A Comparative Study of Pentafluorophenyl and Octadecylsilane Columns in High-throughput Profiling of Biological Fluids Yoong-Soon Yong , a Eric Tzyy Jiann Chong, b Hsin-Chang Chen, c Ping-Chin Lee b and Yee Soon Ling a,d * a Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, 88400, Malaysia b Faculty of Science & Natural Resources, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, 88400, Malaysia c Institute of Occupational Medicine and Industrial Hygiene, College of Public Health, National Taiwan University, Taipei 100, Taiwan d Water Research Unit, Universiti Malaysia Sabah, Kota Kinabalu, Sabah, 88400, Malaysia (Received: December 20, 2016; Accepted: March 25, 2017; DOI: 10.1002/jccs.201600873) 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 perfor- mance of pentafluorophenyl (PFP)- and octadecylsilane (ODS)-based columns in profiling biologi- cal 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 abun- dant perturbed lipidome in overweight participants, followed by sphingolipids and various phos- pholipids. 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. Keywords: Biological fluids; Metabolomics; Octadecylsilane; Pentafluorophenyl; Partial least squares-discriminant analysis. INTRODUCTION Metabolomics is the study of endogenous low- weight metabolites (typically <1500 Da) for clarifying the functional information in biological system states. 1,2 It is widely applied in the study of various diseases, 3,4 biomarker discovery, 1 drug efficacy and toxicity screening, 5 nutrition 6 and environmental exposure. 7,8 Over the decades, researchers have attempted to explore specific low-weight molecules that can serve as diagnos- tic markers for monitoring and predicting disease pro- gression and therapeutic inspection. 9,10 Up to 40% of the genes in the human genome are hypothetical and their functions remain unknown, 11 of which some may be identified as orphan genes. Therefore, functional omics, including transcriptomics, proteomics, and metabolomics, are gaining much attention in academia. Metabolomics is advantageous because it serves as a direct signature of the investigated biochemical activity, enabling the simple correlation of phenotypes and activ- ities. Through biofluid (blood and urine) metabolite profiling, metabolomics summarizes the physiological status of an individual and therefore has high potential in personalized medicine 12 because different individuals metabolize compounds at different rates. Nuclear magnetic resonance (NMR) and mass spectrometry (MS) are the most commonly used meth- odologies in metabolomics. Several studies have high- lighted the benefits of combining multiple platforms while analyzing biofluids and extracts. However, because of the limitations due to cost and instruments, *Corresponding author. Email: lingyeesoon@ums.edu.my J. Chin. Chem. Soc. 2017 1 © 2017 The Chemical Society Located in Taipei & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim JOURNAL OF THE CHINESE CHEMICAL SOCIETY Article