Evaluation of direct analysis in real time ionization–mass spectrometry (DART–MS) in fish metabolomics aimed to assess the response to dietary supplementation Tomas Cajka a , Hana Danhelova a , Antonin Vavrecka b , Katerina Riddellova a,n , Vladimir Kocourek a , Frantisek Vacha b , Jana Hajslova a a Institute of Chemical Technology, Prague, Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, Technicka 3,166 28 Prague 6, Czech Republic b University of South Bohemia in Ceske Budejovice, Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture, Husova tr. 458/102, 370 05 Ceske Budejovice, Czech Republic article info Article history: Received 12 January 2013 Received in revised form 2 April 2013 Accepted 8 April 2013 Available online 15 April 2013 Keywords: Direct analysis in real time (DART) Mass spectrometry Fish Feeding Metabolomic fingerprinting abstract Ambient mass spectrometry employing a direct analysis in real time (DART) ion source coupled to a medium high-resolution/accurate mass time-of-flight mass spectrometer (TOFMS) was used as a rapid tool for metabolomic fingerprinting to study the effects of supplemental feeding with cereals (triticale) on the composition of muscle metabolites of common carp (Cyprinus carpio L.). First, the sample extraction and DART–TOFMS instrumental conditions were optimized to obtain the broadest possible representation of ionizable compounds occurring in the extracts obtained from common carp muscle. To this end, a simultaneous (all-in-one) extraction procedure was developed employing water and cyclohexane mixture as the extraction solvents. Under these conditions both polar as well as non-polar metabolites were isolated within a single extraction step. Next, the metabolomic fingerprints (mass spectra) of a large set of common carp muscle extracts were acquired. Finally, the experimental data were statistically evaluated using principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Using this approach, differentiation of common carp muscle in response to dietary supplementation (feeding with and without cereals) was feasible. Correct classification was obtained based on the assessment of polar and as well as non-polar extracts fingerprints. The current study showed that DART– TOFMS metabolomic fingerprinting represents a rapid and powerful analytical strategy enabling differentiation of common carp muscles according to feeding history by recording metabolomic fingerprints of ionizable components under the conditions of ambient MS. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Most of fish are a rich source not only of high-quality protein, essential vitamins, and minerals but also ω–3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahex- aenoic acid (DHA) [1,2]. The content of “healthy” lipids and other components in particular fish species may largely differ depending on whether the fish is wild or grown under farming conditions. In the latter case, the farming practices are based on the use of various diet supplements what typically results in higher content of neutral lipids [3]. In Central Europe, one of the most important farmed freshwater fish species is common carp (Cyprinus carpio L.). It is traditionally reared in earthen ponds. To increase its production, natural diet is commonly supplemented with cereals, i.e., feed with high proportion of carbohydrates, representing the primary source of energy [4]. Subsequently, specific enzymatic system of fish enables to utilize high amount of carbohydrates, which are depos- ited in lipids. Unfortunately, their fatty acids pattern is not affected in a desirable way, nevertheless, optimization of ω–3 fatty acids composition in carps from different pond production systems can be achieved by addition of plant materials containing higher content of PUFA precursor, α-linoleic acid, ALA [5]. To monitor the effect of feeding practice on fish quality within tested experimental set-ups and to enable traceability of declared production system in case of marketed fish, suitable analytical strategies have to be available. Obtaining as much as possible comprehensive information within a short time is the current trend when choosing laboratory approach. Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/talanta Talanta 0039-9140/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.talanta.2013.04.025 n Corresponding author. Tel.: +420 220 443 218; fax: +420 220 443 184. E-mail address: katerina.riddellova@vscht.cz (K. Riddellova). Talanta 115 (2013) 263–270