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 20μL 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 minutes 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 1.2pg/mL, which allows for the detection of 3.6pg/mL in urine or 72fg in a 20μL sample. Thus resting catecholamine levels are 216 times higher than the LOD. Unconjugated norepinephrine and epinephrine levels were significantly increased 15 minutes after novel cage stress and epinephrine remained elevated after 30 minutes, 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 20μL samples.

Metabolomics of fescue toxicosis in grazing beef steers

“…The steers were maintained on their respective pastures for 28 days. At baseline (time 0), 1, 2, 14, and 28 days post pasture assignment, plasma and urine were collected and processed for HRM (Li et al, 2013;Patel et al, 2015), urinary ergot alkaloid analysis (Hill et al, 2000), and plasma and urine catecholamine analysis (Smith et al, 2013;Youngblood et al, 2004). Additional details of these analyses are provided below.…”

“…Sample processing for catecholamine extraction was adapted from (Youngblood et al, 2004) for plasma and (Smith et al, 2013) for urine. Briefly, for plasma, 50 mg acid activated alumina, 500 µL sample, 500 µL 0.2 M Tris buffer (pH 9.0), and 20 µL of internal standard (epinine, 2 ng) were incubated for 15 min at room temperature on a rocker.…”

“…For the urine (Smith et al, 2013), 50 mg acid activated alumina, 1 mL 0.05 N HCl with 5 mM Na 2 S 2 O 5 , 100 µL urine, 20 µL internal standard, and 1 mL 0.2 M Tris (pH 9.0) were mixed, and the pH was adjusted to 8.5. The mixture was vortexed and incubated at room temperature for 5 min on a rocker.…”

Analysis of Catecholamines and Pterins in Inborn Errors of Monoamine Neurotransmitter Metabolism—From Past to Future

“…Furthermore, alumina has to be washed extensively after sample binding to remove interfering substances. Desorption of catecholamines is usually done by addition of acids like perchloric acid [19], phosphoric acid, formic acid [20,21], and acetic acid [22]. Taken together, the different steps required for preparation and the instability of catecholamines in contact with alumina leads to low extraction yields for l -Dopa, dopamine, DOPAC, norepinephrine, and epinephrine.…”

Comprehensive analysis of nine monoamines and metabolites in small amounts of peripheral murine (C57Bl/6 J) tissues

“…ECD is also much more specific for the monoamines than MS detectors. Therefore, although in recent decades several other detectors have been tested for the quantification of such derivatives, high-performance liquid chromatography (HPLC) with ECD is still considered one of the best choices, indicated by the high number of reports (Allen, Rednour, Shepard, & Pond, 2017;Bidel et al, 2016;Birbeck & Mathews, 2013;Ferry, Gifu, Sandu, Denoroy, & Parrot, 2014;Ganesana, Lee, Wang, & Venton, 2017;Nguyen, Aerts, Van Dam, & De Deyn, 2010;Parrot, Neuzeret, & Denoroy, 2011;Schou-Pedersen, Hansen, Tveden-Nyborg, & Lykkesfeldt, 2016;Smith, Schwartz, & Lucot, 2013;Tsunoda, 2006).…”