Hyphenation & High-Throughput Analysis
The ability to obtain complementary spectroscopic information simultaneously from different analytical platforms is of considerable benefit when attempting to charaterise unknown metabolites.
Substantial investment in techologies to permit efficiet hyphenation of NMR, MS and other analytical techniques has been made over many years, and Biomolecular Medicine has an array of integrated analytical tools to tackle the challenges of characterising complex biofluids and tissues.
Recently, through scientific collaboration with a major NMR instrument manufacturer, we have pioneered the application of directly coupled LC-NMR to the direct structure elucidation of drug metabolites in biofluids and other complex mixtures. LC-NMR represents a considerable technical advance brought about by the direct interfacing of a conventional LC system with a specially developed NMR flow probe (which are now available in multinuclear, inverse detection geometry and gradient configurations).
The whole LC-NMR system is under direct spectrometer control and can be run in continuous flow and stop-flow modes (the former for initial drug metabolite detection and partial structure elucidation, the latter for detailed structural analysis). Overall the NMR/LC-NMR approach to the study of the drug metabolism and pharmacokinetics is very efficient and permits the investigation of structure-metabolism relationships without many of the time and resource limitations of conventional chromatographic studies.
We have pioneered the application of NMR methods to the systematic investigation of structure-metabolism relationships of congeneric compounds and have shown that 1H and 19F NMR together with LC-NMR spectroscopy provides an efficient approach for driving a structure-metabolism research programme.
Further work in hand concerns the structure reactivity relationships of drug glucuronides. We are currently developing (with Bruker GmbH) triple hyphenated LC-NMR-MSn systems and CEC-NMR-MS systems for metabolite structure elucidation in complex mixtures using ion trap multipole MS.
High-resolution chromatographic separations afforded by UPLC systems (Waters) have greatly improved the number of individual species that can be resolved and characterised using spectrometry. Biomolecular Medicine have considerable experience with such instrumentation and recently published extensive protocols for biofluid analysis using hyphenated UPLC-MS platforms (Want et al. (2010)).
Robotic preparation instrumentation can greatly improve sample throughput and have principally been used by researchers in Biomolecular Medicine for consistent preparation of biofluids in studies were large numbers of toxicology samples were generated.
This technology promises to revolutionise analytical procedures for the structure elucidation of unknowns in the pharmaceutical/chemical industry over the next few years, and opens up metabonomics analyses for large sample sets such as those often encountered in molecular epidemiology studies.
Key Recent Publications
Want EJ, Wilson ID, Gika H, Theodoridis G, Plumb RS, Shockcor J, Holmes E, Nicholson JK. 2010. Global metabolic profiling procedures for urine using UPLC-MS. Nat Protoc 5(6):1005-18.
Pearce JT, Athersuch TJ, Ebbels TM, Lindon JC, Nicholson JK, Keun HC. 2008. Robust algorithms for automated chemical shift calibration of 1D 1H NMR spectra of blood serum. Anal Chem 80(18):7158-62.
Lauridsen M, Maher AD, Keun H, Lindon JC, Nicholson JK, Nyberg NT, Hansen SH, Cornett C, Jaroszewski JW. 2008. Application of the FLIPSY pulse sequence for increased sensitivity in 1H NMR-based metabolic profiling studies. Anal Chem 80(9):3365-71.
