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Short-chain fatty acids (SCFA) and lactate in ruminal fluid are products resulting from the microbial fermentation of substrates and can be used to reflect the composition and activity of the ruminal microbiome. Determination of SCFA and D-/L-lactate in ruminal fluid currently requires two separate protocols, which is time-consuming and costly. In this study, we have optimised and validated a simple and unified 3-nitrophenylhydrazine (3-NPH) derivatisation protocol and a 20 min chiral-LC-MS method for the simultaneous quantification of all SCFA and D- and L-lactate in ruminal fluid. This method, which requires no sample pretreatment or purification shows adequate sensitivity (limit of detection (LOD): 0.01 µg/mL), satisfactory accuracy (recovery: 88-103%), and excellent reproducibility (relative standard deviation (RSD) for repeated analyses < 3% for most analytes). The application of this method to a cohort of 24 animals allowed us to reveal a large inter-cow variation in ruminal SCFA and lactate level, the concentration range for each species, the widespread correlation between different SCFA, and the strong correlation between D- and L-lactate.

Short-chain fatty acids (SCFA, C2-C5) in milk and serum are derived from rumen bacterial fermentation and, thus, have the potential to be used as biomarkers for the health status of dairy cows. Currently, there is no comprehensive and validated method that can be used to analyse all SCFAs in both bovine serum and milk. This paper reports an optimised protocol, combining 3-nitrophenylhydrazine (3-NPH) derivatisation and liquid chromatography-mass spectrometry (LC-MS) analysis for quantification of SCFA and β-hydroxybutyric acid (BHBA) in both bovine milk and bovine serum. This method is sensitive (limit of detection (LOD) ≤ 0.1 µmol/L of bovine milk and serum), accurate (recovery 84-115% for most analytes) and reproducible (relative standard deviation (RSD) for repeated analyses below 7% for most measurements) with a short sample preparation step. The application of this method to samples collected from a small cohort of animals allowed us to reveal a large variation in SCFA concentration between serum and milk and across different animals as well as the strong correlation of some SCFAs between milk and serum samples.

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.

A comprehensive survey on phospholipids (PLs), sphingolipids, glycolipids, and ceramides (Cer) of bovine milk was conducted by liquid chromatography tandem-mass spectrometry. A total of 514 species belonging to 15 classes were identified by an automated search tool (LipidSearch) and a manual analysis of MS2 spectra. Over 60 species were detected for the classes of phosphatidylcholine (PC), phosphatidylinositol, phosphatidylserine, and sphingomyelin (SM), between 20 and 45 species for phosphatidylethanolamine (PE), phosphatidic acid (PA), and lactosylceramide (LacCer), and between 7 and 20 species for phosphatidylglycerol (PG), lyso-phosphatidylcholine, lyso-phosphatidylethanolamine, glucosylceramide (GluCer), PC-plasmalogen (PC-P), PE-plasmalogen, ganglioside, and Cer. The structural assignment for PL classes is straightforward based on class-specific product ion/neutral losses and fatty acid-related product ions. By contrast, structural determination of lipid classes containing a backbone of sphingoid bases (SM, GluCer, LacCer, ganglioside, and Cer) is much more challenging, and combining MS2/MS3 information acquired in both positive and negative modes is sometimes needed. The quantification of all 15 classes of lipids was performed at the species level. A large variation in abundance was observed across the lipid classes; PC, PE, and SM are the most abundant polar lipid classes, with concentrations reaching 120, 150, and 90 μmol/L, respectively, whereas PA, PG, and PC-P are present at very low levels (<1.5 μmol/L) and the remaining classes at an intermediate level (2-45 μmol/L). This is the first comprehensive characterization of minor lipid classes from bovine milk and the information provided can be used as a reference for studying milk lipids.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Human milk contains an abundant supply and diverse array of oligosaccharides that are known to impart significant health benefits to the nursing infant including establishment and maintenance of a healthy gut microflora, immune development and protection against gastrointestinal infections. When breastfeeding is not possible or insufficient, infant formulas are commonly used as an alternative. However, limited information is available about the presence of naturally occurring oligosaccharides in these infant formulas and their likely health benefits. The present study examined the presence of naturally occurring oligosaccharides in commercial goats' milk-based stage 1 and stage 2 infant formulas and their prebiotic and anti-infection properties. LC/MS was used to detect and quantify oligosaccharides and their prebiotic potential was assessed by their ability, at concentrations present in reconstituted ready-to-use infant formula, to promote the growth of Bifidobacterium animalis subsp. lactis BB12, B. longum BB536, Lactobacillus acidophilus 4461 and L. casei 2607 in vitro. For anti-infection properties, the ability of goat milk oligosaccharides to prevent the adhesion of Escherichia coli NCTC 10418 and a Salmonella typhimurium isolate to Caco-2 cells was investigated. The results showed the presence of fourteen quantifiable oligosaccharides in stage 1 and stage 2 goats' milk-based infant formula. This was similar to the number of oligosaccharides detected in the fresh goats' milk. Of these, five were structurally similar to those found in human milk. These oligosaccharides were shown to significantly enhance the growth of bifidobacteria and lactobacilli and reduce the adhesion of E. coli NCTC 10418 and S. typhimurium to Caco-2 cells. Together, these results suggest that oligosaccharides naturally present in goats' milk-based infant formula exhibit strong prebiotic and anti-pathogen adhesion properties and may confer gut health benefits to infants.

