## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### J-Resolved NMR Analysis  
The \({}^{1}\)H-\({}^{1}\)H J-resolved (JRES) technique is ideal for resolving many complex overlapping multiplet signals by a dispersion of chemical shift and coupling constant data into two orthogonal frequency domains, an advantage that readily facilitates spectral assignment. If properly optimized through the acquisition of a sufficient, albeit experimental time-restrictive, number of F2 projections, it may be employed for confirming determinationsof the linewidth at half-height (\(\Delta v_{1/2}\)) values of many resonances in complex biofluid samples. As noted above in Section 7, this parameter is, of course, of critical importance for exploring equilibria between the 'free' and'macromolecule-bound' forms of salivary biomolecules, although, as specified, this consideration is of much less importance for human saliva than it is for protein-rich blood plasma, or inflammatory knee-joint synovial fluid, for example, in view of its much lower total protein content [130]. Indeed, the binding of low-molecular-mass metabolites to protein, either electrostatically, or via H-bonding or van der Walls forces, substantially increases the line-widths of such signals, so much so that they are often viewed and termed as being 'NMR-invisible' in CPMG or Hahn spin-echo spectra acquired on protein-rich biofluids [124]. Such a parameter, which is determined by \({}^{1}\)H nuclei T\({}_{2}\) values, is also markedly influenced by differential biofluid viscosities, with greater viscosities leading to higher \(\Delta v_{1/2}\) values in view of reduced resonance T\({}_{2}\) values from the overall lowered molecular mobilities of analytes. Of further value, the \({}^{1}\)H-\({}^{1}\)H JRES technique allows the accurate determination of exact chemical shift values of superimposed \({}^{1}\)H signals present in crowded regions of the 1D profiles obtained on HSS samples. Indeed, this is readily achievable through visualization of the F2'skyline' projection, which generates information that has been found to complement that acquired from the corresponding \({}^{1}\)H-\({}^{1}\)H TOCSY profiles. For example, spectroscopic analysis using the \({}^{1}\)H-\({}^{1}\)H JRES approach was found to be highly valuable for the resolution of a large number of WMS supernatant sample resonances present in the crowded 3.00-4.30 ppm sectors of 1D spectra, particularly the \(\alpha\)-CH function protons of a range of amino acids (leucine, isoleucine, valine, methionine, valine, histidine, phenylalanine, tyrosine, and aspartate), the \(\alpha\)-CH\({}_{2}\) group of glycine, the -CH\({}_{2}\)- functions of taurine, tyrosine, phenylalanine, and histidine, the carbohydrate ring protons of sucrose, galactose, glucose, and N-acetyl sugars, the alditol ring protons of inositol, and the N-CH\({}_{3}\) and -CH\({}_{2}\)-groups of creatinine and creatine, along with the -CHOH- lactate proton. ({}^{1}\)H-\({}^{13}\)C Heteronuclear Multiple Quantum Coherence and Heteronuclear Multiple Bond Coherence Spectroscopies  
For'state-of-the-art' metabolomics analysis of biofluid samples, \({}^{13}\)C NMR analysis is rarely adopted for biomolecular quantification purposes because of its poor sensitivity, which predominantly arises from its low natural abundance (1.11%), with special reference to biomolecules that are present at low concentrations (e.g., \(<\)200 mol./L) and that are more readily determined by the \({}^{1}\)H NMR technique. Indeed, the applications of this technique in such metabolomic profiling experiments is largely restricted to qualitative confirmations of the identities of analytes, including selected biomarkers when detectable at concentrations above their limits of detection.