Nigel J.C. Bailey

Metabolomic analysis of the consequences of cadmium exposure in Silene cucubalus cell cultures via 1H NMR spectroscopy and chemometrics

Several essential and non-essential metals (typically those from periods 4, 5 and 6 in groups 11-15 in the periodic table) are commonly detoxified in higher plants by complexation with phytochelatin. The genetic and gross metabolic basis of metal tolerance in plants is, however, poorly understood. Here, we have analyzed plant cell extracts using 1H NMR spectroscopy combined with multivariate statistical analysis of the data to investigate the biochemical consequences of Cd(2+) exposure in Silene cucubalus cell cultures. Principal components analysis of 1H NMR spectra showed clear discrimination between control and Cd(2+) dosed groups, demonstrating the metabolic effects of Cd(2+) and thus allowing the identification of increases in malic acid and acetate, and decreases in glutamine and branched chain amino acids as consequences of Cd(2+) exposure. This work shows the value of NMR-based metabolomic approaches to the determination of biochemical effects of pollutants in naturally selected populations.

Mass spectrometrically detected directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry for the identification of xenobiotic metabolites in maize plants

Reconstructed ion chromatograms have been used to identify relevant high performance liquid chromatography (HPLC) peaks in a directly coupled high performance liquid chromatography/nuclear magnetic resonance spectroscopy/mass spectrometry (HPLC/NMR/MS) experiment. This has been applied to a study of the metabolism of a model compound, 5-nitropyridone (2-hydroxy-5-nitropyridine), in maize plants grown hydroponically. By monitoring the on-flow reconstructed ion chromatogram corresponding to the 5-nitropyridone fragment at m/z 143, and additional molecular ions corresponding to metabolites identified as products from similar compounds, relevant peaks were identified rapidly for subsequent stopped-flow 1H NMR spectroscopic analysis. The combination of coupled HPLC/NMR/MS enabled the direct identification of three metabolites, namely the N-glucoside, N-malonylglucoside, and O-malonylglucoside. This work demonstrates the power of HPLC/NMR/MS for the structural elucidation of xenobiotic metabolites in complex biological matrices (such as plant material) with minimal sample preparation. In particular, using mass spectrometry for the initial identification of relevant HPLC peaks allows the analysis of complex samples without the necessity for other spectroscopic markers, such as 19F NMR signal for fluorinated compounds or UV spectroscopy for molecules with strong UV chromophores.

Directly coupled CZE-NMR and CEC-NMR spectroscopy for metabolite analysis: paracetamol metabolites in human urine.

Direct coupling of NMR spectroscopic detection with both capillary zone electrophoresis (CZE) and capillary electrochromatography (CEC) was applied to the separation of metabolites of the drug paracetamol in an extract of human urine. Continuous-flow CZE-NMR and CEC-NMR allowed the detection of the major metabolites, the glucuronide and sulfate conjugates of the drug and the endogenous material hippurate. Identification of these substances was achieved by examination of individual rows of the NMR chromatogram and this also gave estimates of the detection limits. For CEC-NMR, spectra were also obtained in the stopped-flow mode including a two-dimensional TOCSY NMR experiment which afforded confirmatory evidence for paracetamol glucuronide. Characterisation of drug metabolites using NMR spectroscopy is therefore possible with nanolitre sample volumes.

On-flow identification of metabolites of paracetamol from human urine using directly coupled CZE–NMR and CEC–NMR spectroscopy

Direct NMR spectroscopic detection on-flow to capillary electrophoresis (CE) or capillary electrochromatography (CEC) was applied to the separation of metabolites of paracetamol from an extract of human urine. The detection and characterisation of the major metabolites, the glucuronide and sulfate conjugates of the drug as well as identification of the endogenous material hippurate was achieved. This demonstrates that NMR detection and identification of drug metabolites is possible with nanolitre volumes of analyte.

Chapter 10 – Biomedical applications of directly-coupled chromatography–nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS)

This chapter describes the biomedical applications of directly coupled chromatography–nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS). The progress in the coupling of high-performance liquid chromatography (HPLC) and related techniques with NMR spectroscopy over the past decade has been very rapid. HPLC remains one of the principal methods for the separation of chemical mixtures and quantitation of components. The numerous applications of the technique in biomedical analysis are evidence of its utility and the number of reports of the use of HPLC–NMR in this area has increased marking the transition of HPLC–NMR from a research technique to a routine analytic methodology. Further developments leading to the use of multiple hyphenation (hypernation) with HPLC–NMR–MS and higher concatenations have also begun to generate applications. With further technological advances in the areas of miniaturized flow probes for use with capillary separations and cryoprobes leading to lower sample requirements, the usefulness of NMR coupled to separations has increased.