Within the NUTRISHIELD project, the first step towards mid-infrared (IR) based determination of the human milk fatty acid composition was performed. Compared to conventional methods, this approach enables significantly faster and less laborious analysis. This major success was enabled by combining the technical expertise of different partner institutions. Technische Universität Wien (TUW) contributed know-how in terms of method development of advanced process analytical tools, whereas the Health and Research Institute La Fe, Hospital La Fe (HULAFE) provided human milk samples and experience regarding their analysis.
Human milk contains various constituents that can be influenced by factors such as the stage of lactation, mothers’ diet, genetics and term/preterm delivery. The different components can either individually or in combination contribute to different health outcomes for the infant. In order to monitor these variations, reliable analytical methods are of high relevancy in clinical environments. In this context, fatty acids represent the human milk macronutrients with the highest variability. The most established analytical method for monitoring individual fatty acid concentrations is gas chromatography (GC), offering reliable results but coming with severe drawbacks such as the necessity of laborious sample preparation steps prior to analysis, application of organic solvents, high costs and significant time consumption.
Mid-IR spectroscopy detects absorption of IR radiation by fundamental vibrations of molecular bonds. In contrast to GC, it can operate in a label free and nondestructive matter. One of the major challenges in analyzing fatty acids in human milk with mid-IR spectroscopy are absorbances arising from its other major components such as water, lactose and proteins that can hide fatty acid related information. For this reason, the first step in developing a proper IR-based method was to find a way of isolating fat from the whole milk samples. For this purpose, an approach based on multiple centrifugation steps was devised that yields representative lipid fractions. Compared to conventional extraction methods, this approach excels with reduced workload, high throughput and completely avoids the use of potentially hazardous solvents. The obtained fat fractions were subsequently measured with attenuated total reflection Fourier transform mid-infrared spectroscopy within less than one minute per sample. Moreover, the fat fractions were analyzed with GC to obtain reference values for the fatty acid concentrations. By applying advanced statistical methods, the mid-infrared spectra were related to the reference concentrations. This way, calibration equations and statistical figures of merit were obtained that provide information about the validity of the calibration models and assess the predictability of the fatty acid concentrations in future samples. These models showed excellent results that indicate high potential for high-throughput human milk fatty acid profiling.
In the future, NUTRISHIELD partner institutions will further investigate the potential of mid-infrared based fatty acid profiling of human milk fatty acids. By combining the knowledge of experts in different fields, the developed techniques could soon represent a serious challenger for established methods such as GC.