Measurement of Nitrite in Urine by Gas Chromatography-Mass Spectrometry

Nitric oxide (NO) is enzymatically produced from l -arginine and has a variety of biological functions. Autoxidation of NO in aqueous media yields nitrite (O = N–O⁻ ). NO and nitrite are oxidized in erythrocytes by oxyhemoglobin to nitrate (NO ₃ ⁻ ). Nitrate reductases from bacteria reduce nitrate to nitrite. Nitrite and nitrate are ubiquitous in nature, they are present throughout the body and they are excreted in the urine. Nitrite in urine has been used for several decades as an indicator and measure of bacteriuria. Since the identification of nitrite as a metabolite of NO, circulating nitrite is also used as an indicator of NO synthesis and is considered an NO storage form. In contrast to plasma nitrite, the significance of nitrite in the urine beyond bacteriuria is poorly investigated and understood. This chapter describes a gas chromatography-mass spectrometry (GC-MS) protocol for the quantitative determination of nitrite in urine of humans. Although the method is useful for detection and quantification of bacteriuria, the procedures described herein are optimum for urinary nitrite in conditions other than urinary tract infection. The method uses [¹⁵ N]nitrite as internal standard and pentafluorobenzyl bromide as the derivatization agent. Derivatization is �performed on 100-μL aliquots and quantification of toluene extracts by selected-ion monitoring of m/z 46 for urinary nitrite and m/z 47 for the internal standard in the electron-capture negative-ion chemical ionization mode.