A Comparative Study of Pentafluorophenyl and Octadecylsilane Columns in High-throughput Profiling of Biological Fluids

Abstract

In high‐throughput metabolomic profiling, chromatographic separation is crucial because a well‐performed chromatographic separation may reduce signal suppression from complex biological matrices and improve the discoverability of low‐abundance metabolites. We compared the performance of pentafluorophenyl (PFP)‐ and octadecylsilane (ODS)‐based columns in profiling biological fluids. Peak resolutions and consistencies were acquired using several reversed‐phase columns and were evaluated. Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than the ODS column. Low relative standard deviations on peak areas and retention times (<10.2 and <0.9%, respectively) acquired using the PFP column evidenced the high reproducibility and consistency of this column. In our study, a PFP column was used for profiling metabolomes extracted from urine and serum samples. Metabolomic study revealed a metabolome difference in normal and overweight participants. In total, 26 lipid species were significantly perturbed and further identified. Choline‐containing lipids were the most abundant perturbed lipidome in overweight participants, followed by sphingolipids and various phospholipids. We recommend the use of PFP columns in high‐throughput metabolomic analysis to promote the development of basic biological and clinical research in the future.

Antiparasitic potential of Nephrolepis biserrata methanol extract against the parasitic leech Zeylanicobdella arugamensis (Hirudinea) and LC-QTOF analysis

“…The MS was calibrated with Tuning Mix (Agilent Technologies, Santa Clara, CA, USA) before each batch analysis. Internal mass calibration standards, purine ( m / z 121.0508) and hexakis-(1H, 1H, 3H-tetrafluoropropoxy)-phosphazine ( m / z 922.0097), were introduced throughout the runs for automated mass correction 39 .…”

Evaluation of Different Stationary Phases in the Separation of Inter-Cross-Linked Peptides

“…Fluorophenyl stationary phases have been shown to provide increased retention for basic hydrophilic compounds. Additional interactive properties of the fluorophenyl column were previously investigated in the applications of off-line fractionations on Hela cells 35 , orthogonality in 2D-LC separations 36 and separation of metabolites in urine samples 37 . Fluorine atoms incorporated in the fluorophenyl stationary phase act as electron withdrawing groups and induce enhanced interactions between the aromatic group and positively charged compounds.…”

“…Additional interactive properties of the fluorophenyl column were previously investigated in the applications of off-line fractionations on Hela cells, 35 orthogonality in 2D-LC separations, 36 and separation of metabolites in urine samples. 37 Fluorine atoms incorporated in the fluorophenyl stationary phase act as electron-withdrawing groups and induce enhanced interactions between the aromatic group and positively charged compounds. More importantly, the shape selectivity from the phenyl columns could be beneficial in separating the "H"-shaped inter-cross-linked peptides from unmodified linear peptides or dead-end cross-linked peptides.…”

A highly sensitive LC-MS/MS method for quantitative determination of 7 vitamin D metabolites in mouse brain tissue

“…The online extraction component of the novel OLE-LC-MS/MS method described with selective elution from the trap column, enables effective bulk decontamination of phospholipids [33]. With the option to loop load and stack, effectively concentrating metabolites of interest, coupled with phospholipid removal, sensitivity appears to be improved orders of magnitude and other metabolites of vit-D with potentially important biological function are realised.…”