The utility of one-dimensional homonuclear Hartmann—Hahn spectroscopy (1D HOHAHA) for identifying the suprofen glucuronides fraction from an HPLC separation

Abstract

Many drug substances that contain carboxylic acid moieties are eliminated from the body via an enzymatic pathway that results in their conjugation with glucuronic acid. Characterization of these acyl glucuronides is complicated because of the ease of acyl migration and hydrolysis. Acyl rearrangements have been well studied and this area has been reviewed by Faed [l]. Liquid chromatography (LC), mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the standard analytical methods used for studying glucuronides [2-61. NMR studies are very well suited for identification of positional isomers of acyl glucuronides because of the large chemical shift changes induced by acylation [7]. Additionally, differentiation of the (Yand the P-anomers generally can be obtained from measurement of the proton-proton coupling constant of the anomeric proton [7]. What is needed is a NMR method that aids in the identification of the positional isomers of glucuronides within a mixture. Analysis of this type reduces the chance of hydrolysis or acyl migration as additional purification and separation steps are not required. Homonuclear Hartmann-Hahn (HOHAHA) NMR spectroscopy is well suited for the analysis of mixtures. The technique was first demonstrated by Davis and Bax [8] to assign the proton resonances from the ribose rings in a trinucleotide. They clearly showed the power of the method as a means for spectral editing and thus simplifying complicated spectra. It uses a selective 180 pulse as a means of accessing the spin network followed by a mixing period to label the protons within the coupling network. Onedimensional HOHAHA requires only one resolved proton resonance to be able to identify all the resonances within a sugar ring. For simple sugars this requirement is generally met using the anomeric proton. If needed the sequential assignment of protons can be had by varying the experimental mixing period (spin lock time) because the relay of magnetization occurs sequentially between coupled protons at a rate dependent on their coupling constant [S]. One-dimensional HOHAHA and in particular its 2D analogue [9, lo] has found wide application for structural studies of small proteins, nucleic acids and polysaccharides. For metabolite studies the 2D version may not be practical because of small sample size. Herein is demonstrated the utility of 1D HOHAHA spectroscopy for the identification of acyl glucuronides within a mixture. The glucuronide fraction isolated from an LC column separating the components of human urine from four healthy male subjects given suprofen, (+)-ol-methyl-4-(2-thienyl-