## 8 Homo- and Heteronuclear Two-Dimensional (2D) NMR Analysis of Human Saliva
### Sample Size Considerations and MV Power Calculations
These prominent resonances consisted of those of acetate, propionate, glycine, methanol, and formate, although future optimizations of this technique at perhaps higher benchtop spectrometer operating frequencies (say 80, 90, or 100 MHz) may lead to the efficient and reliable analysis of perhaps 12 or more salivary biomolecules. We found at least reasonable consistencies and acceptable linear correlations between the salivary levels of acetate, propionate, glycine, and methanol estimated at 60 and 400 MHz operating frequencies (r = 0.97 to >0.99), but less so for formate (r = 0.93). Moreover, for propionate estimations, we also revealed some concentration limit restrictions in view of the overlap of its major \(\delta\) = 1.05 ppm -CH3 function triplet signal (\(J\) = 7.67 Hz [14]) with those of a series of more minor level biomolecules. Because of this, we suggest that, for HSS specimens with propionate concentrations \(\geq\)1.2 mmol./L, samples are diluted somewhat to bring this level below this limit in order to minimize such resonance overlap complications; otherwise, the 60 MHz facility may significantly overestimate this value. Estimated LLOD and LLOQ values for such LF NMR analyses were approximately 100 and 300 \(\upmu\)mol./L, respectively, for major metabolites with prominent -CH3 function singlet resonances, specifically acetate. As expected, these values were somewhat higher for the propionate-CH3 group triplet, and the 2-proton glycine and 1-proton formate singlet signals. Figure 13: (**a**,**b**) Experimental and predictively simulated 60 MHz 1H NMR profiles of a healthy human WMS supernatant sample; the predicted spectrum in (**b**) was computed from metabolite concentrations determined at an operating frequency of 400 MHz. Concentrations of salivary biomolecules featured were propionate (-CH3 resonance 11, 3.25 mmol./L); acetate (-CH3 resonance 30, 19.33 mmol./L); methanol (-CH3 resonance 63, 350 \(\upmu\)mol./L); glycine (-CH2 resonance 74, 750 \(\upmu\)mol./L); and formate (-CH resonance 116, 560 \(\upmu\)mol./L). The lactate-CH3 signal was excluded from this simulation in view of complications arising from overlap with other resonances (typical spectra are shown). Resonance labels correspond to those provided in Table 1 (TSP represents the 3-(trimethylsilyl))propionate-2,2,3,3-d4 chemical shift reference and quantitative internal standard). At this low operating frequency, further spin–spin splitting of propionate’s -CH3 and -CH2 group  
resonances (classically \(t\) and \(q\), respectively) is clearly apparent in both experimentally acquired and simulated 60 MHz spectra. Bruker _TopSpin NMR-SIM_ software was employed to generate simulated spectra. This _NMR-SIM_ program is based on the solution of the quantum mechanical Liouville equation, with the spin system developing during the radiofrequency pulse and FID acquisition process, which features rotating frame magnetization transfer and the execution of \(T_{1}\) and \(T_{2}\) effects. Full details of these simulations will be reported elsewhere. For the readily visible propionate resonances located in the 60 MHz \({}^{1}\)H NMR spectra, i.e., those ascribable to its coupled -CH\({}_{3}\) and -CH\({}_{2}\) groups (\(t\), \(\delta\) = 1.06 ppm, and \(q\), \(\delta\) = 2.18 ppm respectively), a consideration of their \(J\) value indicates that they will enclose 2 \(\times\) 7.67/60 and 3 \(\times\) 7.67/60 = 0.26 and 0.38 ppm, respectively, at 60 MHz (Figure 13), but only 0.026 and 0.038 ppm, respectively, in a corresponding 600 MHz spectrum (Figure 1). This illustrates the precautions that should be taken when attempting to quantify low-molecular-mass salivary metabolites or xenobiotics using this technique in view of inherent signal superimpositional limitations. However, after adopting suitable analytical calibration strategies, such problems may be overcome.