Liquid-chromatographic separation of urinary 5-hydroxy-3-indoleacetic acid, with measurement at 254 nm.
A "high-performance" liquid-chromatographic procedure for 5-hydroxy-3-indoleacetic acid is described and compared with a colorimetric method in which 1-nitroso-2-naphthol is used. The analyte and an internal standard, p-nitrobenzoic acid, were extracted into diethyl ether from urine at pH 4.0 (acidified with HCl) to which sodium chloride had been added, and the ether was back-extracted with acetate buffer, pH 9.2. Aliquots of this extract were injected into a reversed-phase liquid-chromatographic column and eluted with pH 3.5 acetate buffer/methanol (95/5 by vol); the effluent was monitored at 254 nm. The precision (CV) of the method was 11.8% at 1.8 mg/L, 5.5% at 92 mg/L. Analytical recovery averaged 84%. The colorimetric method gave higher values for the analyte than did the chromatographic method for all patients' urines.
D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003124:Colorimetry / Q000379:methods; D006801:Humans; D006897:Hydroxyindoleacetic Acid / Q000652:urine*; D007202:Indicators and Reagents; D009284:Naphthols; D009603:Nitroso Compounds; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D007202:Indicators and Reagents; D009284:Naphthols; D009603:Nitroso Compounds; D006897:Hydroxyindoleacetic Acid; C025341:1-nitroso-2-naphthol