Determination of N-acetylation phenotype using caffeine as a metabolic probe and high-performance liquid chromatography with either ultraviolet detection or electrospray mass spectrometry

A rapid, sensitive method using liquid chromatography-electrospray mass spe ctrometry (LC-ES-MS) was developed and evaluated for the simultaneous quant itative determination of caffeine metabolites 1U, 1X and AAMU in human urin e. This method involved a simple dilution of urine samples. The chromatogra phic separation was achieved on a C-18 reversed-phase column using a gradie nt of acetonitrile in 2 mM, pH 3.0 ammonium formate as mobile phase. After ionisation in an electrospray source, mass spectrometric detection was perf ormed in the negative ion, selected ion monitoring mode. This method yielde d acceptable accuracy and precision within the range 0.25-50 mug/ml. This a nalytical method was applied to investigate the N-acetylator phenotype of H IV infected patients and compared with high-performance liquid chromatograp hy with UV detection. Its specificity was better, which appeared to be abso lutely necessary to prevent errors in metabolic ratios and phenotype interp retation. (C) 2001 Elsevier Science B.V. All rights reserved.