Determination of nitrate by the IE-HPLC-UV method in the brain tissues of Wistar rats poisoned with paraquat

This work was a part of an initial study regarding the involvement of reactive nitrogen species (RNS) in paraquat (PQ) neurotoxicity. The nitrate concentration in the vulnerable regions of the brain (cortex, striatum and hippocampus) of Wistar rats was used as a measure of nitric oxide (NO) production or catabolism of the formed RNS. The tissue homogenates were deproteinized with acetonitrile and then centrifuged. Nitrate was measured in filtrated supernatants by simple and rapid isocratic ion-exchange high performance liquid chromatography with UV detection (IE-HPLC-UV) at 214 nm. The mobile phase (pH 8.5) consisted of borate buffer/gluconate concentrate, methanol, acetonitrile and deionized water (2:12:12:74, v/v/v/v), and the flow rate was 1.3 mL/min. Physiological nitrate levels (18.8 +/- 6.1 nmol/mg of proteins), as well as a diverse range of nitrate concentrations could be determined with good precision (CV= 2.2%) and accuracy (recovery of spiked samples was 99 +/- 4 %) in the brain tissue homogenates. Linearity was achieved in the range of nitrate from 0-80 mu M. The retention time of nitrate anion was 5.3 +/- 0.3 min.