Surrogate analyte approach for quantitation of endogenous NAD+ in human acidified blood samples using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

• An LC–MS/MS assay for the quantitative determination of NAD+ in human whole blood using a surrogate analyte approach was developed and validated.
• Parallelism was established between the surrogate analyte and the authentic analyte in the concentration range of 1.69–25 μg/mL.
• This validated assay was successfully applied to determine the baseline level of endogenous NAD+ in human blood from healthy volunteers.

A high-performance liquid chromatography tandem mass spectrometry (LC–MS/MS) assay for the quantitative determination of NAD+ in human whole blood using a surrogate analyte approach was developed and validated. Human whole blood was acidified using 0.5 N perchloric acid at a ratio of 1:3 (v:v, blood:perchloric acid) during sample collection. 25 μL of acidified blood was extracted using a protein precipitation method and the resulting extracts were analyzed using reverse-phase chromatography and positive electrospray ionization mass spectrometry. 13C5-NAD+ was used as the surrogate analyte for authentic analyte, NAD+. The standard curve ranging from 0.250 to 25.0 μg/mL in acidified human blood for 13C5-NAD+ was fitted to a 1/x2 weighted linear regression model. The LC–MS/MS response between surrogate analyte and authentic analyte at the same concentration was obtained before and after the batch run. This response factor was not applied when determining the NAD+ concentration from the 13C5-NAD+ standard curve since the percent difference was less than 5%. The precision and accuracy of the LC–MS/MS assay based on the five analytical QC levels were well within the acceptance criteria from both FDA and EMA guidance for bioanalytical method validation. Average extraction recovery of 13C5-NAD+ was 94.6% across the curve range. Matrix factor was 0.99 for both high and low QC indicating minimal ion suppression or enhancement. The validated assay was used to measure the baseline level of NAD+ in 29 male and 21 female human subjects. This assay was also used to study the circadian effect of endogenous level of NAD+ in 10 human subjects.