Detection and Semi-Quantification of Lipids on High-Performance Thin-Layer Chromatography Plate Using Ceric Ammonium Molybdate Staining

It is desirable to quickly check the composition of lipids in small size samples, but achieving this is challenging using the existing staining methods. Herein, a highly sensitive and semi-quantitative method is developed for analysis of lipid samples with ceric ammonium molybdate (CAM) staining. The CAM detection method is systematically evaluated with a wide range of lipid classes including phospholipids, sphingolipids, glycerolipids, fatty acids (FA), and sterols, demonstrating high sensitivity, stability, and overall efficiency. Additionally, CAM staining provides a clean yellow background in high performance thin-layer chromatography (HPTLC) which facilitates quantification of lipids using image processing software. Lipids can be stained with CAM reagent regardless of their head group types, position of the carbon–carbon double bonds, geometric isomerism, and the variation in the length of FA chain, but staining is mostly affected by the degree of unsaturation of the FA backbone. The mechanism of the CAM staining of lipids is proposed on principles of the reduction–oxidation reaction, in which Mo(VI) oxidizes the unsaturated lipids into carbonyl compounds on the HPTLC plate upon heating, while itself being reduced to Mo(IV). This method is applied for the separation, identification, and quantification of lipid extracts from porcine brain. Practical applications: The CAM staining method that is developed in this work shows a high sensitivity for diverse lipid classes following HPTLC separation. HPTLC with CAM staining is a promising method for quick assessment of the identity and quantity of diverse lipid classes in lipid extracts of small size biological samples.