Simultaneous pentafluorobenzyl derivatization and GC-ECNICI-MS measurement of nitrite and malondialdehyde in human urine: Close positive correlation between these disparate oxidative stress biomarkers

- Nitrite, nitrate, creatinine and malondialdehyde were derivatized simultaneously.
- Nitrite, nitrate, creatinine and MDA were analyzed simultaneously by GC-ECNICI-MS.
- [15N]Nitrite is useful as a surrogate internal standard for MDA in human urine.
- Nitrite and MDA were found to correlate in urine of elderly healthy subjects.
- A myeloperoxidase-involving mechanism was proposed explaining the nitrite-MDA correlation.

Urinary nitrite and malondialdehyde (MDA) are biomarkers of nitrosative and oxidative stress, respectively. At physiological pH values of urine and plasma, nitrite and MDA exist almost entirely in their dissociated forms, i.e., as ONO− (ONOH, pKa = 3.4) and −CH(CHO)2 (CH2(CHO)2, pKa = 4.5). Previously, we reported that nitrite and MDA react with pentafluorobenzyl (PFB) bromide (PFB-Br) in aqueous acetone. Here, we report on the simultaneous derivatization of nitrite and MDA and their stable-isotope labeled analogs O15NO− (4 μM) and CH2(CDO)2 (1 μM or 10 μM) with PFB-Br (10 μL) to PFBNO2, PFB15NO2, C(PFB)2(CHO)2), C(PFB)2(CDO)2 by heating acetonic urine (urine-acetone, 100:400 μL) for 60 min at 50 °C. After acetone evaporation under a stream of nitrogen, derivatives were extracted with ethyl acetate (1 mL). A 1-μL aliquot of the ethyl acetate phase dried over anhydrous Na2SO4 was injected in the splitless mode for simultaneous GC-MS analysis in the electron capture negative-ion chemical ionization mode. Quantification was performed by selected-ion monitoring (SIM) the anions [M-PFB]−m/z 46 for ONO−, m/z 47 for O15NO−, m/z 251 for −C(PFB)(CHO)2, and m/z 253 for −C(PFB)(CDO)2. The retention times were 3.18 min for PFB-ONO2/PFB-O15NO2, and 7.13 min for −C(PFB)(CHO)2/−C(PFB)(CDO)2. Use of CH2(CDO)2 at 1 μM but not at 10 μM was associated with an unknown interference with the C(PFB)2(CDO)2 peak. Endogenous MDA can be quantified using O15NO− (4 μM) and CH2(CDO)2 (10 μM) as the internal standards. The method is also useful for the measurement of nitrate and creatinine in addition to nitrite and MDA. Nitrite and MDA were measured by this method in urine of elderly healthy subjects (10 females, 9 males; age, 60-70 years; BMI, 25-30 kg/m2). Creatinine-corrected excretion rates did not differ between males and females for MDA (62.6 [24-137] vs 80.2 [52-118] nmol/mmol, P = 0.448) and for nitrite (102 [71-174] vs. 278 [110-721] nmol/mmol P = 0.053). We report for the first time a close correlation (r = 0.819, P < 0.0001) between MDA and nitrite in human urine. This correlation is assumed to be due to involvement of myeloperoxidase which catalyzes the formation of hypochlorite (−OCl) from chloride and hydrogen peroxide. In turn, hypochlorite reacts both with nitrite and with polyunsaturated fatty acids such as arachidonic acid, with the later reaction generating MDA. The proposed mechanisms are supported by the literature but remain to be fully explored.