Human milk (HM) is considered the gold standard for infant nutrition. HM contains macro- and micronutrients, as well as a range of bioactive compounds (hormones, growth factors, cell debris, etc.). The analysis of the complex and dynamic composition of HM has been a permanent challenge for researchers. The use of novel, cutting-edge techniques involving different metabolomics platforms has permitted to expand knowledge on the variable composition of HM. Here, the state-of-the-art in untargeted metabolomic studies of HM, with emphasis on sampling, extraction and analysis steps is presented.
Sample Preparation (1st. step) | Sample Preparation (2nd. step) | Compound Class | Platform | Column/Capillary | References |
---|---|---|---|---|---|
Bligh & Dyer extraction | Deuterated solvent addition to aqueous phase | Polar metabolites | 1H-NMR | - | [13][16][29][32] |
Derivatization of aqueous phase: methoximation and silylation | Polar metabolites and FAs | GC-MS | DB-5ms | [17][18][19] | |
Derivatization of organic phase: methylation | FAs | GC-MS | DB-5ms | [13] | |
Direct injection of aqueous phase | Polar metabolites | LC-QTOF-MS (+) | HILIC | [35] | |
Redissolution of aqueous phase in H2O:ACN (95:5) | Polar metabolites | LC-Orbitrap-MS (+, −) | C18 | [24] | |
Redissolution of organic phase in (ACN:IPA:H2O (65:30:5) |
Lipidic metabolites | LC-Orbitrap-MS (+,−) | C18 | [25] | |
Folch extraction | Deuterated solvent addition to aqueous and organic phases | Hydrophobic and polar metabolites | 1H-NMR | - | [28] |
Redissolution of aqueous phase in formic acid and centrifugation | Polar metabolites (amino acids) | CE-TOF-MS (+) | 60 m × 50 µm I.D. | ||
Redissolution of organic phase in IPA:H2O:ACN (2:1:1) and centrifugation | Lipidic metabolites | UPLC-QTOF-MS (+,−) | C18 | ||
Single phase extraction | Derivatization: methoximation and silylation | Polar metabolites and FAs | GC-MS | DB-5ms | [27][28] |
Direct injection | Lipidic (and polar) metabolites | LC-QTOF-MS (+,−) | C8 | [27][28] | |
UPLC-QTOF-MS (+) | C18 | [15] | |||
Fat extraction with n-hexane/IPA | Deuterated solvent addition | TGs | 13C-NMR; 1H-NMR | - | [20] |
Filtration 3 kDa cutoff spin filter | Deuterated solvent addition | Polar metabolites | 1H-NMR | - | [14][21][22][29][33] |
Protein precipitation | Derivatization: methoximation and silylation | Polar metabolites | GC-MS | DB-5ms | [36] |
Hybrid SPE-Phospholipid extraction and redissolution in diluted organic phase of Bligh & Dyer extraction | Lipidic metabolites | LC-QTOF-MS (+) | C8 | [35] | |
Fat removal with CH2Cl2 and dansylation of aqueous phase | Polar metabolites (amine/phenol submetabolome) | Chemical isotope labelling LC-QTOF-MS (+) | C18 | [4239][4340] | |
Direct injection | Polar metabolites and FAs | UPLC-QTOF-MS (+,−) | C18 | [18] | |
Fat removal by centrifugation | Two additional centrifugations and deuterated solvent addition | Polar metabolites | 1H-NMR | - | [34] |
Filtration 10 kDa cutoff spin filter and deuterated solvent addition | Polar metabolites | 1H-NMR | - | [23][26] | |
Homogenization | Deuterated solvent addition | Polar metabolites | 1H-NMR | - | [31] |
H2O-dilution | NaBH4-reduction and PGC cartridge | Oligosaccharides | UPLC-TQD-MS (+) | Hypercarb® | [24] |
Metabolite class | LC-MS | GC-MS | NMR |
---|---|---|---|
Fatty acyls | Linoleic acid (C18:2) | Oleic acid (C18:1) | - |
Oleic acid (C18:1) | Palmitic acid (C16:0) | ||
Palmitoleic acid (C16:1) | Stearic acid (C18:0) | ||
Glycerolipids | DG (36:1) | - | - |
Glycerophospholipids | LysoPC (16:0) | - | - |
Carbohydrates and carbohydrate conjugates | - | Fructose | Lactose |
Fucose | |||
Ribose | |||
Organic acids and derivatives | - | Malic acid Urea |
Acetate |
Citrate | |||
Lactate | |||
Organo nitrogen compounds | - | - | Choline |
Amino acids, peptides, and analogues | - | Alanine Glutamate Glycine Pyroglutamic acid Serine Valine |
Alanine Creatine Glutamate Glutamine Isoleucine Leucine Tyrosine Valine |
In less than a decade, 26 research papers have been published trying to shed light on the complex and dynamic composition of HM and the feasibility of different options for sample extraction and metabolite detection has been demonstrated. Due to the many factors that influence HM composition, a thorough study design including SOPs for milk extraction, collection, and storage is indispensable for obtaining biologically meaningful results. Multi-platform approaches are encouraged for providing adequate metabolome coverage, as the diversity of compounds contained in HM will not be properly reflected using one single assay. In line with metabolomics workflows tailored to other sample types, the reproducibility of HM metabolomics studies will benefit from the implementation of QA/QC procedures. Automated metabolite annotation and identification with pure chemical standards is warranted and the authors encourage the use of publicly accessible platforms for enabling the exchange of raw data for comparison between studies.