Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high-performance anion-exchange liquid chromatography for their determination

Measurements of nitrite (NO2−) and nitrate (NO3−) in biological fluids are proposed as indices of cellular nitric oxide (NO) production. Determination of NO2− and NO3− in standard solutions is not difficult, however, determinations which reflect accurately cellular NO synthesis represent a considerable analytical challenge. Problems are often encountered arising from background NO2−/NO3− contamination in experimental solutions and laboratory hardware, and with methods for sample extraction. We investigated potential procedures for the extraction and determination of NO2− and NO3− in biological samples. Consequently, a protocol was devised which yielded acceptable results regarding extraction efficiency, assay reproducibility, sample throughput and contaminant minimisation. It entailed rigorous washing of all equipment with water of low NO2− and NO3− content, sample deproteinisation by centrifugal ultrafiltration through a 3K filter and analysis by high-performance anion-exchange liquid chromatography with UV detection. Retention times for NO2− and NO3− in standards and plasma were 4.4 and 5.6 min, respectively. Assay linearity for standards ranged between 31 nM and 1 mM. The limit of detection for NO2− and NO3− in standards was 3 pmol. Recoveries of NO2− and NO3− from spiked plasma (1–100 μM KNO2/KNO3) and from extracted standards (1–250 μM) were approximately 100%. Intra-assay and inter-assay RSDs for NO2− and NO3− in spiked and unspiked plasma were 10.6% or less. Assays on washed platelet supernatants demonstrated collagen-induced platelet generation of NO products and analysis of murine and rat cardiac perfusates was achieved. Our procedure may be suitable for routine determination of NO2− and NO3− in various biological fluids, e.g., plasma.