Mass spectrometry-validated HPLC method for urinary nitrate.

Abstract

The l-arginine/NO biosynthetic pathway is involved in many physiologic and pathologic processes, including vasodilation and inhibition of platelet aggregation and adhesion. Nitrate (NO3−) and nitrite (NO2−) are the most abundant circulating and excretory NO metabolites in humans. In humans on a standardized low nitrate/nitrite diet, circulating nitrite reflects constitutive NO synthase activity (1), whereas measurement of nitrate in urine is a reliable noninvasive method to assess whole-body NO synthesis in humans (2). Current analytical methods for the measurement of nitrate and nitrite include spectrophotometry, chemiluminescence, capillary electrophoresis, assays based on the Griess reaction, HPLC, and gas chromatography-mass spectrometry (GC-MS) (3). Among these assays, GC-MS methods form the basis for reference methods (4). Numerous HPLC methods using ultraviolet, electrochemical, or fluorescence detection have been reported for the analysis of nitrate and nitrite (3). The majority of them, however, have not been validated against MS, and they are mostly limited to human plasma (5) or rat urine (6). Recently, a commercially available HPLC-Griess system has been used for the analysis of urinary nitrite/nitrate in humans (7), but it has not been externally validated, and analysis of nitrate requires its reduction to nitrite. Here I report on a simple, rapid, GC-MS-validated anion-pairing HPLC method with ultraviolet absorbance detection at 205 nm for the accurate measurement of nitrate in human urine without sample pretreatment other than dilution of samples with the mobile phase. This HPLC method is not suitable to measure nitrate in human plasma. HPLC analyses were performed with a Pharmacia LKB pump Model 2248 and an analytical column [250 × 4 mm (i.d.)] packed with Nucleosil 100–5C18 AB (Macherey-Nagel). The mobile phase consisted of water-acetonitrile (90:10 by volume) contained 10 mmol/L tetrabutylammoniumhydrogen sulfate (Merck), which serves as the anion-pairing agent. The pH of the mobile …