Validating regulatory-compliant wide dynamic range bioanalytical assays using chip-based nanoelectrospray tandem mass spectrometry

Automated chip‐based infusion nanoelectrospray ionization coupled to tandem mass spectrometry (nanoESI‐MS/MS) was used to validate a bioanalytical assay conforming to United States Food and Drug Administration (FDA) regulatory guidelines and Good Laboratory Practices (GLP). Reboxetine was used as the analyte fortified in dog plasma along with an analog internal standard (IS). The best nanoESI response for reboxetine was observed with 90% acetonitrile (ACN)/water without any mobile phase modifiers. The analyte and IS were extracted from dog plasma samples by liquid‐liquid extraction (LLE). The supernatant was concentrated to dryness and redissolved in 90% ACN/water for nanoESI. Selected reaction monitoring (SRM) data were collected for all samples to generate ion current profiles with a base width of approximately 20 s. Selectivity experiments showed no interferences in blank plasma samples. Interferences as a result of in‐source collision‐induced dissociation of metabolites were not an issue due to the previously documented metabolism of reboxetine. Matrix suppression was evaluated across multiple lots of dog plasma as well as over different animal species (rabbit, rat, mouse) and different anticoagulants (heparin, EDTA). Matrix suppression ranged from approximately 30–60% across the different lots, species etc.; however, in all instances, the analyte and the IS were suppressed by similar amounts, suggesting the similarity in ionization properties between the two. A three‐batch validation was performed (each batch consisting of four different concentrations, six replicates of each concentration) and demonstrated inter‐assay accuracy (% relative error; RE) of less than ±8% and an inter‐assay precision (% relative standard deviation; RSD) of less than 7%, thus meeting regulatory guidelines. A comparison of analyses by nanoESI‐MS/MS and liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS) showed that nanoESI‐MS/MS had a greater slope for the calibration standard curve compared to LC/MS/MS, indicating greater sensitivity for the former technique. It is also noteworthy that the amount of sample infused during nanoESI‐MS/MS was approximately 80‐fold less compared to the amount of sample injected during LC/MS/MS. The absence of carryover (attributed to the lack of a common fluid path) in the nanoESI technique enabled the extension of the assay linear dynamic range to 500 000‐fold, and the possibility of analyzing samples in a single batch without