EC-SPE-stripline-NMR analysis of reactive products: a feasibility study.

Flow-through electrochemical conversion (EC) of drug-like molecules was hyphenated to miniaturized nuclear magnetic resonance spectroscopy (NMR) via on-line solid-phase extraction (SPE). After EC of the prominent p38α mitogen-activated protein kinase inhibitor BIRB796 into its reactive products, the SPE step provided preconcentration of the EC products and solvent exchange for NMR analysis. The acquisition of NMR spectra of the mass-limited samples was achieved in a stripline probe with a detection volume of 150 nL offering superior mass sensitivity. This hyphenated EC-SPE-stripline-NMR setup enabled the detection of the reactive products using only minute amounts of substrate. Furthermore, the integration of conversion and detection into one flow setup counteracts incorrect assessments caused by the degradation of reactive products. However, apparent interferences of the NMR magnetic field with the EC, leading to a low product yield, so far demanded relatively long signal averaging. A critical assessment of what is and what is not (yet) possible with this approach is presented, for example in terms of structure elucidation and the estimation of concentrations. Additionally, promising routes for further improvement of EC-SPE-stripline-NMR are discussed.

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays, high resolution mass spectrometry and off-line NMR.

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)alpha and hERbeta integrating target-ligand interactions and liquid chromatography-high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-(1)H, 2D-COSY, 2D-NOESY, and (1)H-(13)C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.

A microfluidic high-resolution NMR flow probe.

A microfluidic high-resolution NMR flow probe based on a novel stripline detector chip is demonstrated. This tool is invaluable for the in situ monitoring of reactions performed in microreactors. As an example, the acetylation of benzyl alcohol with acetyl chloride was monitored. Because of the uncompromised (sub-Hz) resolution, this probe holds great promise for metabolomics studies, as shown by an analysis of 600 nL of human cerebrospinal fluid.