Simultaneous quantification of free and esterified cholesterol extracted from plasma by 13C NMR spectroscopy

An efficient strategy for the simultaneous quantification of free and esterified cholesterol in a mixture by 13C NMR spectroscopy is described. It is based on the choice as internal reference of cholesteryl bromide, which has a similar chemical structure and 13C NMR spectral features to those of the analytes in the mixture. In this way, it is possible to obtain accurate results using a short recycle time and benefiting from the nuclear Overhauser enhancement. The analytical procedure has been used to quantify both free and esterified cholesterol in samples of known concentration. The measured values are in good agreement (R2 > 0.998) with the expected values, thanks to the use of several resonance's within the target molecules, yielding a statistical average of the final concentration of each analyte. The method is shown to be suitable for the quantification of esterified and free cholesterol in a crude lipid mixture extracted from human blood.