## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### J-Resolved NMR Analysis
However, the development of inverse geometry probes and supporting pulse sequences has now largely circumvented this limitation, and \({}^{1}\)H-\({}^{13}\)C heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) spectroscopies, and related analytical strategies, provide a substantial enhancement in sensitivity over that of the conventional 1D spectroscopic technique. These inverse \({}^{1}\)H-\({}^{13}\)C correlational spectroscopies feature the detection of \({}^{13}\)C to \({}^{1}\)H connectivities (although heteronuclei other than \({}^{13}\)C may also be employed). The HMQC technique is selective for direct \({}^{1}\)H-\({}^{13}\)C couplings only, whereas HMBC spectroscopies provide valuable molecular information through longer, 2-4 bond range couplings. Hence, the HMQC and HMBC techniques represent the \({}^{1}\)H-\({}^{13}\)C equivalents of the homonuclear \({}^{1}\)H-\({}^{1}\)H COSY and TOCSY approaches, respectively. Most modern high-resolution NMR facilities implement gradient-selected forms of HMQC and HMBC, which serve to remove unwanted resonance artefacts and hence improve the quality of the 2D spectra acquired. A total of 600 MHz \({}^{1}\)H-\({}^{13}\)C inverse-detected HMQC spectra of HSS samples have been found to contain a series of projecting cross-resonances for many \({}^{1}\)H NMR-detectable biomolecules, including organic acid anion bacterial catabolites and amino acids. Spectra acquired confirmed the advantages offered by the 2D HMQC technique, notably its ability to resolve overlapping \({}^{1}\)H NMR resonances in corresponding 1D spectra. Moreover, employment of an 8 mm \({}^{13}\)C NMR probe in these experiments facilitated the detection of a range of biomolecules present at only low salivary levels. Access to available databases aided verifications of the correspondences of metabolite \({}^{1}\)H and \({}^{13}\)C NMR assignments, and therefore this process assisted with analyte identification and clarification. Furthermore, \({}^{13}\)C assignments and related connected HMQC signals may be confirmed via reference to established reference chemical shift values for authentic model compounds (e.g., [PERSON] et al., 1999 [131]). Preprocessing, Bucketing, Normalization, Transformation, and Scaling of \({}^{1}\)H NMR Profiles of HSS Specimens: Bioanalytical Considerations for Metabolite Quantification and Metabolomics Analysis  
In general, the intensities of salivary biomolecule resonances are determined by electronic integration of the appropriate spectral regions through application of spectrometer proprietary software (e.g., XWIN-NMR), and maintenance of the exact integral regions is ensured for all spectra included in studies. Concentrations of all possible \({}^{1}\)H NMR-detectable metabolites, and, where appropriate, also those of xenobiotics such as drugs ingested, are computed by comparisons of their resonance areas with that of internal TSP, which in our studies is usually added at a final level of ca. 250 mol./L. Since the protein content of human saliva [130] is substantially lower than that of human blood plasma or serum [19, 132], the 'envelopes' generated by any broad macromolecule resonances do not significantly superimpose upon the great majority of sharper low-molecular-mass biomolecule signals. Therefore, any potential obscurement of these sharper resonances is predominantly negligible and has been found not to significantly interfere with their electronic integration and metabolite quantification [3], an observation confirmed in [31]. Of course, the concentrations of salivary biomolecules determined represent only the non-macromolecule-bound fraction of these and therefore are expected to be somewhat lower than their total salivary concentrations. Unfortunately, this certainly appears to be a little known or appreciated fact within the salivary NMR-based metabolomics community.