou rn al hom epage: www.elsevier.com/locate/chromb Short communication Liquid–liquid extraction and liquid chromatography–mass spectrometry detection of curcuminoids from bacterial culture medium Suryani Tan a,b,c , Thusitha W.T. Rupasinghe c,e , Dedreia L. Tull c,e , Mary Ann Augustin a , Sally L. Gras b,c,d,∗ a CSIRO Food, Nutrition and Bioproducts Flagship, 671 Sneydes Road, Werribee, Vic 3030, Australia b Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Vic 3010, Australia c Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Vic 3010, Australia d ARC Dairy Innovation Hub, The University of Melbourne, Parkville, Vic 3010, Australia e Metabolomics Australia, The University of Melbourne, Parkville, Vic 3010, Australia a r t i c l e i n f o Article history: Received 27 November 2014 Received in revised form 16 February 2015 Accepted 17 February 2015 Available online 24 February 2015 Keywords: Curcumin LC–MS Curcuminoids Ethyl acetate Recovery Cell medium a b s t r a c t Liquid chromatography–mass spectrometry (LC–MS) has been used to detect polyphenolic curcuminoids found in turmeric but studies of metabolism by bacterial and mammalian cells in vitro are compromised by poor recovery from the culture medium. We report a liquid–liquid extraction procedure with ethyl acetate and use LC–MS to quantify extracted curcuminoids. Ethyl acetate allows recoveries of ∼80–86% of curcuminoids from the bacterial growth medium, bacterial cell lysate and combined bacterial cell and growth medium matrices; a clear improvement over acetonitrile where recoveries were ∼25–66%. This optimised method will enable studies of curcuminoid metabolism and may be applicable to other hydrophobic polyphenolic compounds. © 2015 Elsevier B.V. All rights reserved. 1. Introduction Curcuminoids are polyphenolic compounds found in Curcuma rhizomes [1,2]. Interest is growing in curcuminoids due to their potential biological activities including anti-inflammatory [3], can- cer chemopreventative [4] and antioxidant properties [5]. LC–MS has been widely applied to study polyphenols [6] and can be used to examine curcuminoid structure and degradation [7,8], including the detection of metabolites produced by bacterial and mammalian cells. Extraction and quantification are affected by several factors, however, including: the polyphenolic chemi- cal structure, the extraction method and solvent, the stability of polyphenolic compounds, the matrix structure and presence of co- extracted substances that interfere with LC–MS analysis [9]. Many procedures have been developed to quantify break- down products that occur within phase I and II metabolism of ∗ Corresponding author at: Department of Chemical and Biomolecular Engineer- ing, The University of Melbourne, Parkville, Vic 3010, Australia. E-mail address: sgras@unimelb.edu.au (S.L. Gras). curcuminoids in humans and animals (Table 1). The solvents employed form either a single water miscible phase or partition into an organic phase and aqueous phase allowing liquid–liquid extraction (LLE), with recoveries varying from 49% to 100%. In one study, curcumin was extracted with ethyl acetate from Roswell Park Memorial Institute medium (Table 1), containing phosphate and other salts. A low recovery of 39% was reported [14]. The differences in recovery between sample matrices (Table 1) illustrate the need to optimize both the solvent and extraction pro- cedure [15], the solvent:sample ratio and use of quenching agents such as methanol also require consideration. This study aims to develop an improved protocol for extracting curcuminoids from bacterial culture medium. We report the extrac- tion from three matrices: bacterial growth medium, bacterial cell lysate and a combined matrix of bacterial cell lysate and growth medium. These matrices allow an assessment of extracellular cur- cuminoid metabolites, intracellular curcuminoid metabolites and total curcuminoid metabolites respectively. The recovery of cur- cuminoids using two common solvents: acetonitrile and ethyl acetate is also assessed in combination with methanol as a quench- ing agent. http://dx.doi.org/10.1016/j.jchromb.2015.02.024 1570-0232/© 2015 Elsevier B.V. All rights reserved.