Catecholamine Analysis in Plasma or Urine
Here we outline our highly sensitive method for the detection of norepinephrine (NE) and epinephrine (E) from plasma, tissue, or urine samples.
This method uses a simple eight-step sample preparation protocol which also includes an internal standard (dihydroxybenzylamine) to account for the recovery rate. We add the sample to a solution containing aluminum oxide, which under the basic conditions bonds the catecholamines to the aluminum. This complex is then washed repeatedly to remove any remaining contaminants from the sample. In the last step of the protocol, we recover the catecholamines using acetic acid. This sample preparation allows the concentration of NE, E, and dopamine when the sample volume is more than a few hundred microliters.
During high-stress conditions, such as blood sampling from an animal, NE and E levels are significantly elevated. For such cases, the extraction process may not be necessary because the concentrations of NE and E are much higher than the concentrations of the other analytes present in the sample. However, with human blood sampling, stress levels are normally lower and therefore, the levels of NE and E are significantly lower than compared to the animals’. The extraction process is necessary to identify the NE and E peaks among many unknown peaks present in the sample. Human blood contains more unknown compounds not found in animal blood. Thus, the extraction process is more important for the detection of NE and E from blood samples at lower concentrations. The extraction process is always required for urine samples.
This method reliably allows for a detection limit of 0.5 fmol for both NE and E in a 20 min analysis time. Typical NE and E levels in human plasma are approximately 200 pg/ml (1 pmol/ml) and 10 pg/ml (0.1 pmol/ml), respectively. Dopamine detection is also possible using this method.
To minimize the stress level on animals during blood sampling, Eicom recommends using the DR-II. This automated blood sampling system samples via a catheterized implant which significantly reduces the stress level of the animal by removing the human and handling component.
Human Blood Sampling
Healthy volunteer. Blood samples are collected from the volunteer’s arm and immediately mixed with EDTA (ethylenediaminetetraacetic acid). The samples are centrifuged within 2 hours of collection at 5,000G for 20 min. The plasma supernatant can then be stored for one day at -20˚C.
Rat Blood Sampling
Trunk blood (750 μL) from a male Sprague-Dawley rat (~300 g) was mixed in EDTA tubes. Samples were then centrifuged at 5000G for 15 min. The plasma supernatant was then used in the eight-step protocol (detailed in the Application Manual, “Sample Preparation for Catecholamine Analysis in Plasma or Urine”).
Fig. 1 – Chromatogram for a 200 pg standard mixture of NE, E, DA, and DHAB (internal standard) and a typical profile of a human plasma sample at reduced stress levels.
Fig. 2 – Typical profile of rat plasma sample with trunk blood sampling.