## 7 NMR Pulse Sequences: Recent Advances and Future Prospects
### 'State-of-the Art' Advances beyond the CPMG Pulse Sequence  
Generally, 2D filters such as the CPMG pulse sequence is combined with a water/HOD presaturation one, and this combination, known as the presat-CPMG pulse sequence, has now been employed in biofluid NMR analysis for a considerable period of time, and remains a stalwart of NMR-based metabolomics techniques. Although this composite pulse sequence has a logical historical basis, it often does not achieve perfect water suppression, requiring a high radiofrequency power, and consequently it endures a diminishing level of performance with enhancements in magnetic field strength. However, in view of these problems experienced, a new combinatorial pulse sequence known as 'WASTED' (**WA**ter **S**uppression with a **T**ransverse relaxation filter that Eliminates Distortions) has been developed [127], and this represents a composite of the CPMG-superior PROJECT sequence with the Robust-5 water presaturation one. The PROJECT sequence is particularly valuable, since it has the ability to recover resonances suppressed by that which vitiates the intense water signal, notably, but not exclusively, those localized close to its presaturation frequency. Moreover, PROJECT's performance is predominantly independent of magnetic field strength. Hence, WASTED pulse sequences circumvent some of the hurdles associated with the presat-CPMG strategy, and hence offers benefits regarding the multicomponent analysis of biofluids, for which the suppression of the ca. 50 mol./L water signal and a broad'spectrum' of biomacromolecules simultaneously during spectral acquisition is an essential requirement. Therefore, the carefully planned application of WASTED pulse sequences, including the sequential order of application of the PROJECT and Robust-5 components, can successfully attenuate water resonance intensities from ca. 50 mol./L down to as little as 0.90 mmol./L [127]. Additionally, the T\({}_{2}\) filters of WASTED permits inter-pulse delays (\(\gamma\)) that are an order of magnitude greater than those countenanced by the CPMG sequence, a process yielding a lower power input for successful operation. In view of these lowered power depositions, pulse sequences such as WASTED provide opportunities for the employment of T\({}_{2}\) encoding methods to distinguish between differential metabolite 'pools' or the structures of macromolecules. An example of our laboratories' use of the WASTED pulse sequence is available in the pilot study spectra shown in Figure 2.  
Case Study 1: Investigation of the Influences of Sample Donor, Metabolite Identity, and Doublet Resonance Line & Value on the \(\Delta\
u_{1/2}\) Values of Lactate and Alanine in Single-Pulse \({}^{1}\)H NMR Spectra Acquired on WMS Supernatants--A Design of Experiments Approach  
From our single-pulse _metnoses_ 1D \({}^{1}\)H NMR spectra acquired on WMS supernatants (Figure 4a-c), we found that the mean \(\pm\) SEM \(\Delta\
u_{1/2}\) value for the lactate-CH\({}_{3}\) signal was 1.67 \(\pm\) 0.03 Hz (range 1.33-2.64 Hz; n = 7 samples, each with 2 \(\times\)\(\Delta\
u_{1/2}\) values per doublet resonance), although these values appeared to be both sample donor- and metabolite-dependent. Indeed, that for alanine was significantly higher (mean \(\pm\) SEM 1.98 \(\pm\) 0.03 Hz, range 1.63-2.65 Hz). In order to explore this further, we elected to perform an analysis-of-variance (ANOVA)-based experimental design model incorporating the 'between-participants (samples)', 'between-metabolites' (i.e., lactate vs. alanine), and 'between-lines-nested-within-resonances' fixed sources of variation, and also a metabolite x participant first-order interaction effect. This UV analysis revealed that the 'between-participants' and 'between-metabolites' effects were found to be statistically significant (\(p<10^{-6}\) and \(<\)10\({}^{-5}\) respectively), whereas that 'between-lines-nested-within-resonances' was not.