Table 3.

The most commonly used 2D NMR techniques with their application in metabolomics

Method	How it works	What can we do with it?	Subtypes, other types of this method
COSY (homonuclear correlation spectroscopy)	The first 2D NMR method used for the analysis of extracts Identifies spins that are coupled to one another	Facilitates identification of: GABA (the pattern of its 3 methylene protons), glycerophosphocholine, phosphoethanolamine and myoinositol (spin connectivities) imidodipeptides (those including proline or hydroxyproline) [56]	Double Quantum Filtered shift-COrrelated SpectroscopY (DQF-COSY)-enhanced spectral resolution, better determination of the coupling constant, suppression of large singlets [21] COCONOSY (NOE and shift-correlated spectroscopy): confirmation of malate peak assignment [26]
HSQC (Heteronuclear Single Quantum Coherence)	Uses magnetization transfer between nuclei, usually between hydrogen and carbon atoms (1H–13C) [57] Not a quantitative technique T1 noise artifacts present, but can be corrected with algorithms [58]	Confirmed identification of: Numerous aminoacids (isoleucine, leucine, valine, threonine, lysine, glutamate, proline, glutamine, asparagine, phenylalanine) Lactate and glucose Ethanolamine and glycerol Glycogen Lipids [59]	Adiabatic pulsing—generates quantitative data gHSQC [60] Offset-compensated, CPMG-adjusted HSQC (Q-OCCAHSQC) [61] Q-HSQC QQ-HSQC (shorter total acquisition time) Q-CAHSQC HILIC (2D Hydrophilic Interaction Chromatography), can replace LC-NMR to identify certain metabolites, e.g., in urine [62] HSQC0 (time-zero 2D spectrum) signal intensities are proportional to compounds’ concentrations, which facilitates their identification [63]
HMQC (Heteronuclear Multiple-Quantum Correlation)	Broader peak because of homonuclear proton J-coupling (worse resolution) More difficult metabolite identification [54]	Assigning peaks of 1- and 3-methylhistidine
NOESY (Nuclear Overhauser Effect Spectroscopy)	Has additional peaks that are not informatory (they can be eliminated by reversing the phase) The resonances of nuclei which are closely located in space are coupled to each other (not those which are connected by covalent bonds)	Confirmation of peak assignment in 1D spectra [64] For certain solute concentrations better water suppression than the one achieved by standard water presaturation techniques [65]
DQF-COSY (Double Quantum Filtered shift-COrrelated SpectroscopY)	Uses the phenomenon of double quantum coherence between scalar-coupled protons	Improved spectral resolution Identification of sugar protons and coupled methylene protons Suppression of big singlet signals, and thanks to it, reduction of spectral artifacts from T1 noise Facilitates determination of the spin multiplicity and the coupling constant [26]
HMBC (Heteronuclear Multiple Bond Coherence)		Less frequently used than HSQC Suppression of the HSQC-like one-bond interaction is not always achieved, which might result in artifacts [66]
INADEQUATE (Incredible Natural Abundance Double Quantum Transfer Experiment)		Reduced overlap [55] Identification of taurine, myoinositol, serine thanks to diagonal transparency (not possible in TOCSY/COSY) [67] Less effective resonance discrimination than HSQC [68]
TOCSY (TOtal Correlation SpectroscopyY	Cross peaks are formed for both directly and indirectly coupled nuclei	A clearer spectra than in 1D NOESY spectra with reduced spectral overlap [69] Facilitation of determination of nucleotides (e.g., uridine nucleotides) thanks to reduction of spectral overlap [70]
STOCSY (Statistical TOCSY)		Generates a pseudo-2D spectrum, which shows the relationships between the intensities of different peaks [71]	SHY-statistical heterospectroscopy for the coanalysis of multispectroscopic data of a number of samples [71]
2D JRES NMR (two-dimensional J-resolved NMR spectroscopy)		It is a reliable and fast technique Measures isotopic patterns of compounds labeled with 13C carbon Suitable for reproducible isotopic profiling (e.g., of isotopomers of alanine) [72]	p-JRES NMR (projections of 2D J-resolved NMR have more uniform baseline and the peaks are less crowded than in 1D spectra) [72]

Modified according to Refs. [26, 54, 56–72]