The use of on-line liquid chromatography/mass spectrometry and stable isotope techniques for the identification of budesonide metabolites

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Abstract
A moving belt interface was used to identify budesonide metabolites, obtained from rat and mouse liver incubations, by liquid chromatography/mass spectrometry (LC/MS). The metabolites were separated on a small-bore C18 column with an ethanol/water gradient as mobile phase at a flow rate of 0.2 ml min−1. A spray device was used for deposition of the aqueous solvent on to the belt. Chemical ionization mass spectra were obtained with methane as the reagent gas. Deuterium-labelled budesonide, which was used to facilitate metabolite identification by the isotope cluster technqiue, was found to be slightly separated from the unlabelled analogue on the LC column. Incubations were also performed under 18O2 to elucidate the mechanism of a new metabolic pathway (16α, 17α-acetal splitting) and to confirm the oxidative nature of reactions leading to hydroxylated metabolites. The moving belt LC/MS technique afforded higher sensitivity, and gave more abundant MH+ ions of the compounds studied, than previously found by direct probe mass spectrometry. Phthalate ester background, partly from the polymide belt, complicated the identification of minor metabolites.