2062 J. Sep. Sci. 2014, 37, 2062–2068 Helena Zahradn´ ıˇ ckov ´ a 1 Ale ˇ s Tom ˇ cala 1,2 Petra Berkov ´ a 1 Ivana Schneedorferov ´ a 1,3 Jan Okrouhl´ ık 3 Petr ˇ Simek 1 Magdalena Hodkov ´ a 1 1 Biology Centre, Academy of Sciences of the Czech Republic, ˇ Cesk ´ e Bud ˇ ejovice, Czech Republic 2 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Czech Republic 3 Faculty of Science, University of South Bohemia, ˇ Cesk ´ e Bud ˇ ejovice, Czech Republic Received February 5, 2014 Revised April 7, 2014 Accepted April 24, 2014 Research Article Cost effective, robust, and reliable coupled separation techniques for the identification and quantification of phospholipids in complex biological matrices: Application to insects The quantification of phospholipid classes and the determination of their molecular struc- tures are crucial in physiological and medical studies. This paper’s target analytes are cell membrane phospholipids, which play an important role in the seasonal acclimation pro- cesses of poikilothermic organisms. We introduce a set of simple and cost-effective analytical methods that enable efficient characterization and quantification of particular phospholipid classes and the identification and relative distribution of the individual phospholipid species. The analytical approach involves solid-phase extraction and high-performance thin-layer chromatography, which facilitate the separation of particular lipid classes. The obtained fractions are further transesterified to fatty acid methyl esters and subjected to gas chro- matography coupled to flame ionization detection, which enables the determination of the position of double bonds. Phospholipid species separation is achieved by high-performance liquid chromatography with mass spectrometry, which gives information about the head- group moiety and attached fatty acids. The total content of each phospholipids class is as- sessed by phosphorus determination by UV spectrophotometry. The simultaneous analysis of phosphorus, fatty acid residues, and phospholipid species provides detailed information about phospholipid composition. Evaluation of these coupled methods was achieved by application to an insect model, Pyrrhocoris apterus. High correlation was observed between fatty acid compositions as determined by gas chromatography and high-performance liquid chromatography analysis. Keywords: Insects / Mass spectrometry / Phospholipids / Thin-layer chromatog- raphy DOI 10.1002/jssc.201400113  Additional supporting information may be found in the online version of this article at the publisher’s web-site 1 Introduction Researchers in the fields of physiology, pharmacology, and medicine desire quick, cheap, and reliable methods for polar lipid analysis. We present such a technique using the insect model Pyrrhocoris apterus. Insect tissues possess similar lipid Correspondence: Dr. Helena Zahradn´ ıˇ ckov ´ a, Biology Centre ASCR, v.v.i., Braniˇ sovsk ´ a 31, ˇ Cesk ´ e Bud ˇ ejovice 370 05, Czech Re- public E-mail: helenaz@bclab.eu Fax: +420387775287 Abbreviations: CL, cardiolipin; FA, fatty acid; FAME, fatty acid methyl ester; FID, flame ionization detector; HPTLC, high- performance TLC; MP, mobile phase; NL, nonpolar lipid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipid; PS, phosphatidylser- ine; SM, sphingomyelin compositions of polar lipids and nonpolar lipids (NLs) to other animals including mammalian tissues [1], therefore insects provide a suitable system for testing this technique. Moreover, obtaining insect samples is cheap and safe and no legal restrictions are involved. Temperature is one of the most important abiotic factors for poikilothermic organisms, especially insects. Biological membranes assembled from phospholipids (PLs) are respon- sible for a wide variety of vital functions and their primary structure is influenced by temperature. Since membranes have to maintain structural and functional cell integrity dur- ing fluctuations of environmental temperature, the study of the effects of temperature on membrane lipids is particularly important for understanding organism temperature adapta- tion mechanisms. Changes in distribution of individual PLs involving fatty acid (FA) acyl chain and PL head group re- structuring seem to be an integral part of the adaptation of cell membranes to environmental changes in temperature C  2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com