Dual flow-path HPLC method for time-optimised quantification of physicochemically different metabolite classes in human serum samples using a single mass spectrometer

Biomedical studies often involve the analysis of a wide range of analytes. The varying physicochemical properties of these analytes frequently hinder their detection by a single method, making it necessary to employ different analytical instruments or chromatographic approaches. This process often requires considerable time. In this study, we present a time-optimised LC-MS-based approach using only one mass spectrometer, but utilisation of two orthogonal chromatographic techniques, namely hydrophilic interaction liquid chromatography (HILIC) and reversed phase (RP)-HPLC in parallel. The established method allowed short-chain fatty acids (SCFA) and amines (such as trimethylamine N-oxide and carnitine) to be recovered from one human serum sample. SCFA were derivatized to their corresponding 3-nitrophenylhydrazones for improved RP-HPLC separation and electrospray ionisation efficiency, whereas amines could be separated and detected in their native forms on a zwitterionic HILIC phase. The combination of RP and HILIC methods through a six-port valve was achieved with a dual split sampler, enabling re-equilibration of one chromatographic column during the separation step of the other method. This approach ensured optimal capacity use of the MS and resulted in a 37 % analysis time reduction compared to the analysis with both individual methods. Application to human serum samples confirmed the robustness and reproducibility of the method, with quantification results showing relative standard deviations ≤ 5.8 % for all analytes as well as stable retention times and chromatographic resolutions for both methods. By allowing the combination of any chromatographic columns and methods with only one detector, this setup is well suited for quantification of extensive sample batches applying customised chromatographic conditions, while ensuring the optimal separation performance for both methods as well as maintaining efficient detector utilisation.