Metabolic profiling workflow for cell extracts by targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry

A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method

“…Concerning the two chromatographic modes mostly used in metabolomics, namely reversed phase and Hydrophilic interaction chromatography (HILIC), it was reported that there was a higher ME in the second one [ 16 , 17 ]. The comparison of the measured ME from the present work and Serafimov et al showed similar high values for the ME (from 42 to 110% for Serafimov et al) [ 6 ] while reversed-phase and HILIC were used. Although an ESI source is more prone to the ME than atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) sources, it remains the largest ion source used in metabolomics [ 18 ].…”

“…[ 1 ]. Although several authors applied LC-MS metabolomics methods on in vitro cell extracts [ 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ], data on the methodology used to evaluate analytical performances considering the cellular matrix remain only scarcely detailed. Serafimov et al used standard addition experiments to evaluate the performances of an LC-MS/MS method [ 6 ], whereas other works performed method evaluation on a surrogate matrix or water [ 4 , 5 ].…”

“…The direct consequence is variability in the number of endogenous metabolites from one culture batch to another. Furthermore, as shown in previous works conducted on in vitro cell extracts, the number and the abundance of metabolites extracted vary according to several pre-analytical parameters, including the detachment of adherent cells by scraping or trypsinization, nature, and volume of the lysis solvent and the duration of contact with the lysis solvent [ 6 , 7 , 12 ]. However, the specific impact of the in vitro cellular matrix on analytical performances has been barely reported.…”

“…Indeed, the matrix effect (ME) related to in vitro cell extracts has only been studied in two papers, and the values reported depend on the approach used to evaluate the ME. While Serafimov et al showed an important ME for some metabolites (up to 42%) based on a standard addition approach, Zhu et al observed a ME of less than 20% using a surrogate matrix approach [ 4 , 6 ]. Despite these data, the knowledge about the specific impact of the in vitro cellular matrix on the analytical performances of LC-MS/MS methods appears to be insufficient, and several questions remain.…”

From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography–mass spectrometry bioanalysis

“…However, several calibration curves using different representative pooled matrices can be prepared to overcome these drawbacks and use the calibration that best covers the concentration to be analyzed [92]. Matrix-matched EC cannot always correct the matrix effect when it varies between study samples, emphasizing the importance of using an IS to correct these discrepancies, as discussed at the end of the following section (3.1.2) [93,94].…”