A novel fully automated method for the measurement of sulphoconjugated catecholamines in urine using the Gilson ASTED-XL sample preparation system and high-performance liquid chromatography.
Dopamine is produced in the kidney where it causes sodium excretion. Dopamine sulphate is deconjugated in vivo, and may be a physiological reservoir for this active renal dopamine. To investigate the role of dopamine and dopamine sulphate in sodium homeostasis we have developed a fully automated HPLC assay for free, total and sulphoconjugated dopamine. Using the Gilson ASTED-XL sample preparation unit, with temperature controlled racks, urinary free and total dopamine are measured pre-and post-incubation with arylsulphatase. Dopamine sulphate is calculated from the difference between free and total measurements. Acidified 24 h urines are processed automatically. Free dopamine assay follows buffering to pH 7.0, addition of internal standard, addition of EDTA to stabilize free catecholamines at neutral pH, and incubation at 37 degrees C for 30 min. This mixture is trace enriched on a HEMA-SB TEC prior to ion-paired HPLC with amperometric detection. To measure total dopamine the entire process is automatically repeated with addition of arylsulphatase (400 mU/mL urine) at the beginning of the 37 degrees C incubation. The working range of the assay is up to 7 micromol/L total dopamine. Within-and between-run imprecision for dopamine sulphate is less than 3 and 7% respectively. Median dopamine sulphate excretion in 12 normotensive subjects was 4.3 micromol/24 h.
Bayly
G R
GR
Department of Clinical Biochemistry, Birmingham Heartlands Hospital, UK.
Barlett
W A
WA
Harper
L V
LV
Jones
A F
AF
D000328:Adult; D001331:Automation; D002395:Catecholamines / Q000737:chemistry / Q000652:urine*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004563:Electrochemistry; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D012016:Reference Values; D015203:Reproducibility of Results; D013464:Sulfuric Acids / Q000737:chemistry*
D002395:Catecholamines; D013464:Sulfuric Acids