Novel application of differential ion mobility spectrometry-tandem mass spectrometry for improved assay selectivity and sensitivity in the quantitative determination of total NNN total NNAL and 2- / 3-HPMA in human urine
Differential Ion Mobility Spectrometry (DMS) has recently emerged as an orthogonal gas-phase ion separation technique which, when interfaced between liquid chromatography (LC) and Tandem Mass Spectrometry (MS/MS), promises improvements in both assay selectivity and sensitivity by differentiating analyte from interference based upon physical cross-section. In the current research, we have established the potential for DMS technology to address existing LC-MS/MS assay limitations associated with the quantitation of the urinary biomarkers 2-/3-Hydroxypropylmercapturic acid (HPMA), total N-nitrosonornicotine (NNN), and total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Leveraging a SCIEX TripleQuad 6500+ with SelexION differentail mobility separation device in an LC-DMS-MS/MS workflow, total NNN method detection limits could be lowered tenfold from those previously reported in the literature, to 0.20 pg/mL. This represents a relevant improvement in sensitivity since 25-30% of smoker urine contains baseline NNN concentrations ˂ 2.00 pg/mL. Achieving a 0.20 pg/mL detection limit required only a fivefold concentration of extract, accomplished without concomitant matrix effect or interference. Further, the additional selectivity offered by the LC-DMS-MS/MS approach suggested that previous reports of augmented NNN response in urine left at room temperature were due to a chromatographically unresolved isobaric interference, which proved separable from NNN when leveraging ion mobility. For total NNAL, a fivefold improvement in signal-to-noise ratio (SNR) using LC-DMS-MS/MS allowed extracts to be reconstituted without concentration, resulting in an absence of ionization suppression and increased assay robustness for a 5.0 pg/mL detection limit (SNR 40:1). In the determination of 2-/3-HPMA, the sensitivity for 2-HPMA by LC-DMS-MS/MS was increased twofold compared to LC-MS/MS, whilst an interference at the retention time of 3-HPMA could be eliminated by ion mobility, reducing complex and prolonged sample preparation and chromatographic separation. Each of the three LC-DMS-MS/MS methods was successfully validated according to the criteria established by the U.S. FDA in the 2018 Bioanalytical Method Validation Guidance document for small molecule quantitation.