Improved HPLC Determination of Plasma and Urine Oxalate in the Clinical Diagnostic Laboratory

Abstract

An improved HPLC method for the measurement of oxalate as 2.3-dihydroxyquinoxaline derivative in plasma and urine has been developed. A reversed phase C18 column with isocratic elution and UV detection at 314 nm was used. Plasma samples immediately were deproteinized by acetonitrile with addition of phosphate buffer pH 7, to avoid the conversion of blood constituents to oxalate; acetonitrile, present in the supernatant, was dried. No pretreatment was necessary for urine. The derivati-zing procedure with 1.2-diaminobenzene was performed by heating at 140° C for 25 min. The detection limits at a signal/noise ≥ 3 ranged from 0.15 mg/L for plasma to 0.5mg/L for urine. The within run reproducibility (n=20) evaluated for urine at 12 mg/L) was 3.4% ± 0.3%; for plasma at 1.5 mg/L was 5.6 ± 0.5%; the between run reproducibility (n=10) was 5.0 ± 0.4% for urine and 7.5 ± 0.5% for plasma. The reference ranges (n=20) were: 10 to 30 mg/L for urine: 0.9 to 1.8 for plasma. Oxalate levels were measured in urine of exposed workers (n=200, mean 56.0 ± 5 mg/L) and in plasma of patients with renal damage (n=10, mean 4.5 ± 0.5 mg/L). The procedure is accurate, rapid and allows the analysis of samples collected at different times, without pre-analycal errors.