In this report, we present a detailed comparison of the lipid composition of human milk (HM) and formula milk (FM) targeting different lactation stages and infant age range. We studied HM samples collected from 26 Polish mothers from colostrum to 19 months of lactation, along with FM from seven brands available on the Polish market (infant formula, follow-on formula and growing-up formula). Lipid extracts were analysed using liquid chromatography coupled to high-resolution mass spectrometry (LC–Q-TOF–MS). We found that the lipid composition of FM deviates significantly from the HM lipid profile in terms of qualitative and quantitative differences. FM had contrasting lipid profiles mostly across brands and accordingly to the type of fat added but not specific to the target age range. The individual differences were dominant in HM; however, differences according to the lactation stage were also observed, especially between colostrum and HM collected in other lactation stages. Biologically and nutritionally important lipids, such as long-chain polyunsaturated fatty acids (LC-PUFAs) containing lipid species, sphingomyelines or ether analogues of glycerophosphoethanoloamines were detected in HM collected in all studied lactation stages. The observed differences concerned all the major HM lipid classes and highlight the importance of the detailed compositional studies of both HM and FM.

Commercial formula milk (FM) constitutes the best alternative to fulfill the nutritional requirements of infants when breastfeeding is precluded. Here, we present the comparative study of polar metabolite composition of human breast milk (HBM) and seven different brands of FM by nuclear magnetic resonance spectroscopy. The results of the multivariate data analysis exposed qualitative and quantitative differences between HBM and FM composition as well as within FM of various brands and in HBM itself (between individual mothers and lactation period). Several metabolites were found exclusively in HBM and FM. Statistically significant higher levels of isoleucine and methionine in their free form were detected in FM samples based on caprine milk, while FM samples based on bovine milk showed a higher level of glucose and galactose in comparison to HBM. The results suggest that the amelioration of FM formulation is imperative to better mimic the composition of minor nutrients in HBM.

Metabolomics is the field of omics research that offers valuable insights into the complex composition of biological samples. It has found wide application in clinical diagnostics, disease investigation, therapy prediction, monitoring of treatment efficiency, drug discovery, or in-depth analysis of sample composition. A suitable study design constitutes the fundamental requirements to ensure robust and reliable results from the study data. The study design process should include a careful selection of conditions for each experimental step, from sample collection to data analysis. The pre-analytical variability that can introduce bias to the subsequent analytical process, decrease the outcome reliability, and confuse the final results of the metabolomics research, should also be considered. Herein, we provide key information regarding the pre-analytical variables affecting the metabolomics studies of biological fluids that are the most desirable type of biological samples. Our work offers a practical review that can serve and guide metabolomics pre-analytical design. It indicates pre-analytical factors, which can introduce artificial data variation and should be identified and understood during experimental design (through literature overview or analytical experiments).