Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA),and oleylethanolamide (OEA), Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution, A linear correlation (r(2) = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of:anandamide, PEG and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were-in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs. (C) 2000 Academic Press.