We optimized this deacylation procedure and quantified the sphingoid bases and lysoglycosphingolipids after derivatization with ophthaldialdehyde (OPA) by HPLC with fluorometric detection (24).
To maximize deacylation and minimize decomposition, the optimal condition for this microwave oven proved to be an exposure time of 60 min at 85% power and a fixed setting of 7 for the exhaust fan (see Results).
Peak identification was based on comparison of the retention times with those of authentic calibrators of the lysocompounds (sphingosine, sphinganine, lyso-G1cCer, and lyso-CTH) and those of Cer, G1cCer, and CTH after microwave-assisted deacylation.
Because Cer, G1cCer, and CTH consist of a mixture of different molecular species, we prepared calibrator solutions of these compounds used for the recovery studies using an estimated average molecular weight and exactly measured each concentration by quantification after deacylation.
conditions for complete deacylation with minimal decomposition--for the alkaline hydrolysis of Cer and G1cCer in lipid extracts of plasma.
To measure these, we applied lipid extracts to the column without deacylation and with and without the addition of the internal calibrator sphinganine.