A novel endogenous retention-index for minimizing retention-time variations in metabolomic analysis with reversed-phase ultrahigh-performance liquid-chromatography and mass spectrometry.

Consistent retention time (t R) of metabolites is vital for identification in metabolomic analysis with ultrahigh-performance liquid-chromatography (UPLC). To minimize inter-experimental t R variations from the reversed-phase UPLC-MS, we developed an endo genous r etention- i ndex (endoRI) using in-sample straight-chain acylcarnitines with different chain-length (L C , C0–C26) without additives. The endoRI-corrections reduced the t R variations caused by the combined changes of mobile phases, gradients, flow-rates, elution time, columns and temperature from up to 5.1 min–0.2 min for most metabolites in a model metabolome consisting of 91 metabolites and multiple biological matrices including human serum, plasma, fecal, urine, A549 cells and rabbit liver extracts. The endoRI-corrections also reduced the inter-batch and inter-platform t R variations from 1.5 min to 0.15 min for 95 % of detected features in the above biological samples. We further established a quantitative model between t R and L C for predicting t R values of acylcarnitines when absent in samples. This makes it possible to compare metabolites' t R from different t R databases and the UPLC-based metabolomic data from different batches. [Display omitted] • An endo genous r etention- i ndex derived from in-sample acylcarnitines for minimizing inter-experimental t R variations in metabolomics with RPLC-MS. • The endoRI-correction reduced inter-batch t R variations to below 0.2 min for metabolites in multiple biological samples. • A mathematical model enables accurate prediction of t R for acylcarnitines with no standards. [ABSTRACT FROM AUTHOR]