Table 1.
Sample preparation steps and platforms employed in untargeted analysis of HM metabolome.
| 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 | [45,46] | |
| 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] |
CE, capillary electrophoresis; FAs, fatty acids; GC, gas chromatography; HILIC, hydrophilic interaction liquid chromatography; IPA, 2-propanol; I.D., inner diameter; LC, liquid chromatography; MS, mass spectrometry; 13C-NMR, carbon-13 nuclear magnetic resonance; 1H-NMR, proton nuclear magnetic resonance; PGC, porous graphitic carbon; QTOF, quadrupole time of flight; TGs, triacylglycerols; TQD, triple quadrupole; UPLC, ultraperformance liquid chromatography; +, positive ionization mode; -, negative ionization mode.