A NEW PRE-COLUMN DERIVATIZATION METHOD FOR DETERMINATION OF NITRITE AND NITRATE IN HUMAN PLASMA BY HPLC
Nitrite and nitrate are metabolites of nitric oxide (NO) which its fundamental role in biological systems is emphasized. Therefore, accurate measurement of these ions is profoundly important. Here, a simple, accurate, and sensitive method based on reversed phase liquid chromatography is developed. This HPLC technique includes pre-column derivatization with Griess reagents and detection at 540 nm. Sample preparation is easy and involves liquid-liquid back extraction which purifies the analyte and improves sensitivity. Lower limit of quantification for nitrite was found to be 66.7 nM which provides enough sensitivity to determine nitrite in many biological samples. Furthermore, reduction of nitrate with vanadium chloride (III) was optimized. An internal standard was used to enhance method's repeatability, making the correlation variations of intra and inter-day assays below 8.77% with less than 17.00% error at all concentrations. The method was linear in the range of 0.1-50 mu M for sodium nitrite and 1-500 mu M for sodium nitrate. This technique was successfully applied to assay plasma nitrite/nitrate levels in patients with chronic kidney disease and healthy volunteers.