Analytic performance characteristics of the chemical-reduction gas phase-chemiluminescence assay for the quantification of nitric oxide metabolites.

Nitric oxide metabolites (NOm) in biological fluids have been widely used as surrogate markers to inform changes in nitric oxide-mediated signaling, which is critical for many biological functions. One method for quantifying NOm employs chemical reduction to convert the various metabolites into nitric oxide, followed by ozone-based chemiluminescence detection. Herein, we tested and quantified the bioanalytical parameters of the reduction-chemiluminescence assay and propose conditions for optimizing the assay. The slopes of the standard curves used for quantifying the metabolites showed a 3.8 % intra-assay and 7.6 % inter-assay relative standard deviation (%RSD). An 8 % and 8.5 % RSD was measured for two separate quality control serum samples, and a Levey-Jennings plot showed that all individual values fell within 1 standard deviation of the mean of all measurements. Repeat analysis of human serum samples (n = 51) resulted in an average incurred sample reanalysis of 7.7 %, with 49 samples having repeated results above the original value, indicating no loss of NOm over the four-week period. This was further confirmed by recovery experiments, which indicated average recovery rates of 100.4 ± 8.2 % and 99.3 ± 4.3 % for two operators, respectively. ANOVA of data from two operators indicated that sample-to-sample variability was the main contributor to the total variance. Overall, our results demonstrate that the assay provides appropriate and reproducible quantification of NOm, supporting its use for both exploratory and routine laboratory applications.