Isomers Recognition in HPLC-MS/MS Analysis of Human Plasma Samples by Using an Ion Trap Supported by a Linear Equations-Based Algorithm

The tandem mass spectrometry (MS/MS) approach employing an ion trap mass analyzer (IT) was evaluated in isomers recognition. The proposed approach consists of sole, simple, and rapid liquid chromatographic separation (HPLC) without requiring resolution between the analytes. Then, the MS/MS properties were optimized to solve the signal assignment using post-processing data elaboration (LEDA). The IT-MS/MS experiment uses the same site, helium as collision gas, and different time steps to modify the applied conditions on the studied ions. Nevertheless, helium cannot ensure the quick energization of the precursor ion due to its small cross-section. Then, different combinations between excitation amplitude (ExA) and excitation time (ExT) were tested to achieve the activation of the fragmentation channels and the formation of the MS/MS spectrum. Usually, the IT-MS/MS acquisition cycle is longer for other multistage instruments, decreasing the frequency of sample data collection and influencing the chromatographic profile. To solve these problems, two time segments were set up, and the elution conditions were optimized with a compromise between peaks distinction and run time reduction. The developed HPLC-MS/MS method was checked and applied to analyze a series of human plasma samples spiked with an equimolar mixture of pair of isomers.

Automated summary

The study evaluated the use of tandem mass spectrometry with ion trap mass analyzer for isomers recognition. The approach involved simple and rapid liquid chromatographic separation without resolving the analytes. The MS/MS properties and data processing were optimized to solve the signal assignment. Different combinations of excitation amplitude and time were tested to achieve fragmentation and MS/MS spectrum formation. The developed HPLC-MS/MS method was applied to analyze human plasma samples spiked with an equimolar mixture of isomers.