N-Alkylpyridinium sulfonates for retention time indexing in reversed-phase-liquid chromatography-mass spectrometry-based metabolomics

Chromatographic retention time information is valuable, orthogonal information to MS and MS/MS data that can be used in metabolite identification. However, while comparison of MS data between different instruments is possible to a certain degree, retention times (RTs) can vary extensively, even when nominally the same phase system is used. Different factors such as column dead volumes, system extra column volume, and gradient dwell volume can influence absolute retention times. Retention time indexing (RTI), routinely employed in gas chromatography (e.g., Kovats index), allows compensation for deviations in experimental conditions. Different systems have been reported for RTI in liquid chromatography, but none of them have been applied to metabolomics to the same extent as they have with GC. Recently, a more universal RTI system has been reported based on a homologous series of N-alkylpyridinium sulfonates (NAPS). These reference standards ionize in both positive and negative ionization modes and are UV-active. We demonstrate the NAPS can be used for retention time indexing in reversed-phase-liquid chromatography-mass spectrometry (RP-LC-MS)-based metabolomics. Having measured >500 metabolite standards and varying flow rate and column dimension, we show that conversion of RT to retention indices (RI) substantially improves comparability of retention information and enables to use of RI for metabolite annotation and identification.

Automated summary

Chromatographic retention time information is crucial for metabolite identification, but variations in retention times can occur due to factors like column dead volumes and gradient dwell volume. A new universal retention time indexing system based on N-alkylpyridinium sulfonates has been developed for liquid chromatography, showing potential for improving comparability and enabling metabolite annotation and identification. This system has the advantage of being applicable to both positive and negative ionization modes, with UV activity, making it useful for reversed-phase liquid chromatography-mass spectrometry-based metabolomics.