Plasma and urinary catecholamines concentration
In each treatment period, for plasma catecholamines concentration assessment, venous blood samples (10 mL) were drawn into K2EDTA tubes, on day -1 and day 10, 30±5 minutes before the rest phase of CPT and within 2 minutes after the recovery phase of CPT. The determination of the plasma-free fractionated catecholamines DA, epinephrine (EPI), and NE was performed by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The lower limit of quantification (LLOQ) and the upper limit of quantification (ULOQ) of the method were 17.4 and 1116.8 ng/L, for DA, 17.6 and 1124.5 ng/L, for EPI, and 63.0 and 3966.2 ng/L, for NE, respectively.
For the determination of the urinary creatinine and catecholamines excretion profile, urine was collected for 24 hours, starting within 15 minutes before breakfast on days -1, 3, 6, 8, and 10, on each treatment period. The determination of urinary concentrations of total catecholamines and their metabolites was performed by high-performance liquid chromatography (HPLC) with electrochemical detection.
The catecholamines metabolites determined were: i) normetanephrine; ii) metanephrine; iii) 4-hydroxy-3-methoxy-phenylacetic acid, also named homovanillic acid (HVA); and iv) 4-hydroxy-3-methoxy-mandelic acid (HMMA), also named vanillylmandelic acid (VMA).
The 24-hour urinary catecholamines excretion profile was normalised by using the 24-hour creatinine concentration.