Determination of Free and Sulfoconjugated Catecholamines in Plasma and Urine by High-Performance Liquid Chromatography
14 March 2008 (online)
The determination of free and sulfoconjugated catecholamines (CA) in serum and urine with amperometric detection after HPLC separation is described. The reliability of this method has been extensively investigated. Since in men 69-90% of the total CA are sulfoconjugated, an enzymatic hydrolysis of these conjugates with arylsulfatase VI has been elaborated and optimized for a routine assay. The reproducibility with coefficients of variation between 1% and 3% for free and 5% and 10% for conjugated CA and recovery rates of 65%-75% for both were found. The calibration plots were linear between 10 and 5000 ng/1 and the smallest detectable amount of CA was 0.1 nmol/1. The sample amount of 0.75 to 1.0 ml for free and 0.2 ml for conjugated CA was lower than with the extraction method (18) which needs 3 ml of serum. Using an automatic sampler, the sampling rate was 50-60 p.d. With the AI2O3 adsorption and the same buffer eluent system, L-dopa can also be detected at the same voltage of 700 mV. The values obtained for the HPLCA described method correlated well with those of the radio enzyme assays according to Da Prada et al. (7). The HPLCA detection of free and sulfoconjugated CA in urine was carried out after an ion exchange column procedure on Biorex 70. The validity of the urine assay was at least as reliable as the determination of free and conjugated CA in plasma.
catecholamines - HPLC - amperometric detection - sulfoconjugates - deconjugation by sulfatase - AV differences