Development and validation of a liquid chromatographic-tandem mass spectrometric method for the determination of caffeic acid phenethyl ester in rat plasma and urine.

ABSTRACT

Caffeic acid phenethyl ester (CAPE) is one of the most bioactive compounds of propolis, a resinous substance collected and elaborated by honeybees. A new liquid chromatography-electrospray ionisation tandem mass spectrometric method was developed and validated for its determination in rat plasma and urine, using taxifolin as internal standard. After sample preparation by liquid/liquid extraction with ethyl acetate, chromatographic separations were carried out with an ODS-RP column using a binary mobile phase gradient of acetonitrile in water. Detection was performed using a turboionspray source operated in negative ion mode and by multiple reaction monitoring. The method was validated, showing good selectivity, sensitivity (LOD = 1 ng/ml), linearity (5-1000 ng/ml; r > or = 0.9968), intra- and inter-batch precision and accuracy (< or =14.5%), and recoveries (94-106%) in both plasma and urine. Stability assays have shown that CAPE is rapidly hydrolysed by plasmatic esterases, which are however inhibited by sodium fluoride. The method was applied to the determination of CAPE levels in rat plasma and urine after oral administration, showing that CAPE is rapidly absorbed and excreted in urine both as unmodified molecule and as glucuronide conjugate.