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Milk is a rich source of lipids, with the major components being triglycerides (TAG) and phospholipids (mainly phosphatidylcholine (PC), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)). Liquid chromatography-mass spectrometry (LC-MS) is the predominant technique for lipid identification and quantification across all biological samples. While fatty acid (FA) composition of the major lipid classes of milk can be readily determined using tandem MS, elucidating the regio-distribution and double bond position of the FA remains difficult. Various workflows have been reported on the quantification of lipid species in biological samples in the past 20 years, but no standard or consensus methods are currently available for the quantification of milk phospholipids. This study will examine the influence of several common factors in lipid analysis workflow (including lipid extraction protocols, LC stationary phases, mobile phase buffers, gradient elution programmes, mass analyser resolution and isotope correction) on the quantification outcome of bovine milk phospholipids. The pros and cons of the current LC-MS methods as well as the critical problems to be solved will also be discussed. [ABSTRACT FROM AUTHOR]

Abstract Determination of the fatty acid profile of milk fat generally involves a protocol that comprises total lipid extraction, transesterification and GC analysis. The lipid extraction step is time-consuming and often employs toxic solvents such as chloroform. A novel protocol is presented here that skips the lipid extraction step and allows the determination of fatty acid composition via direct methylation of milk fat isolated after centrifugation of raw milk. This new method is reliable for relative quantification of fatty acids in raw milk fat and offers a much higher throughput compared with the classical methods. [ABSTRACT FROM AUTHOR]

Three acid- and alkaline-catalysed transesterification methods were compared with the aim to validate a simple yet reliable protocol for fatty acid (FA) profiling of milk fat. While both the acid- and alkaline-catalysed methods were able to convert completely triglycerides and phospholipids into fatty acid methyl esters (FAMEs), the acid catalyst caused significant degradation of conjugated linoleic acid C18:2c9t11 at high temperature. Although a milder temperature can mitigate this negative impact, a long reaction time (2 h) is required to achieve full methylation. By contrast, despite being unable to methylate free fatty acids (FFA), the alkaline-catalysed transesterification yielded comparable results for all major FA due to the very low level of FFA in milk. The alkaline-catalysed methylation is benign for C18:2c9t11. We recommend here a simple one-step protocol based on 0.2 M methanolic KOH, a short reaction time (20 min) and a mild reaction temperature (50 °C) for milk FAME preparation. [ABSTRACT FROM AUTHOR]

Bovine milk contains 3–5% of fat, of which the dominant portion (about 98%) is in the form of triacylglycerols, whereas polar lipids such as glycerophospholipids and sphingolipids are minor components (0.5–1%) of milk fat. Milk fat is thought to contain several thousand lipid species, making it the most complex material in nature in terms of lipid composition. Significant progress has been made in the past two decades in the identification and quantification of lipid species of milk thanks to the advance in analytical tools especially high-resolution mass spectrometers (MS). Currently, milk lipids are characterized mainly in two ways, i.e. global fatty acid composition profiling by gas chromatography and lipid molecular species identification and quantification by liquid chromatography tandem MS; the former provides information related to the physicochemical properties and nutritive quality of milk fat, whereas the latter provides the detailed chemical structure of lipid species. Until now, only about 400 lipid species have been described in bovine milk, with many low-abundance species remaining to be discovered. The merits and limitations of various separation techniques and different MS methodologies applied to lipid structural elucidation and quantification are critically reviewed and the challenging problems to be solved in milk lipidomic analysis highlighted. [ABSTRACT FROM AUTHOR]

• First adoption of Paterno-Buchi reaction in studying milk lipid fine structure. • Unsaturated fatty acids show a non-specific regio-distribution in phospholipids. • Regioisomers are widespread in milk phospholipid species. • Double-bond isomers of C18:1 and C18:2 are widely detected in milk phospholipids. Hundreds of phospholipid (PL) species with defined fatty acid (FA) composition have been identified previously in bovine milk using liquid chromatography tandem mass spectrometry (LC-MS/MS). Paterno-Buchi photochemical reaction coupled with LC-MS/MS was applied in this study to further unravel the regio-distribution and double bond (DB) locations of FAs. Using SPE-purified PLs and 2-acetylpyridine as the photochemical derivatization reagent, we were able to reveal the non-specific regio-distribution of unsaturated FAs and the widespread occurrence of regioisomers in milk PLs. Although Δ9 and Δ9,12 were found to be the predominant DB location(s) for C18:1 and C18:2 respectively, other DB positional isomers such as C18:1Δ11, C18:1Δ12 and C18:1Δ13 and C18:2Δ9,11 were widely detected in PL structures, implying that the minor isomers of C18:1 and C18:2 equally participate in the synthesis of PLs. Our study provides novel information on the fine structure of milk PLs and further underlines the complexity of milk lipid composition. [ABSTRACT FROM AUTHOR]

Regioisomers (or positional isomers) of triacylglycerols (TAGs) of milk are known to show differential outcome in relation to human absorption. Quantitation of TAG regioisomers remains a big challenge due to the lack of facile chromatographic separation technique. The feasibility of using fragment ion intensity ratio to determine the ratio of co-eluting AAB/ABA-type regioisomer pairs was confirmed in this study. The ability of C30 stationary phase in resolving interfering TAG isomers was demonstrated for the first time. This allowed us to reveal the complexity of using fragment ion intensity to quantify 1,2-olein-3-palmitin (OOP), 1,3-olein-2-palmitin (OPO), 1,2-olein-3-stearin (OOS), and 1,3-olein-2-stearin (OSO) regioisomers in milk samples. A novel algorithm was proposed to consider the contribution of OPO/OOP and OSO/OOS double bond (DB)-isomers and to eliminate the interference of isobaric ions from other isomers, an aspect overlooked in previous studies. This liquid chromatography-mass spectrometry method that requires no pre-fractioning and a moderate chromatographic separation time of 36 min is simple and, thus, suitable for screening a large number of samples for genetic analysis of this trait. Preliminary results using a small cohort of animals showed that OPO/OOP ratio differs significantly between Jersey and Holstein cows, and a large variation was also observed across individual Holstein cows. [ABSTRACT FROM AUTHOR]

• Two new methods for fatty acid methylation without lipid extraction were validated. • Dried milk can be reliably used for in situ alkali-catalysed transesterification. • Acidic methylation of fresh milk is feasible with optimised reaction parameters. • The new methods are applicable to both homogenised and raw milk samples. Determination of the fatty acid (FA) profile of milk fat generally involves total lipid extraction from liquid milk, transesterification and GC analysis. The lipid extraction step is time consuming and often employs toxic solvents such as chloroform. Two alternative methods are presented here that skip the lipid extraction step and allow the determination of FA composition via direct transesterification of dried milk and liquid milk respectively. We have shown that dried milk can be used directly in alkaline-catalysed methylation, whereas direct transesterification of both dried milk and fresh milk is feasible with acidic methanol. Both methods generate similar results as compared to the classical two-step method (i.e. lipid extraction and FA methylation) when optimised methylation parameters (temperature, time, milk and reagent volume) are followed. By omitting the lipid extraction step, these simplified one-step methods offer a much higher throughput and a reduced cost in FA composition analysis of milk samples. [ABSTRACT FROM AUTHOR]