Simultaneous GC-ECNICI-MS measurement of nitrite, nitrate and creatinine in human urine and plasma in clinical settings

Abstract

Creatinine in urine is a useful biochemical parameter to correct the urinary excretion rate of endogenous and exogenous substances. Nitrite (ONO^-) and nitrate (ONO₂^-) are metabolites of nitric oxide (NO), a signalling molecule with multiple biological functions. Under certain and standardized conditions, the concentration of nitrate in the urine is a suitable measure of whole body NO synthesis. The urinary nitrate-to-nitrite molar ratio (U_NOxR) may indicate nitrite-dependent renal carbonic anhydrase (CA) activity. In clinical studies, urine is commonly collected by spontaneous micturition. In those cases the nitrate and nitrite excretion must be corrected for creatinine excretion. Pentafluorobenzyl (PFB) bromide (PFB-Br) is a useful derivatization reagent of numerous inorganic and organic compounds, including urinary nitrite, nitrate and creatinine, for highly sensitive and specific quantitation by GC-MS. Here, we report on the simultaneous PFB-Br derivatization (60min, 50°C) of ONO^-, O¹⁵NO^-, ONO₂^-, O¹⁵NO₂^-, creatinine (d_o-Crea) and [methylo-²H₃]creatinine (d₃-Crea) in acetonic dilutions of native human urine and plasma samples (4:1, v/v) and their simultaneous quantification by GC-MS as PFBNO₂, PFB¹⁵NO₂, PFBONO₂, PFBO¹⁵NO₂, d_o-Crea-PFB and d₃-Crea-PFB, respectively. Electron capture negative-ion chemical ionization (ECNICI) of these derivatives generates anions due to [M-PFB]^-, i.e., the starting analytes. Quantification is performed by selected-ion monitoring (SIM) of m/z 46 (ONO^-), m/z 47 (O¹⁵NO^-), m/z 62 (ONO₂^-), m/z 63 (O¹⁵NO₂^-), m/z 112 (d_o-Crea), and m/z 115 (d₃-Crea). Retention times were 2.97min for PFB-ONO₂/PFB-O¹⁵NO₂, 3.1min for PFB-NO₂/PFB-¹⁵NO₂, and 6.7min for d_o-Crea-PFB/d₃-Crea-PFB. We used this method to investigate the effects of long-term oral NaNO₃ or NaCl (serving as placebo) supplementation (each 0.1mmol/kg body weight per day for 3 weeks) on creatinine excretion and U_NOxR in 17 healthy young men. Compared to NaCl (n=8), NaNO₃ (n=9) supplementation increased U_NOxR (1709±355 vs. 369±77, P<0.05). Creatinine excretion did not differ between the groups (6.67±1.34mM vs. 5.72±1.27mM, P=0.57). The method is also applicable to human plasma. In 78 adults patients newly diagnosed for cerebrovascular disease (CVD), there was a close correlation (r=0.9833) between the creatinine concentrations measured in plasma by GC-ECNICI-MS and those measured in serum by an enzymatic assay. Creatinine-corrected plasma nitrate and nitrite concentrations (P=0.035 and P=0.004, respectively) but not their concentrations (P=0.68 and P=0.40, respectively) differ between male (n=54) and female (n=24) CVD patients. No such differences were found between preterm newborn boys (n=25) and girls (n=22). Like in urine, circulating creatinine may be useful to correct for gender-specific differences in plasma nitrite and nitrate in adults. Chronic NaNO₃ supplementation to healthy young men does not affect renal CA-dependent nitrite excretion or creatinine synthesis and excretion.

Keywords: Clinical studies; Creatinine; Nitric oxide; Quantification; Renal carbonic anhydrase; Supplementation.