Human milk contains abundant oligosaccharides (OS) which are believed to have strong health benefits for neonates. OS are a minor component of bovine milk and little is known about how the production of OS is regulated in the bovine mammary gland. We have measured the abundance of 12 major OS in milk of 360 cows, which had high density SNP marker genotypes. Most of the OS were found to be highly heritable (h 2 between 50 and 84%). A genome-wide association study allowed us to fine-map several QTL and identify candidate genes with major effects on five OS. Among them, a putative causal mutation close to the ABO gene on Chromosome 11 accounted for approximately 80% of genetic variance for two OS, N-acetylgalactosaminyllactose and lacto-N-neotetraose. This mutation lies very close to a variant associated with the expression levels of ABO. A third QTL mapped close to ST3GAL6 on Chromosome 1 explaining 33% of genetic variation of an abundant OS, 3'-sialyllactose. The presence of major gene effects suggests that targeted marker-assisted selection would lead to a significant increase in the level of these OS in milk. This is the first attempt to map candidate genes and causal mutations for bovine milk OS.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Seasonal change of milk composition could offer opportunities for dairy manufacturers. A systematic survey on seasonal variation of six classes of polar lipids was conducted with 19 Holstein cows over the entire milking season using liquid chromatography-mass spectrometry technique. This study revealed that most polar lipid classes were positively correlated with the total fat content, but negatively correlated with fat globule size. All polar lipid classes displayed a large cow-to-cow variation as well as seasonal variation. All of the six classes showed a gradual increase over the milking season with the highest concentration observed in May (autumn). However, the proportion of different polar lipid classes remained constant during the entire milking season. This finding suggests that the production of polar lipids is highly regulated in the mammary gland. The implication of such a seasonal variation of polar lipids in the nutritional and technological aspects of milk is discussed., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Human milk oligosaccharides (OS) play an important role in protecting the neonate. In addition to fructo-oligosaccharides and galacto-oligosaccharides, bovine milk OS have great potential to be used in pediatric food products to mimic the functions of human milk OS. Currently, little is known about the accumulation of OS in bovine milk in relation to genetic and environmental factors. A systematic survey on seasonal variation of 14 major OS was thus conducted with 19 cows over the entire milking season using a liquid chromatography-mass spectrometry technique. This study revealed a number of significant correlations between structurally related and structurally nonrelated OS and a substantial individual animal difference for all 14 OS. Most of the 14 OS displayed a remarkable seasonal variation in abundance (up to 10-fold change), with the highest abundance observed in April and May (i.e., autumn) for the majority of the 19 cows.

