HPLC Urinary Organic Acid Profiling: Role of the Ultraviolet and Amperometric Detectors

Abstract

The profiling of urinary organic acids is an important aspect of the diagnosis of inborn metabolic disorders. The carboxylic acids of interest may contain additional functional moities such as phenyl, hydroxyl, oxo, etc. The state-of-the-art method of organic acid analysis is by GC/MS. Prior to GC/MS analysis, the carboxylic acids must be isolated from urine by extraction or ion exchange chromatography and made volatile by derivatization. This is a lengthy procedure that does not lend itself to rapid analysis. We have developed a rapid procedure for the profiling of urinary α, B-unsaturated, aromatic and α-ketocarboxylic acids.

Urine containing an internal standard, 3-hydroxy-4-methoxy-benzyl alcohol, is filtered through a 0.3 um Millipore filter and injected on to an HPX-87 organic acid HPLC column (Bio-Rad). The mobile phase, 4.5 mN H₂SO₄, is passed through the column at 0.8 ml/min. Detection is effected by an UV detector at 200 nm in series but upstream from an electrochemical detector with a glassy carbon working electrode at +1.15V vs. an Ag/AgCl reference electrode. At this electrode potential, phenolic, methoxyphenyl, eneolic and α-ketocarboxylic acids are oxidized and can be electrochemically detected with a glassy carbon electrode.