Chromatography with Spectrophotometric Detection

We describe a method for determining uric acid in serum by reversed-phase liquid chromatography with spectro- photometric detection at 280 nm. Serum, 100 ulis mixed with 100 u1of a solution containing, per liter, 70 ml of acetonitrile in sodium acetate (20 mmol/Iiter, pH 4.0) and 500 mg of the internal standard, adenine. The mixture is allowed to stand in an ice bath for 3 mm, then centrifuged. A 7.5-�tl portion of the supernate is chromatographed on a "�Bondpak C18" column, with a 35 mI/liter solution of acetonitrile in sodium acetate (10 mmol/liter, pH 4.0) as the mobile phase. For 10 runs of duplicates, the within-run CV was 1.2% and the day-to-day CV (10 days) was 2.5% for a uric acid concentration of 53 mg/liter. Sera from 100 patients were analyzed for uric acid by the proposed method, a continuous-flow (SMA 12/60) method, and a uricase method; mean values for the 100 sera were 64, 71, and 64 mg/liter, respectively. Correlations were as follows: r = 0.987 for proposed method vs. SMA 12/60 and r = 0.997 for proposed method vs. the uricase method. The proposed method is sensitive and specific and we think it will be useful for evaluating other i.ilc acid methods for interference and specificity. Additional Keyphrases: suggested reference method 1n termethod comparison The most common automated method for uric acid determination in use today depends on the ability of uric acid to reduce phosphotungstate to "tungsten blue," a procedure that can give falsely high results in the presence of certain drugs or of abnormal concen- trations of endogenous metabolites. The enzyme uricase (urate oxidase, EC 1.7.3.3) offers greater specificity. More recently, a specific procedure based on anion- exchange liquid chromatography and electrochemical detection has been developed (1, 2). We present in this paper a new, alternative, high-performance liquid- chromatographic procedure, involving a bonded alkyl stationary phase and spectrophotometric detection. The method was derived from a specific procedure for serum theophylline that is used in many laboratories (3). Our method has been extensively compared to an automated uricase procedure and we have used it to investigate further the specificity of the automated phosphotung-