Extraction and separation of urinary catecholamines as their diphenyl boronate complexes using C 18 solid-phase extraction sorbent and high-performance liquid chromatography

Abstract

The clinical utility of a one-step extraction procedure based on the retention of a diphenyl boronate–catecholamine complex on a C 18 solid-phase extraction sorbent was investigated for the measurement of urinary catecholamines. Although recoveries with the extraction procedure were optimal over a relatively broad pH range (7.5–9.5), analytical factors such as sample loading and elution flow-rates, wash step and elution conditions, the concentration of catecholamines in urine to be extracted and the type of C 18 sorbent used for extraction were found to influence the efficiency of this procedure and would therefore need to be controlled for optimal recoveries. Under optimal conditions the recovery of noradrenaline, adrenaline and dopamine from spiked urine was high and reproducible (mean recoveries were >85% for all catecholamines). The effectiveness of sample clean-up step was demonstrated by reverse phase, ion pair high-performance liquid chromatography with electrochemical detection. The method described was found to be suitable for the routine measurement of catecholamines in urine in clinical biochemistry laboratories. It has a high sample extraction throughput (40/h) and has adequate precision (between batch C.V.<8%) and sensitivity (LOD<30 nmol/l; LOQ<65 nmol/l) for all the catecholamines measured. The method has acceptable accuracy, showing a mean bias of 6.6% for noradrenaline, 7.3% for adrenaline and 6.8% for dopamine from the mean value of laboratories ( N=69) participating in an External Quality Assurance scheme for greater than 12 months.