For LC-MS-based lipidomic analysis of milk, total lipid extraction from raw milk is generally conducted with Folch or Bligh and Dyer methods; both methods are based on two-phase partition of lipids, and thus time-consuming. In this work, three solvent systems for one-phase extraction of milk lipids were compared with the standard Folch method. Two of the solvent systems (butanol/methanol, 3:1 and 1:1) previously tested for lipid extraction from plasma were found to provide adequate extraction for polar lipids, but incomplete extraction for triglycerides, especially highly lipophilic species. By contrast, our newly designed solvent mix composed of butanol, methanol and chloroform (at a 3:5:4 ratio) provided similar extraction efficiency for triglycerides and higher yield for some of the phospholipids, as compared to the Folch method. This new one-phase extraction method is very simple yet comprehensive and thus suitable for high throughput lipid analysis of milk samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Long-chain polyunsaturated fatty acids (LC-PUFA) are an important nutritional lipid and have potential in being able to promote human health. Docosahexaenoic acid (DHA, C22:6ω3) is often added in infant formulas to meet the nutritional requirement of formula-fed infants. A comprehensive survey on DHA-containing triacylglycerol (DHA-TAG) molecular species has been conducted for seven infant formulas (IFs) sourced from Australia, Europe, and the USA as well as bovine milk and human milk. Using LC-triple quadrupole MS and LC-LTQ-orbitrap MS we were able to identify and quantify 56 DHA-TAG species in these samples; the fatty acid structure of these species was assigned using their MS(2) spectra. The species composition of DHA-TAG was found to be different between bovine milk, human milk, and IFs and also between different brands of IFs. Bovine milk and human milk contain DHA-TAG of smaller molecular size (728-952 Da), whereas five out of the seven IF samples contain species of broader mass range (from 728 to 1035 Da). Our study indicates that two types of DHA were used in the seven IF products surveyed and that there is very large difference in molecular species distribution in different IF products that may influence the fine nutritional profile and biological functions of IF products.

The n-3 long-chain polyunsaturated fatty acids (LC-PUFA) are low-abundance components in milk fat, but have great potential in promoting human health. A comprehensive survey on triacylglycerol (TAG) molecular species in milk that contain at least one type of n-3 LC-PUFA, namely eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid, was conducted in this work using HPLC-linear trap quadrupole-Orbitrap and HPLC-triple quadrupole mass spectrometry techniques. A total of 51 TAG species that contain n-3 LC-PUFA have been identified in bovine milk and their structures assigned. The TAG species containing docosahexaenoic acid were found in much smaller number and at much lower abundance compared with the other 2 types of TAG. An HPLC-triple quadrupole mass spectrometry-based method was developed, which provides relative quantification of all these TAG species in a run of 36 min. Application of this method to the quantification of n-3 LC-PUFA-incorporated TAG in 32 individual animal milk samples allowed us to determine variation between animals, identify strong metabolic relationships between TAG species, and reveal negative effect of a grape marc supplement on the accumulation of eicosapentaenoic acid in milk., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Polar lipids (PLs) are a significant functional component of milk that are difficult to quantitate. A simple method for comprehensive identification and quantitative analysis of all essential PL species using bovine milk is described. The lipid fraction was extracted by a mix of chloroform and methanol and the extract was directly used for PL identification and quantification. PLs were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyser in positive mode. The structure of PLs was established or confirmed by tandem MS in both positive and negative modes. The method is sensitive (with a LOD for all PL classes ≤0.1 ng) and reproducible, enabling simultaneous quantification of 70 PL species within a run of 45 min. Application of this method to the quantification of PLs in 32 bovine milk samples revealed the relative abundance of different PL classes, significant variation of PL content between individual samples and the correlation between the major PL classes. The method provides a tool for investigating the variation and metabolism of important PL components in bovine and human milk and in diverse mammalian species., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Free oligosaccharides (OS) are a significant functional component of milk that are difficult to quantitate. A simple method for quantitative analysis of the major free OS in bovine milk is described. Following a defatting step, protein elimination was performed by ultrafiltration. OS were separated by hydrophilic interaction liquid chromatography (HILIC) and detected by an Orbitrap mass analyzer in negative mode. The method is sensitive [with a limit of detection (LOD) for all representative OS of <0.1 ng] and reproducible, enabling simultaneous quantification of 13 major OS within a single run. Application of this method to the quantification of major OS in 32 milk samples collected after three different feeding treatments allowed us to reveal the relative abundance of different OS species, the variation of the OS content between individual cows, and the correlations between some of the major OS.

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]