Multiple lots of matrix were assessed to determine if any additional variability found during this method validation could be attributed to the analysis of multiple lots of matrix. Human acidified blood inter-lot chromatographic selectivity was evaluated by preparing control blanks and zero samples in six lots of human control acidified blood samples and comparing the peak area ratios (PAR) of each zero sample to the mean PAR of the LLOQ calibration standard samples analyzed within the same run. The results from the acidified blood inter-lot chromatographic selectivity experiment for 13C5-NAD+ in six different lots of human acidified blood showed no response for 13C5-NAD+ in the control (blank acidified blood). But there was a trace amount response of 13C5-NAD+ in the zero sample with IS (13C5D3-NAD+). This indicated that there was some contribution from the possible impurity of IS. The peak area was measured and it was smaller than 20% of the LLOQ peak area, which was acceptable from regulatory standards. The selectivity of the method was also evaluated during each regressed validation run by LC–MS/MS to monitor for possible interfering peaks at the same chromatographic retention times as the analyte and IS. No significant chromatographic interferences were detected at the retention time of the analyte or IS as shown in Fig. 2.