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Bang D.Y.,Yonsei University | Bang D.Y.,Lotte R and nter | Byeon S.K.,Yonsei University | Moon M.H.,Yonsei University
Journal of Chromatography A | Year: 2014

A simple and fast lipid extraction method from human blood plasma and urine is introduced in this study. The effective lipid extraction from biological systems with a minimization of the matrix effect is important for the successful qualitative and quantitative analysis of lipids in liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The method described here is based on the modification of the quick, easy, cheap, effective, rugged and safe (QuEChERS) extraction method, which was originally developed for pesticide residue analysis in food, for the purpose of isolating lipids from biological fluids. Applicability of QuEChERS method for lipids was evaluated by varying organic solvents for the extraction/partitioning of lipids in MgSO4/CH3COONa for the removal of water and by varying sorbents (primary secondary amines, graphitized carbon black, silica, strong anion exchange resins and C18 particles) for the dispersive solid-phase extraction (dSPE) step. This study shows that 2:1 (v/v) CHCl3/CH3OH is effective in the extraction/partitioning step and that 50mg of C18 particles (for 0.1mL plasma and 1mL of urine) are more suitable for sample cleanup for the dSPE step of the QuEChERS method. Matrix effects were calculated by comparing the recovery values of lipid standards spiked to both plasma and urine samples after extraction with those of the same standards in a neat solution using nanoflow LC-ESI-MS/MS, resulting in improved MS signals due to the decrease of the ion suppression compared to the conventional Folch method. The modified QuEChERS method was applied to lipid extracts from both human urine and plasma samples, demonstrating that it can be powerfully utilized for high-speed (<15min) preparation of lipids compared to the Folch method, with equivalent or slightly improved results in lipid identification using nLC-ESI-MS/MS. © 2014 Elsevier B.V. Source

Seo J.-H.,Seoul National University | Hwang J.-Y.,Seoul National University | Hwang J.-Y.,Amore Pacific | Seo S.-H.,Seoul National University | And 5 more authors.
Bioscience, Biotechnology and Biochemistry | Year: 2012

ω-Aminotransferase (ω-AT) is an important class of enzymes for the synthesis of chiral amines or β-amino acids. Family profile analysis was applied to screen putative ω-ATs from Mesorhizobium loti MAFF303099, a nitrogen fixation bacterium that has a larger number of ATs than other microorganisms. By family profile analysis, we selected 10 putative ω-ATs according to E-value. The functions of the putative ω-ATs were investigated by examining activities towards amines and/or β-amino acids. 10 putative proteins were found to have ω-AT activity with narrow or broad substrate specificity. Structure analysis using crystal structure of mll7127 and homology models of mll1632 and mll3663 indicated that the structures of active sites of the enzymes were very similar and highly conserved, but their substrate specificities appreared to be determined by residues positioned at the entrance region of the active site binding pockets. Source

Bang D.Y.,Yonsei University | Bang D.Y.,Lotte R and nter | Lim S.,Yonsei University | Moon M.H.,Yonsei University
Journal of Chromatography A | Year: 2012

The effect of ionization modifiers added to the mobile phase of nanoflow liquid chromatography-tandem mass spectrometry (nLC-ESI-MS 3) on the simultaneous analysis of all phospholipid (PL) classes in negative ion mode has been investigated. While MS analysis of most PL classes is carried out in negative ion mode, analysis of neutral polar (polar but electrically neutral) lipids like phosphatidylcholine (PC) and sphingomyelin (SM) is highly efficient in positive ion mode. Therefore, analysis of PL mixture samples often requires two separate runs in both positive and negative ion mode. In order to establish run conditions to carry out a single nLC-ESI-MS-MS for all PLs, the ionization efficiency of 13 different types of PL molecules in nLC-ESI-MS has been evaluated in negative ion mode by varying the modifiers and their concentrations. Experiments demonstrated that a mixture of 0.05% ammonium hydroxide and 1mM ammonium formate added to the mobile phase provided effective ionization for all classes of PLs. The optimized conditions were applied to the analysis of a phospholipid mixture extracted from a human urine sample, yielding the identification of a total of 85 PL species. Analysis of the same sample with dual nLC-ESI-MS 2 runs in both positive and negative ion mode confirmed that nLC-ESI-MS 3 with the mixed modifier run only in negative ion mode gave comparable results. © 2012 Elsevier B.V. Source

Bang D.Y.,Yonsei University | Bang D.Y.,Lotte R and nter | Moon M.H.,Yonsei University
Journal of Chromatography A | Year: 2013

An on-line two-dimensional liquid chromatography method was developed for comprehensive lipid profiling by coupling strong anion exchange (SAX) and nanoflow reversed-phase liquid chromatography (nRPLC) prior to electrospray ionization-tandem mass spectrometry (2D-SAX/nRPLC-ESI-MS/MS). Lipids can be classified into four different types according to the electrical propensities of the lipids: anionic, weak anionic, neutral polar, and special lipids. In 2D-SAX/nRPLC, various lipids can be fractionated in the first dimension (SAX: 5. μm to 100. Å, 5.0. cm. ×. 75. μm i.d.) by step elution (methanol and salt solution), followed by the molecular separation of lipids in the second dimension (RP: 3. μm to 100. Å, 7.0. cm. ×. 75. μm i.d.) with binary gradient LC. Since the elution of lipids from SAX can be achieved with a very small volume of eluent delivered from an autosampler, it can be simply implemented with an LC-ESI-MS instrument for full automation, and the salt step elution, including the two-step injection procedure, can be used for the selective analysis of the desired lipid fraction. For nRPLC-ESI-MS/MS run in either positive or negative ion mode, a common ionization modifier (0.05% ammonium hydroxide with 5. mM ammonium formate) was introduced into the binary mobile phase solutions so that 2D-LC-MS could be operated in both ion modes without changing the mobile phase solutions. The developed on-line 2D-SAX/nRPLC-ESI-MS/MS was evaluated with 22 different standard lipids for the optimization of the salt step elution and was applied to a healthy human plasma lipid extract, resulting in the identification of a total of 303 plasma lipids, including 14 different classes. © 2013 Elsevier B.V. Source

Lim S.,Yonsei University | Bang D.Y.,Yonsei University | Bang D.Y.,Lotte R and nter | Rha K.H.,Yonsei University | Moon M.H.,Yonsei University
Bulletin of the Korean Chemical Society | Year: 2014

Ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLCESI-MS/MS) provides a high-speed method to screen a large number of samples for small molecules with specific properties. In this study, UPLC-ESI-MS/MS with multiple reaction monitoring (MRM) was employed to screen urinary phospholipid (PL) content for biomarkers of prostate cancer. From lists of urinary PLs structurally identified using nanoflow LC-ESI-MS/MS, 52 PL species were selected for quantitative analysis in urine samples between 22 cancer-free urologic patients as controls and 45 prostate cancer patients. Statistical treatment of data by receiver operating characteristic (ROC) analysis yielded 14 PL species that differed significantly in relative concentrations (area under curve (AUC) > 0.8) between the two groups. Among PLs present at higher levels in prostate cancer urine, phosphatidylcholines (PCs) and phosphatidylinositols (PIs) constituted the major head group PLs (3 PCs and 7 PIs). For technical reasons, PL species of low abundance may be underrepresented in data from UPLC-ESI-MS/MS performed in MRM mode. However, the proposed method enables the rapid screening of large numbers of plasma or urine samples in the search for biomarkers of human disease. Source

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