Conventional NMR Spectroscopy

Solution state NMR spectroscopy is the study of molecules by recording the interaction of radiofrequency electromagnetic radiation with the nuclei of molecules in solution when placed in a strong magnetic field. The circulation of electrons around nuclei in different electronic environments creates local magnetic environments. These local magnetic fields give rise to characteristic chemical shifts suitable for functional group identification. Moreover, the presence of coupling between pairs of nuclei extending over 1-3 bonds establishes the connectivity between atoms and provides a powerful tool for structural elucidation including stereochemical information. However, the quantum basis of NMR, first described in 1946 by Bloch and Pur-cell (awarded a Nobel prize in 1952), is beyond the scope of this review. The interested reader is referred to several excellent textbooks on the principles of NMR [20, 21]. Here, the pivotal features that pertain to toxicology are presented in Table 8.1. Some milestones in NMR and the most important experiments routinely used for the assignment of metabolites in biofluid spectra are outlined in Table 8.2.

In brief, the goal is to elucidate the molecular structure by interpreting the relationships of NMR-active nuclei, typically protons (XH) or carbon (13C). Compared to protons, the relative sensitivity of carbon is only 0.16%, which imposes time restrictions on NMR experiments to determine carbon data. For drug discovery programmes where fluorine is present in the drug, the fluorine nucleus (with a high relative sensitivity of 83 % relative to the proton) provides exquisite selectivity for drug-related material in urine and plasma, due to the lack of endogenous fluorine present in mammalian biofluids. To measure an NMR spectrum the biofluid (commonly

Tab. 8.2 Some significant dates in the evolution of NMR and toxicology.


Associated development


The detection of proton NMR signals is reported independently by Bloch in

California and Purcell at Harvard.


Nobel Prize for Physics is jointly awarded to Bloch and Purcell.

Mid 1950s

13C NMR signals are first observed.


NMR is mostly the domain of organic synthetic chemists and NMR physicists.


Fourier transform (greater sensitivity) and 2D NMR (higher resolution) are



Conversion of glucose to lactate is observed in erythrocytes by Brown et al.


Nicholson and coworkers report urinalysis of normal and fasting diabetics by

proton NMR and continue to develop this field over the next 20 years.


Bales et al. report analysis of acetyl aminophen metabolites from urine, and the

first applications of NMR in toxicology start to appear in the literature.

Late 1980s

First reports of LC-NMR although, due to technical limitations, the technique

does not become routine until the mid 1990s.


Nobel Prize for Chemistry to Ernst for pioneering work in 2D NMR.


HRMAS is introduced for the analysis of tissues. Anthony et al. report NMR

pattern-recognition techniques that provide new insights into predictive models



Spraul and coworkers develop flow-injection NMR, which greatly facilitates

screening ofbiofluids.


In the post-genomic age, Nicholson, Lindon, and Holmes propose metabonomics.


The first cryoflow probe is announced and applied to the analysis of APAP

metabolites in urine (the gold standard for testing new hyphenated NMR

methods). Online solid-phase extraction provides for greatly enhanced sensitivity.

100-500 ^L of sample) is placed in a 5-mm glass tube and inserted into the bore of a superconducting magnet at field strengths ranging from 9.4 to 18.7 T. These are commonly referred to as 400-800-MHz NMR systems on the basis of the proton resonance frequency. The relaxation of nuclei following a train of RF pulses is then measured and translated into chemical shifts, coupling constants, and integration data. The resulting signals are presented typically as a 1-dimensional (1D) proton NMR spectrum (Figure 8.1). The term 1D refers to one dimension of frequency, as the y axis is the signal intensity. The spectrum is representative of a urine sample from a healthy volunteer; the most common urinary and plasma metabolites are catalogued elsewhere [17, 22]. The presence of a metabolite tends to be confirmed by spiking available standards into the biofluid sample. Thus, the main signals are often readily assigned. However, overlapping signals and multiplicity patterns tend to complicate assignments in 1D spectra. When abnormal and disease states differ

Fig. 8.1 A representative 500-MHz 1H NMR spectrum of normal human urine, showing some assigned resonances.

Abbreviations: Ala, alanine; Cit, citrate; Cn, creatinine; DMA, dimethylamine; Gly, glycine; Hip, hippurate; IS, internal standard (TSP); Lac, lactate; Phe, phenylalanine; TMAO, trimethylamine-N-oxide; Ur, urea; Val, valine.

Fig. 8.1 A representative 500-MHz 1H NMR spectrum of normal human urine, showing some assigned resonances.

Abbreviations: Ala, alanine; Cit, citrate; Cn, creatinine; DMA, dimethylamine; Gly, glycine; Hip, hippurate; IS, internal standard (TSP); Lac, lactate; Phe, phenylalanine; TMAO, trimethylamine-N-oxide; Ur, urea; Val, valine.

in metabolite composition, spiking of standards may aid assignment. However, it is often necessary to identify metabolites not encountered previously. In these instances 2D NMR experiments may enable deconvolution of latent information.

Throughout the 1960s and 1970s the introduction of Fourier transform to NMR and higher-dimensional NMR spectroscopy provided further incentives to use the technique for complex mixture analysis. The second Nobel Prize associated with the field of NMR was awarded to Richard Ernst in 1991 for his development of 2D NMR. The term refers to two dimensions of frequency, with the intensity of the NMR signals as the third dimension. Higher dimensionality can often simplify spectra and aid signal assignment. However, even with higher dimensionality, when signal overlap remains or concentrations of metabolites are below the detection limit (depending on the strength of magnet used, but at 800 MHz detectable concentrations tend to be >100 nM), the information in 2D NMR is often insufficient. Spectral editing can be used to simplify spectra; this comprises methods based on spin-echo, quantum coherence filters, and molecular differences in relaxation times and diffu sion rates [17]. However, when 2D and spectral-edited experiments fail to solve the structure of an analyte present in a complex mixture, a sample purification step is often required. This typically includes solvent extraction, solid-phase chromatographic extraction (SPE), or freeze-drying. Alternatively, a chromatographic step followed by off-line NMR is commonly used.

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