Collision energy consistency between analyte and internal standard: A critical factor for accurate quantification of...

Simultaneous quantification of steroid hormones with vastly differing physiological concentrations using LC-MS/MS often necessitating compromised collision energy (CE) settings, potentially introducing inaccuracy. This study investigates the critical impact of CE consistency between analytes and their stable isotope-labeled internal standards (SIL-IS) on quantification accuracy using a triple quadrupole LC-MS/MS system. We analyzed six hormones in 61 plasma samples using a novel dual-CE acquisition method, monitoring SIL-IS response at both low and optimal CE. The method was reapplied to 70 patient samples following collision cell replacement to assess instrument dependency. Significant instrument-specific bias (15-92%) were observed for high-concentration analytes when CE settings between native analytes and SIL-IS were mismatched (LCE-HCE). These biases were markedly amplified on a degraded instrument. Synchronizing CE settings (LCE-LCE) or utilizing consistently optimized isotopic transitions restored accuracy, reducing mean bias to below 15% post-optimization. The instrument in optimal condition showed minimal CE-dependent bias. Inconsistent CE application is a hidden source of quantitative inaccuracy in multi-analyte steroid panels, exacerbated by suboptimal instrument conditions. While these findings provide important methodological insights, further studies on different LC-MS/MS platforms are warranted to establish their generalizability. We strongly recommend verification of CE consistency during method validation and instrument troubleshooting.