Outcome measure of plasma anandamide levels
Levels of anandamide were measured at baseline (day 0) and treatment days 14 and 28. These were determined by liquid chromatography-tandem mass spectrometry, conducted by ABS laboratories (Hertfordshire, UK). In order to extract plasma to assess plasma anandamide levels, blood was obtained using a needle and a lithium heparin anticoagulant vacuum. Plasma was obtained by immediately centrifuging blood samples using a Clifton centrifuge for five minutes at 2800 rpm, before immediate storage at −80 °C. Through this method, the lower limit of quantification for anandamide levels in plasma was 0.1 ng/ml.