Why is the sum of my NAD and NADH extraction readings greater than my Total NAD/NADH reading?

(I’m working with Amplite® Fluorimetric NAD/NADH Ratio Assay Kit *Red Fluorescence*) Why does my Total NAD/NADH reading not match the total of my individual NAD/NADH readings? 

The most likely cause for this situation is an inaccurate NADH extract reading. Depending on the initial NAD concentration, the NADH extraction solution may not be able to eliminate all the NAD from the sample well, meaning that some NAD will remain and conflate readings. Given that the detection probe is highly sensitive, even small residual NAD may influence fluorescence measurements. That is why we recommend users to measure Total NAD/NADH and NAD extract. Then from there, the NADH concentration can be calculated as Total NAD/NADH minus NAD extract. This will generally give a more accurate measurement of concentration, and by extension, NAD/NADH ratio.