A Solid Phase Method for the Extraction and Measurement of Anandamide, from Multiple Human Bio-matrices

N-Arachidonoylethanolamine (anandamide, AEA) was the first endocannabinoid to be identified and has since become associated with the mediation of several physiological functions and disease states. AEA has been isolated from numerous tissues and bio-fluids, in the low nM range, using lipid extraction techniques utilising organic solvents. These techniques require the drying down of relatively large volumes of solvents, which make them unsuitable for high-throughput analysis. Here we describe a solid-phase extraction (SPE) method for the investigation of AEA concentrations in human plasma, serum, milk, urine, amniotic fluid, peritoneal fluid, saliva, follicular fluid and fluid from an ovarian cyst. AEA (mean ± standard deviation) was detected in serum and plasma from blood isolated from 20 adult women (0.68 ± 0.29 and 0.64 ± 0.28 nM, respectively), from pregnant women at term (1.37 ± 0.42 nM) and from umbilical vein (1.26 ± 0.33 nM) and umbilical artery (1.14 ± 0.35 nM), in milk (0.12 ± 0.05 nM) and from amniotic (0.03 ± 0.02 nM), peritoneal (0.93 ± 0.27 nM), follicular (1.17 ± 0.51 nM) and ovarian cyst (0.32 ± 0.01 nM) fluids. AEA was undetectable in saliva and urine. The 60% AEA extraction efficiency achieved with SPE from plasma was superior to the 19% efficiency achieved using the existing organic solvent extraction method. Limits of quantification and detection for AEA were also dramatically improved using SPE (8 fmol/mL and 4 fmol/mL) compared with organic extraction (25 fmol/mL and 18.75 fmol/mL plasma). These improvements allow the use of smaller plasma samples with SPE. Intra- and inter-day variability were comparable and the mean AEA concentrations of pooled plasma samples (n = 15, 1.18nM) were identical with the two techniques. Similarly, when 56 plasma samples from labouring and non-labouring women were analysed using both techniques no extraction method-dependent differences were observed. Consequently, we provide evidence for a robust SPE technique for the extraction of AEA from bio-matrices to replace the existing liquid extraction methods which is superior in terms of speed, extraction efficiency and sample size required.