Journal of Chromatography A, 1218 (2011) 5470–5479 Contents lists available at ScienceDirect Journal of Chromatography A j our na l ho me p ag e: www.elsevier.com/locate/chroma Measurement of phospholipids by hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry: The determination of choline containing compounds in foods Yuan-Yuan Zhao, Yeping Xiong, Jonathan M. Curtis ∗ Department of Agricultural, Food and Nutritional Sciences, University of Alberta, 318F Agriculture/Forestry Centre, Edmonton, Alberta T6G 2P5, Canada a r t i c l e i n f o Article history: Received 19 December 2010 Received in revised form 14 April 2011 Accepted 8 June 2011 Available online 17 June 2011 Keywords: Hydrophilic interaction chromatography (HILIC) Tandem mass spectrometry (MS/MS) Choline Egg yolk Phospholipids a b s t r a c t A hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC LC–MS/MS) method using multiple scan modes was developed to separate and quantify 11 compounds and lipid classes including acetylcholine (AcCho), betaine (Bet), choline (Cho), glycerophosphocholine (GPC), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phos- phatidylethanolamine (PE), phosphatidylinositol (PI), phosphocholine (PCho) and sphingomyelin (SM). This includes all of the major choline-containing compounds found in foods. The method offers advan- tages over other LC methods since HILIC chromatography is readily compatible with electrospray ionization and results in higher sensitivity and improved peak shapes. The LC–MS/MS method allows quantification of all choline-containing compounds in a single run. Tests of method suitability indicated linear ranges of approximately 0.25–25 g/ml for PI and PE, 0.5–50 g/ml for PC, 0.05–5 g/ml for SM and LPC, 0.5–25 g/ml for LPE, 0.02–5 g/ml for Cho, and 0.08–8 g/ml for Bet, respectively. Accuracies of 83–105% with precisions of 1.6–13.2% RSD were achieved for standards over a wide range of concentra- tions, demonstrating that this method will be suitable for food analysis. 8 polar lipid classes were found in a lipid extract of egg yolk and different species of the same class were differentiated based on their molecular weights and fragment ion information. PC and PE were found to be the most abundant lipid classes consisting of 71% and 18% of the total phospholipids in egg yolk. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Choline is a dietary component essential for the normal function of all cells [1]. The major form of choline in the body and in the diet is the phospholipid phosphatidylcholine. Extreme dietary deficiency of choline can result in liver dysfunction, impaired growth, abnor- malities in bone formation, kidney failure, and anemia [2]. Choline is critical during fetal development [1], when it affects brain and spinal cord structure and function and thereby the risk for neural tube defects and lifelong memory function. Choline acts as a source of methyl groups for synthetic reactions throughout the body the greatest need for which is during gestation and the early post-natal period. Although choline can be biosynthesized by humans, a large dietary intake is also required. This has been recognized in various countries, e.g. in 1998 the US Institute of Medicine Food and Nutri- tion Board established dietary reference intake levels of 550 mg/day for men and for women 425 rising to 450 and 550 mg/day during pregnancy and lactation. ∗ Corresponding author. Tel.: +1 780 492 6364. E-mail address: jcurtis1@ualberta.ca (J.M. Curtis). Although small amounts of choline are present in many foods, egg yolks are among the richest dietary sources providing ∼680 mg choline per 100 g yolk (or 126 mg per 50 g egg) [3]. Considering the newly emerging understanding of the importance of choline in nutrition, there exists an opportunity to promote eggs as an excellent and convenient source. In addition to phosphatidylcholine (PC), the dominant form of choline in egg yolk, other important and related species present include phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), acetylcholine (AcCho), phosphocholine (PCho), glycerophosphocholine (GPC), phos- phatidylinositol (PI), sphingomyelin (SM), choline (Cho), betaine (Bet) and lysophosphatidylcholine (LPC) (Fig. 1). Many analytical methods have been proposed to identify and quantify phospho- lipids, choline and betaine with various levels of sophistication and specificity. A QC assay for total choline has been developed [4] using combined acid hydrolysis with enzymatic release of choline, derivitisation and colorimetric determination. However, digestion on hydrolysis loses all information on the individ- ual choline-containing compounds. In contrast, phosphorus-31 nuclear magnetic resonance ( 31 P NMR) has been used to quantify phospholipids including PC, PE, and PI in soybean and egg lecithin 0021-9673/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2011.06.025