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.

Characterization of in Vitro Modified Human Very Low-Density Lipoprotein Particles and Phospholipids by Capillary Electrophoresis

“…Recently, we have developed a MEKC with a high-salt stacking method [32]. In this study, we analyzed phospholipids isolated from native, in vitro oxidized and glycated VLDL particles using the MEKC with high salt-stacking method.…”

Contemporary sample stacking in analytical electrophoresis

“…The LOD for both warfarin enantiomers in urine was 1.5 μg/L. Chong et al developed a new MEKC method with high‐salt stacking, and used it to characterize native, in vitro oxidized and glycated human high‐density lipoprotein phospholipids. The BGE used was a mixture of 50 mM bile salts, 30% v/v 1‐propanol and 10 mM sodium phosphate at pH 8.5, and the sample buffer was a mixture of 100 mM NaCl and 20% 1‐propanol.…”

Rapid detection of dysfunctional high‐density lipoproteins using isoelectric focusing‐based microfluidic device to diagnose senescence‐related disease

“…To the best of our knowledge, however, there has been no report on methods to detect modified proteins, either structurally or functionally, to compare between healthy state and disease state. Recently, Liu group reported a micellar electrokinetic chromatography (MEKC) method to analyze HDL phospholipids [21]. Although the method was simple and fast, it has a limitation in that it can analyze phospholipid, but not HDL particle itself.…”