title: Quantification of endogenous metabolites by the postcolumn infused-internal standard method combined with matrix normalization factor in liquid chromatography-electrospray ionization tandem mass spectrometry.
abstract: Quantification of endogenous metabolites has enabled the discovery of biomarkers for diagnosis and provided for an understanding of disease etiology. The standard addition and stable isotope labeled-internal standard (SIL-IS) methods are currently the most widely used approaches to quantifying endogenous metabolites, but both have some limitations for clinical measurement. In this study, we developed a new approach for endogenous metabolite quantification by the postcolumn infused-internal standard (PCI-IS) method combined with the matrix normalization factor (MNF) method. MNF was used to correct the difference in MEs between standard solution and biofluids, and PCI-IS additionally tailored the correction of the MEs for individual samples. Androstenedione and testosterone were selected as test articles to verify this new approach to quantifying metabolites in plasma. The repeatability (n=4 runs) and intermediate precision (n=3 days) in terms of the peak area of androstenedione and testosterone at all tested concentrations were all less than 11% relative standard deviation (RSD). The accuracy test revealed that the recoveries were between 95.72% and 113.46%. The concentrations of androstenedione and testosterone in fifty plasma samples obtained from healthy volunteers were quantified by the PCI-IS combined with the MNF method, and the quantification results were compared with the results of the SIL-IS method. The Pearson correlation test showed that the correlation coefficient was 0.98 for both androstenedione and testosterone. We demonstrated that the PCI-IS combined with the MNF method is an effective and accurate method for quantifying endogenous metabolites.
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taiwan.
authors.forename: Hsiao-Wei
authors.initials: HW
authors.lastname: Liao
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taiwan; The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taiwan.
authors.forename: Guan-Yuan
authors.initials: GY
authors.lastname: Chen
authors.affiliation: Department of Pharmacy, National Taiwan University Hospital, Taiwan; Department of Internal Medicine, College of Medicine, National Taiwan University, Taiwan.
authors.forename: Ming-Shiang
authors.initials: MS
authors.lastname: Wu
authors.affiliation: Department of Pharmacy, National Taiwan University Hospital, Taiwan; Department of Internal Medicine, College of Medicine, National Taiwan University, Taiwan.
authors.forename: Wei-Chih
authors.initials: WC
authors.lastname: Liao
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taiwan; The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taiwan.
authors.forename: I-Lin
authors.initials: IL
authors.lastname: Tsai
authors.affiliation: School of Pharmacy, College of Medicine, National Taiwan University, Taiwan; The Metabolomics Core Laboratory, Center of Genomic Medicine, National Taiwan University, Taiwan; Department of Internal Medicine, National Taiwan University Hospital, Taiwan. Electronic address: kuoch@ntu.edu.tw.
authors.forename: Ching-Hua
authors.initials: CH
authors.lastname: Kuo
mesh_terms: D000735:Androstenedione / Q000097:blood / Q000592:standards; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D006801:Humans; D055432:Metabolomics / Q000379:methods*; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods* / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards; D013739:Testosterone / Q000097:blood / Q000592:standards
chemical_list: D013739:Testosterone; D000735:Androstenedione
keywords: Endogenous metabolites; Liquid chromatography–electrospray ionization tandem mass spectrometry; Matrix effects; Matrix normalization factor (MNF); Postcolumn infused-internal standard (PCI-IS)