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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. [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]