Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography, using evaporative light-scattering and electrospray mass spectrometry for detection João M. Rocha a,b , Paavo J. Kalo b , Velimatti Ollilainen b , F. Xavier Malcata c,d,∗ a CBQF/Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal b Department of Food and Environmental Sciences, P. O. Box 27 (Latokartanonkaari 11), FIN-00014 University of Helsinki, Finland c ISMAI – Instituto Superior da Maia, Avenida Carlos Oliveira Campos, P-4475-690 Avioso S. Pedro, Portugal d ITQP – Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, P-2781-901 Oeiras, Portugal Keywords: Simple lipids Acylglycerols Free sterols Fatty acids Normal phase liquid chromatography Evaporative light-scattering detection Positive electrospray ionization tandem mass spectrometry abstract A novel method was developed for the analysis of molecular species in neutral lipid classes, using sepa- ration by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 m-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identi- fication of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour. Despite being minor constituents of flours, lipids play important roles in baking. Several authors have indeed reported on the specific effects of cereal lipids upon the final bread quality: previous defat- ting of flours impairs loaf volume and crumb grain of bread obtained therefrom, owing to favourable interactions of lipids with proteins and starch granules in flour. Furthermore, changes in protein–lipid interactions caused by wetting and mechanical work during dough mixing are important aspects for a full understanding of the func- tional role of lipids upon baking features, and in achieving baked products with appropriate final textures [1–10]. A large array of chromatographic methods has been used for identification and quantification of molecular species in simple (neutral) lipid classes. Reversed-phase (RP) HPLC separation of molecular species of neutral lipids is based mainly on partition between the non-polar bonded stationary and the polar mobile phases. Separation of molecular species of triacylglycerols (TAG), diacylglycerols (DAG) and monoacylglycerols (MAG) in reversed- ∗ Corresponding author. Tel.: +351 225 580 004; fax: +351 225 090 351. E-mail address: fmalcata@ismai.pt (F.X. Malcata). phase columns has been successfully achieved [11,12]. However, TAG with identical equivalent carbon number (ECN) (=CN - 2n, where CN is the number of acyl group carbons and n the number of double bonds) tend to co-elute, and are thus called critical pairs [13]. In silver ion HPLC, separation is based on the weak interaction between the silver ions and the -electrons of double bonds; cation exchange silver ion HPLC is considered the most efficient [14]. Unlike RP-HPLC, silver ion HPLC separates TAG species according to their number of double bonds [15], and holds a potential to separate geometrical isomers of TAG and regioisomers of unsaturated TAG as well. Furthermore, regioisomers of MAG and DAG acetates have been successfully separated by silver ion HPLC [16]. Separation in normal phase (NP) HPLC is based on the adsorption of analytes on a polar stationary phase. Plattner and Payne-Wahl [17], as well as Rhodes and Netting [18] showed that, in NP-HPLC, TAG elute in the order of descending number of acyl group carbons, and ascending number of double bonds. Christie [19,20] introduced separation of lipid classes by NP-HPLC with evaporative light-scattering detec- tion (ELSD); this technique has been claimed to separate molecular species of simple lipids in low erucic acid rapeseed oil [21]. In order to identify molecular species of TAG, DAG and MAG, Duffin et al. [22] performed fundamental studies on collision- induced decay (CID) of their ammonium adducts produced by