DETERMINATION OF LIPIDS
This assay is quantitative and rapid and enables to get reliable results with acylglycerols as well as other lipids, including waxes, sterols and long-chain bases, the lipids being converted to carbon by charring in concentrated sulfuric acid. The limit of sensitivity is about 2-5 mg according to the lipid. The linearity of the response is observed for all lipids up to about 100 mg but the slopes differ for different lipids. Under precise conditions, the optical density of the reaction mixture appears directly proportional to the carbon content of the lipid under assay (Marsh JB et al., J Lipid Res 1966, 7, 574).
In glass tubes containing lipid samples thoroughly dried or scraped silica gel spots, add 1 ml of concentrated sulfuric acid.
Heat the tubes at 200°C for 15 min in a sand bath (or a heating bloc).
Cool rapidly the tubes (in a cold metal block or in water) during 2 min and transfer the tubes in an ice bath.
Add 1.5 ml water , vortex thoroughly and replace in the ice for 5 min.
Remove the tubes from the ice and left standing for 10 min.
If silica gel is present, centrifuge the tubes at low speed for 5 min.
Transfer the reaction mixture in optical cuvettes and measure the optical density at 375 nm. A solvent blank is included in the assay together with tubes containing known amounts of standard lipids.
An alternate procedure has been evaluated using sulfuric acid, anisaldehyde or vanillin (Johnsson R et al., J Chromatogr A 2007, 1164, 298). Practical conditions have been compared before scanning the TLC plates.
A highly sensitive and semi-quantitative method has been proposed for analysis of lipid samples with ceric ammonium molybdate (CAM) staining after HPTLC chromatography (Asressu KH et al., Eur J Lipid Sci Technol 2023, 125, 2200096). The CAM detection method was systematically evaluated with a wide range of lipid classes including phospholipids, sphingolipids, glycerolipids, fatty acids, and sterols, demonstrating high sensitivity, stability, and overall efficiency. 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 was shown to be mostly affected by the degree of unsaturation of the FA backbone. Interestingly, lipid classes such as MG, DG, FA, and cholesterol were detected at 0.13 μg amount with high intensity, indicating the high sensitivity of CAM staining.
Compared to other literature reports, the CAM detection method demonstrated a higher sensitivity, stability and overall efficiency for a wide range of lipid classes such as phospholipids, glycerolipids, fatty acids, sterols and even sphingolipids. It must be noticed that to get more accurate quantitation, relatively well matching in composition (unsaturation level) between the reference standards and the sample is required.