Quantitative determination of non-lactose milk oligosaccharides

Abstract

A method for the determination of non-lactose oligosaccharides (NLO) in milk using liquid chromatography has been developed. Oligosaccharides were labelled with a fluorescent tag, 2-aminobenzamide (2AB), and were identified by comparison of their retention times to those of oligosaccharide standards, their mass (as measured by mass spectrometry) and their fragmentation patterns in the mass spectrometer. The concentrations of the NLO in milk have been determined using 2 different approaches: (1) by preparing a calibration curve using genuine standards of each oligosaccharide. (2) by preparing a calibration curve using maltotriose as a universal standard for all NLO, and assuming all 2AB labelled oligosaccharides give an equimolar response in the detector. The accuracy of the method was assessed by spike-recovery experiments. Using genuine NLO standards for calibration, recoveries were in the range 96-114%. Using maltotriose as a universal calibrant, recoveries were in the range 86-120%. Method precision was assessed by determining the relative standard deviation of the results under repeatability (RSD(r)) and intermediate reproducibility (RSD(iR)) conditions. In most cases RSD(r) and RSD(iR) were below 5% irrespective of calibration method, but increased when NLO levels were close to LoQ.

Keywords: Fluorescent label; Human milk oligosaccharide; Liquid chromatography; Mass spectrometry; Milk; Validation.