Application of thin-layer chromatography image analysis technique in quantitative determination of sphingomyelin

A sensitive and convenient method for sphingomyelin determination was developed based on thinlayer chromatography (TLC) and image-processing analysis. The mobile phase composition, detection and quantification conditions were systematically investigated through several trials. The molybdenum blue reaction allowed specific detection of the phospholipid with a high sensitivity and a wide linear range. Digital images of TLC plate chromatograms were captured with flatbed scanner and converted into peak chromatograms using TLSeeA (R) software and quantitative analysis was conducted. The linearity, sensitivity, accuracy and precision of the system were evaluated. The limits of detection and quantification were 0.5 and 1.7 mu g/spot, respectively. Separation of the mixture consisting of sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine was also carried out.