Measurement of urinary catecholamines in small samples for mice

Abstract Introduction Analysis of catecholamines in small samples of urine is difficult and sensitive to stress. Current techniques require pooling of samples or expensive separation by double mass spectrometry. A method for extraction of unconjugated catecholamines in 20 μL urine samples has been developed using alumina extraction prior to separation by high performance liquid chromatography (HPLC) and electrochemical detection (ECD). Methods Three murine experiments tested the application of the procedure. In the first, collection occurred in the morning and evening prior to handling, and in the morning after three days of handling. In the second, passively obtained urine was compared to stressfully obtained urine in the same mice. Finally, basal collections were compared to urinary catecholamine levels 15 and 30 min into novel cage stress. Urine was extracted alongside 2,3-dihydroxybenzoic acid (DHBA) internal standard via alumina and brought to pH 8.5 with tris buffer. The mixture underwent two wash steps for depuration and eluted with perchloric acid for analysis on HPLC with ECD. Results This novel extraction method using low amounts of urine yielded 48% recovery in the samples and 60% recovery in the standard extraction on average. With a signal to noise ratio of 3:1, the limit of detection (LOD) of a standard is 1.2 pg/mL, which allows for the detection of 3.6 pg/mL in urine or 72 fg in a 20 μL sample. Thus resting catecholamine levels are 216 times higher than the LOD. Unconjugated norepinephrine and epinephrine levels were significantly increased 15 min after novel cage stress and epinephrine remained elevated after 30 min, but did not show significant differences when comparing collection time, handling exposure, or specific collection technique. Discussion The technique is an effective measure for sympathetic activity in micro samples, with a limit of detection in the attomole range for 20 μL samples.