Quantitation of plasma catecholamines by on-line trace enrichment high performance liquid chromatography with electrochemical detection.

A sensitive and specific procedure for quantifying plasma catecholamines using on-line trace enrichment high performance liquid chromatography with electrochemical detection (HPLC-EC) is described. Plasma catecholamines are off-line extracted using aluminum oxide and then on-line enriched on cation exchange enrichment HPLC columns and back-eluted using a potassium enriched mobile phase for further separation by ion-pair, reverse phase HPLC. The salient features of this method are (1) the on-line combination of distinct HPLC retention mechanisms for enrichment and separation of the catecholamines to yield optimal assay specificity, (2) a lower working limit of detection of 25 pg norepinephrine and epinephrine per milliliter plasma based upon an assayed plasma volume of 1 ml, (3) the generation of calibration curves using standard additions to a 'like' matrix, (4) a high daily sample throughput due to the lack of late-eluting chromatographic interferences and (5) use of an optimized aluminum oxide prepurification procedure. The advantages of this method relative to other HPLC-EC assays for plasma norepinephrine and epinephrine are discussed as are the general considerations to be addressed when defining performance characteristics of an assay.