Purine Metabolites in Gout and Asymptomatic Hyperuricemia: Analysis by HPLC–Electrospray Tandem Mass Spectrometry

Abstract

Hyperuricemia, a serum urate concentration >0.45 mmol/L (7.0 mg/dL) in men and 0.36 mmol/L (6.0 mg/dL) in women, is the biochemical hallmark of gout (1)(2)(3), but many individuals with life-long hyperuricemia do not develop gouty arthritis (4). Conversely, serum urate concentrations may be within reference values in some patients with acute gout, particularly during the early phases of the disorder. We hypothesized that gout patients may have a different profile of purine precursors than do asymptomatic people with hyperuricemia and that concentrations of these compounds may reflect disorders of purine metabolism. To our knowledge, purine metabolites have not been studied and compared in gout and asymptomatic hyperuricemia. With established methods for measuring purine compounds in blood and urine (5)(6)(7)(8)(9), retention time is not always adequate for identification of every peak because urine and blood usually contain many interfering compounds. Quantification by tandem mass spectrometry (MS/MS) (10)(11) may require an internal standard, which often is hard to obtain. In this study of 10 purine compounds in the blood of gout patients and hyperuricemia patients with no gout symptoms, we used HPLC for serum sample separation, MS/MS for peak identification, and ultraviolet (UV) detection for quantification. Purines were purchased from Fluka and Sigma. The HPLC-MS experiment was performed on an Agilent 1100 series liquid chromatograph with a mass spectrometric detector trap. Separation was carried out on a Supelcosil™ LC-18-DB column [25 cm × 4.6 mm (i.d.); 5-μm film thickness]. The column temperature was maintained at 25 °C. The mobile phases were as follows: 10 mmol/L ammonium acetate, adjusted to pH 6.5 with glacial acetic acid (eluant A), and methanol (eluant B). The elution gradient was as follows (flow rate, 1 mL/min): 0 to 10 …