Catecholamine determination by three methods: HPLC-ECD, HPLC-THI and REA.

Abstract

Three methods for the determination of plasma catecholamine (CA) were compared. The first method (HPLC-ECD) employed a HPLC equipped with an electrochemical detector. The second method (HPLC-THI) consisted of a fluorometric technique using HPLC. The third method (REA) was based on radioenzymatic assay. 1) HPLC-ECD: Sensitivity to norepinephrine (NE), epinephrine (E) and dopamine (DA) was between 5 and 10 pg/ml for each, with recoveries for each ranging from 65 to 85%. The coefficients of variation (CVs)(intra assay) for NE, E and DA were 5.0, 4.7 and 10.5%, respectively. The CVs (inter assay) were 11.5, 15.4 and 34.0%, respectively. 2) HPLC-THI: Sensitivity to NE and E was between 5-10 pg/ml for each, with recoveries for each ranging from 60 to 85%. The CVs for NE and E were approximately 10% each for intra assay and 15% each for inter assay. 3) REA: Sensitivities for NE, E and DA were all 1 pg/50 μl (20 pg/ml). Recovery for each CA ranged from 90 to 100%. The CVs (intra assay) for NE, E and DA were 9.6, 10.2 and 35.7%, respectively, and the CVs (inter assay) for NE, E and DA were 15.0, 16.3 and 40.5%, respectively. 4) There was a positive correlation between the results of these different methods. Because both the HPLC-ECD and HPLC-THI methods require at least 1 ml of plasma, one advantage of the REA method is that comparatively smaller sample volumes (50μl) produce results with approximately equal sensitivity.