A simple solid‐phase extraction method for the analysis of red cell phospholipids by liquid chromatography‐ tandem mass spectrometry

摘要

Background There has been increasing interest in the analysis of phospholipids in red blood cells as potential long‐term biomarkers of different disease states. Here, we describe a simple method for the analysis of two phospholipids: 1‐Palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphoethanol ( PE 16:0/18:1) and 1‐Palmitoyl‐2‐linoleoyl‐sn‐glycero‐3‐phosphoethanol ( PE 16:/0/18:2) in erythrocytes by liquid chromatography‐tandem mass spectrometry ( LC ‐ MS / MS ). Methods Whole blood samples were removed free of plasma and washed in isotonic saline. Red cells were lysed with ultrapure water. Lysate samples were processed using a hybrid solid‐phase extraction ( SPE ) phospholipid cartridge (1 mL , 30 mg). Both PE 16:0/18:1 and PE 16:0/18:2 and their deuterated internal standards were separated on an ACE C4 (150?mm × 2.1?mm, 2.7?μm particle size) by gradient elution at a flow rate of 0.5 mL per minute using mobile phases consisting of 0.01 mol/L ammonium acetate in: water (A), methanol (B), and isopropanol (C). The phospholipid species were quantified by the following transitions: PE 16:0/18:1: 701.5→281.3 and PE 16:0/18:2: 699.5→279.3. Results Both PE species displayed linearity ranging from 10 to 500?μg/L. The coefficient of variation ( CV %) of PE 16:0/18:1 concerning intraday and interday precision was between 1.9%‐2.6% and 3.0%‐4.3%, respectively. For PE 16:0/18:2, this was between 1.8%‐3.4% and 3.7%‐4.1%, respectively. Both phospholipid species had accuracy ( PE 16:0/18:1: 91%‐98% and PE 16:0/18:2: 94%‐103%) and extraction recovery ( PE 16:0/18:1: 95%‐106% and PE 16:0/18:2: 92%‐102%) exceeding 90% over the analytical range. The limit of detection was 5?μg/L. Conclusion Here we propose a simple SPE LC ‐ MS / MS method for analyzing phospholipids in erythrocytes, which can be easily adopted.