Robustness of the Discovery® HS F5 HPLC Column in the Study of Metabolomics

By: Nathaly Reyes-Garcés, Barbara Bojko, Janusz Pawliszyn, Department of Chemistry, University of Waterloo, Ontario, Canada N2L 3G1, Reporter US Volume 34.4

Introduction

The performance of the Discovery HS F5 HPLC column was monitored during the metabolomics study executed on a liquid chromatograph – high resolution mass spectrometer (LC/MS) platform. The comparison of chromatograms of selected compounds was done before and after 170, 450, 730, 1125, and 1525 injections of tissue extracts obtained with solid phase microextraction. The results showed excellent robustness, stability, and reproducibility of the column in combination with highly efficient sample clean-up offered by SPME.

Metabolomics is currently one of fastest developing areas of bioanalysis. Its main goal is to characterize the full profile of small molecules in the system under study. In order to cover analytes with a very wide range of different physical and chemical properties, the elements of an analytical protocol should be carefully optimized and validated. High method precision is required in order to avoid false positive results and enhance proper identification of the extracted analytes. Herein, the performance of the Discovery HS F5 HPLC column after using solid phase microextraction as a sample preparation tool will be evaluated for use in the study of metabolomics.

Experimental

Human brain tumors and piglet lung were sampled (30 min extraction time) with 7 mm mix-mode SPME fibers in vitro and in vivo, respectively. The extracted analytes were subsequently desorbed in acetonitrile:water, 1:1 v/v and extracts were analyzed via LC/MS containing the following components: Accela autosampler with cooled system tray, Accela LC Pumps, and Exactive Orbitrap mass spectrometer (Thermo, San Jose, CA, US). The MS experiments were performed in both positive and negative ionization mode. A 10 cm × 2.1 mm, 3 μm particle size PFP column connected to a Discovery HS F5 guard column cartridge (2 cm × 2.1 mm, 3 μm) were used in the study.

For analysis in positive ionization mode, mobile phase A consisted of water:formic acid, 99.9:0.1, v/v and mobile phase B consisted of acetonitrile:formic acid (99.9:0.1, v/v). For analysis in negative ionization mode, mobile phase A consisted of a 1 mM NH4F solution and mobile phase B consisted of acetonitrile. The flow rate of the mobile phase was 300 μL/min. The following gradient elution was used: 100% A from 0 to 3.0 min, followed by a linear gradient to 10% A from 3.0 to 25.0 min, and an isocratic hold at 10% A until 34.0 min. The total run time was 40 min per sample, including a 6 min column re-equilibration time. The injection volume was 10 μL.

The QC used for monitoring of column performance in ESI+ consisted of tranexemic acid, trans-4-(Aminomethyl)cyclohexanecarboxylic acid (ACHCA), phenylalanine, phenylalanine-d5, tryptophan and progesterone. Taurocholid acid, cholic acid, and chenodeoxycholic acid-d4 in ESI-.

Results

Solid phase microextraction is a sample preparation method, which combines sampling, sample preparation, and extraction in one step. The biocompatibility of the mix-mode extraction phase achieved by the use of polyacrylonitrile as a binding glue prevents occlusion of the sample matrix constituents other than small molecules thus providing excellent sample clean up. This, in turn, counteracts phospholipid buildup of compounds on the column, which is common issue for solvent-based extraction methods, and prolongs column lifetime. Monitoring of QC samples containing standards which cover abroad range of retention times, during the long-term use of the Discovery HS F5 column confirmed this finding and demonstrated the robustness of the column when operated under optimum conditions for compounds of different physical and chemical properties.

Chromatogram of Selected Compounds Obtained

Chromatogram of Selected Compounds Obtained

Chromatogram of Selected Compounds Obtained

Chromatogram of Selected Compounds Obtained

Chromatogram of Selected Compounds Obtained

Materials

     
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