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

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involv-ing the calculation of individual metabolite recoveries (pre/post extraction spiking U -13 C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode. One hundred sixty-one (161) metabolites were successfully detected in ESI + mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three differ-ent biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U -13 C-labeled Pichia pastoris (Ko-magataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter-and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process effi-ciencies were evaluated following the Matuszewski protocol with U -13 C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.(c) 2022 Elsevier B.V. All rights reserved.