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Randolph Glickman, J. Christian Berry, Neeru Kumar; Effect of Storage Temperature and Antioxidant Concentration on Catecholamine Stability. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6332. doi: https://doi.org/.
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Disturbances of dopamine metabolism may be involved in several retinal disorders. Accurate measurement of dopamine and its metabolites is complicated by the lability of these compounds due to auto-oxidation. In order to support research in this area, we carried out a systematic study of the stability during storage for up to one week of dopamine (DA) and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as 3,4-dihydroxybenzylamine (DHBA, a compound used as an internal standard in catecholamine analysis), as a function of storage temperature and the presence or absence of ascorbic acid as a protective antioxidant.
The reagents used in this study were obtained from commercial vendors. The test samples consisted of mixtures of DA, DOPAC, and DHBA, 50 ng/ml each, diluted in PBS from freshly prepared stock solutions. Three groups of samples were each tested at 25°C (RT), 4°C, and -75°C. Each group contained four samples with 0, 1, 10, and 40 µg/ml ascorbic acid added, respectively. The concentration of the analytes was measured daily using HPLC with electrochemical detection at a working potential of +460 mV.
After one day at RT in the absence of AA, there was a loss of ~90% of the DA, and nearly all of the DOPAC. At one day stored at 4°C, the corresponding loss was 80% of DA and 40% of DOPAC, and at -75°C only 15% of DA and 12.5% of DOPAC degraded after one day. After six days of storage at RT, about 95% of the DA and 99% of the DOPAC had degraded, while at -75°C only 23% of the DA and 30% of the DOPAC were lost. Addition of AA improved the stability of DA and DOPAC in a concentration-dependent manner at RT and 4°C, with maximal protection at 40 µg/ml of AA: over 64% of the DA and 58% of DOPAC remained at 6 days of storage. At -75°C, after six days of storage, all concentrations of AA provided excellent protection, with virtually no DA loss, and only 15-20% loss of DOPAC. DHBA was slightly more stable than the other compounds, and was completely protected by AA plus cold storage.
Our observations show that catecholamine-containing samples are sufficiently stable at -75°C that addition of AA is not necessary, at least for storage periods of up to 1 week. In the case of longer storage periods, addition of 1 µg/ml AA is recommended for optimal stability of these labile compounds, particularly DOPAC.
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