Wu and colleagues developed a liquid chromatography tandem mass spectrometry assay for the EGFR inhibitor AZD-3759 in rat plasma. Diazepam served as the internal standard. The calibration related the AZD-3759 peak area to the diazepam peak area, using known AZD-3759 concentrations to establish the relationship.
The method monitored separate mass transitions for the drugs and reported retention times of 1.19 minutes for AZD-3759 and 2.21 minutes for diazepam. Here the diazepam peak was expected because the method called for adding it during sample preparation. Its presence in the chromatogram, by itself, would provide no evidence that an animal received diazepam.
Check which compound the method quantifies, which material was spiked into each tube and whether the internal standard can be distinguished from any diazepam already in the specimen. A non-isotopic standard can behave differently from the target during extraction or ionization, so examine the actual method validation. Validation for AZD-3759 in rat plasma does not transfer to an assay intended to report diazepam in a human specimen.