title: Micellar electrokinetic chromatography profiles of human high-density lipoprotein phospholipids.
abstract: A simple and fast micellar electrokinetic chromatography (MEKC) method was developed to investigate phospholipids isolated from human high-density lipoproteins (HDL). To optimize the MEKC conditions, several factors including bile salt concentration and organic modifier concentration in the separation buffer as well as temperature have been examined. The optimal separation buffer chosen was a mixture of 50 mM bile salts, 30% v/v 1-propanol and 10 mM sodium phosphate (pH 8.5). The applied voltage and temperature selected were 25 kV and 40°C, respectively. Meanwhile, high-salt stacking has been performed for sample pre-concentration to enhance peak sensitivity. Several factors including organic modifier concentration and salt concentration in the sample matrix as well as sample injection time have been optimized. The optimal sample buffer selected was a mixture of 100 mM NaCl and 20% 1-propanol, and the optimal sample injection time selected was 32 s under a pressure of 0.5 psi. Several phospholipid standards including lysophosphatidyl choline, phosphatidyl choline (PC), sphingomyelin, phosphatidyl ethanolamine, phosphatidyl inositol, phosphatidyl serine and phosphatidic acid have been studied using the optimal MEKC method. The MEKC profile of the mixed phospholipid standards showed good separation and reproducibility. The linear ranges for PC and sphingomyelin were 0.025-1.2 and 0.025-2.0 mg/mL, respectively. The concentration limits of detection of PC and sphingomyelin were 0.0156 and 0.0199 mg/mL, respectively. Using phosphatidic acid as an internal standard, precision and accuracy have been measured for PC and sphingomyelin. The intraday and interday quantitative analysis showed good results. The new MEKC method has been used to characterize native, in vitro oxidized and glycated human HDL phospholipids within 16 min. At absorbance 200 nm, two similar peaks were observed for native and oxidized HDL phospholipids, but three peaks were observed for glycated HDL phospholipids. Interestingly, at absorbance 234 nm, distinctively different MEKC profiles were observed for the three HDL phospholipids.
authors.affiliation: Department of Chemistry, National Changhua University of Education, Changhua, Taiwan.
authors.forename: Chin-Pong
authors.initials: CP
authors.lastname: Chong
authors.forename: Ting-Yu
authors.initials: TY
authors.lastname: Lin
authors.forename: Chia-Liang
authors.initials: CL
authors.lastname: Chang
authors.forename: Ying-Ling
authors.initials: YL
authors.lastname: Yang
authors.forename: Ming-Hua
authors.initials: MH
authors.lastname: Tsai
authors.forename: Yu-Shan
authors.initials: YS
authors.lastname: Yu
authors.forename: Mine-Yine
authors.initials: MY
authors.lastname: Liu
mesh_terms: D000433:1-Propanol / Q000737:chemistry; D050197:Atherosclerosis / Q000097:blood / Q000378:metabolism; D001647:Bile Acids and Salts / Q000737:chemistry; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D003920:Diabetes Mellitus / Q000097:blood / Q000378:metabolism; D020404:Glycerophospholipids / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D008075:Lipoproteins, HDL / Q000097:blood / Q000737:chemistry*; D010084:Oxidation-Reduction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012965:Sodium Chloride / Q000737:chemistry; D013696:Temperature
chemical_list: D001647:Bile Acids and Salts; D020404:Glycerophospholipids; D008075:Lipoproteins, HDL; D012965:Sodium Chloride; D000433:1-Propanol