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Abstract:
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Analysis of catecholamines in small samples of urine is difficult and is greatly affected by collection methods. It requires either pooling of samples or expensive separation by double mass spectrometry. Peripheral catecholamines are increased by stress thus a reduction of stress during collection of samples from subjects is essential to decrease variability. A method for extraction of unconjugated catecholamines in 20 µL samples of urine was developed using alumina prior to analysis by high performance liquid chromatography (HPLC) with electrochemical detection (ECD). Collection occurred in the morning and evening prior to handling, and in the morning after three days of handling. After the urine was passively obtained, it was added to alumina with 0.05N hydrochloric acid with 5mM sodium metabisulfite, 2,3-dihydroxybenzoic acid (DHBA) internal standard, and brought to pH 8.5 with 0.2M tris buffer. The mixture underwent two wash steps for purification and 0.1N perchloric acid was added to elute the catecholamines. Samples were run on HPLC with ECD. This novel extraction method using low amounts of urine yielded on average between 30 to 43% recovery in the samples and between 50 to 70% recovery in the standard extraction. Unconjugated catecholamine levels did not show significant differences between the different collection times, nor between handled and unhandled techniques. Experiments are currently being conducted to compare this technique to an active collection method. |
