Determination of oxalate in urine and plasma using reversed-phase ion-pair high-performance liquid chromatography with tris(2,2'-bipyridyl)ruthenium(II)-electrogenerated chemiluminescence detection.

Abstract

Oxalate is quantitated in both urine and plasma samples using reversed-phase ion-pair high-performance liquid chromatography (HPLC) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)2+(3)]-electrogenerated chemiluminescent (ECL) detection. Underivatized oxalate was separated on a reversed-phase column (Zorbax ODS) using a mobile phase of 10% methanol in 100 mM phosphate buffer at pH 7.0. The eluted compounds were combined with a stream of 2 mM Ru(bpy)2+(3) at a mixing tee before the ECL flow-cell. In the flow-cell, Ru(bpy)2+(3) is oxidized to Ru(bpy)3+(3) at a platinum electrode, and reacts with oxalate to produce chemiluminescence (CL). Urine samples were filtered and diluted prior to injection. Plasma samples were deproteinized before injection. A 25-microliters aliquot of sample was injected for analysis. Possible interferants, including amino acids and indole-based compounds, present in biological samples were investigated. Without the separation, amino acids interfere by increasing the total observed CL intensity; this is expected because they give rise to CL emission on their own in reaction with Ru(bpy)3+(3). Indole compounds exhibit a unique interference by decreasing the CL signal when present with oxalate. Indoles inhibit their own CL emission at high concentrations. By use of the indicated HPLC separation, oxalate was adequately separated from both types of interferants, which thus had no effect on the oxalate signal. Urine samples were assayed by both HPLC and enzymatic tests, the two techniques giving similar results, differing only by 1%. Detection limits were determined to be below 1 microM (1 nmol/ml) or 25 pmol injected. The working curve for oxalate was linear throughout the entire clinical range in both urine and plasma.