HPLC chromatograms of filtered human plasma alone and spiked with 20 µM NAD⁺ and NADH (A). Enzymatic amplification reaction for pure NADH, unheated and heated plasma samples (B). Intra-assay variances of the NAD enzymatic amplification reaction for NADH, unheated and heated plasma measured fourfold (C). Chromatograms taken at 260 and 340 nm are shown as overlay. Data are shown as mean ± SD. ^a calculated from the NADH standard at 1.25 µM for all assay plates performed (n = 15), ^b calculated from all samples analyzed (n = 205).