The Application of Weak-Anion Exchange Chromatography for the Analysis of Organic Zwitterions Using LC/MS/MS

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r 2 ≥ 0.995), accuracy (85– 115%), precision (C.V. < 12%), sample preparation stability (≤ 5%, 72h), and established patient ranges. The method was found to be both efficient and accurate for the analysis of urinary zwitterionic organic acids.