Urinary neopterin quantification by reverse-phase high-performance liquid chromatography with ultraviolet detection

• Published in: Journal of biochemical and biophysical methods Vol. 59; no. 3; pp. 275 - 283
• Main Authors: de Castro, Marcel Ribeiro, Di Marco, Giovana Seno, Arita, Danielle Yuri, Teixeira, Luciana Cristina, Pereira, Aparecido Bernardo, Casarini, Dulce Elena
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 30.06.2004
• Subjects: assay; Biomarkers - urine; Chromatography, High Pressure Liquid - methods; disease-activity; HPLC; Humans; Lupus Erythematosus, Systemic - diagnosis; Lupus Erythematosus, Systemic - urine; markers; Neopterin; Neopterin - urine; rejection; Reproducibility of Results; Reverse phase; Sensitivity and Specificity; serum neopterin; Spectrophotometry, Ultraviolet - methods; Systemic lupus erythematosus; Urinalysis - methods; Urine; UV detection; Urine; UV detection; Reverse phase; Systemic lupus erythematosus; Neopterin; HPLC
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/j.jbbm.2004.03.004

Neopterin plays an important role in the malignant disease diagnostics. However, the methods employed in neopterin determination are generally difficult and/or time consuming. The aim of this work was to standardize a practical method to quantify neopterin using high-performance liquid chromatography-ultraviolet (HPLC-UV) and quantify it in patients with systemic lupus erythematosus (SLE). Urine was collected from healthy subjects ( n=49), patients with inactive ( n=15), active ( n=28), and highly active SLE ( n=6). The HPLC was performed using two coupled reverse-phase columns eluted with 150 mM sodium phosphate, pH 4.0, under a flow rate of 0.8 ml/min, with UV detector set at 353 nm and 100-fold diluted urines. The inter- and intra-assay studies presented an imprecision of 12.5% and 12.9% for quality controls of 3.94 and 1.1 μmol/ml, respectively. Recovery from 79.5% to 82% was observed throughout the assay's linear range. Subjects with active (874.2±165.38 μmol/mol creatinin) and highly active SLE (1753.8±453.9 μmol/mol creatinin) showed three- and sixfold increased neopterin levels, respectively, compared to subjects with inactive SLE (314.3±121.3 μmol/mol creatinin) and healthy subjects (294.6±178.6 μmol/mol creatinin) ( P<0.05). Briefly, the proposed method was precise, specific, and reproducible, not invasive and allows the urinary neopterin quantification only with UV detection.