Dopamine plasma assays

The use of HPLC to analyze biogenic amines and their acid metabolites is well documented. HPLC assays for classical biogenic amines such as norepinephrine (NE), epinephrine (E), dopamine (DA), and 5-hydroxytryptamine (5-HT, serotonin) and their acid metabolites are based on several physicochemical properties that include a catechol moiety (aryl 1,2-dihydroxy), basicity, easily oxidized nature, and/or native fluorescence characteristics (Anderson, 1985). Based on these characteristics, various types of detector systems can be employed to assay low concentrations of these analytes in various matrices such as plasma, urine, cerebrospinal fluid (CSE), tissue, and dialysate. 

Besides the poor specificity of many of the assays used to determine plasma drug concentrations, another problem which has arisen from these studies has been the length of the "wash-out" period necessary before the patient is given the neuroleptic under investigation. As a result of the prolonged duration of blockade of dopamine receptors in the brain by conventional neuroleptics and their metabolites, it is necessary to allow a wash-out period of several weeks before the patients are subject to a pharmacokinetic study. This raises serious ethical questions. Perhaps with the advent of new imaging techniques it may be possible in the near future actually to determine the rate of disappearance of neuroleptics from the brain of the patient. This may enable the relationship between plasma concentration and clinical response to be accurately determined. 

The determination of catecholamines requires a highly sensitive and selective assay procedure capable of measuring very low levels of catecholamines that may be present. In past years, a number of methods have been reported for measurement of catecholamines in both plasma and body tissues. A few of these papers have reported simultaneous measurement of more than two catecholamine analytes. One of them utilized Used UV for endpoint detection and the samples were chromatographed on a reversed-phase phenyl analytical column. The procedure was slow and cumbersome because ofdue to the use of a complicated liquid-liquid extraction and each chromatographic run lasted more than 25 min with a detection Umit of 5-10 ng on-column. Other sensitive HPLC methods reported in the literature use electrochemical detection with detection limits 12, 6, 12, 18, and 12 pg for noradrenaline, dopamine, serotonin, 5-hydroxyindoleace-tic acid, and homovanillic acid, respectively. The method used very a complicated mobile phase in terms of its composition while whilst the low pH of 3.1 used might jeopardize the chemical stability of the column. Analysis time was approximately 30 min. Recently reported HPLC methods utilize amperometric end-point detection. 

Highly sensitive, specific, and reliable assay methods are required for measuring the normally very low concentrations of epinephrine, norepinephrine, and dopamine in plasma. Both unconjugated (free) and sulfoconjugated catecholamines circulate in human plasma and are increased in plasma from patients with pheochromocytomas. Clinical... 

Benjonothan, N. and Porter, J. C., A sensitive radioenzymatic assay for dopamine, norepinephrine and epinephrine in plasma and tissue. Endocrinology, 98, 1497-1507, 1976. 

Female rats kept under controlled environmental conditions were given a single intraperitoneal injection of saline solutions of isoproterenol (80 pg), epinephrine (80 or I60 pg), norepinephrine (80 or 160 p.g), dopamine (l mg), vasopressin (500 mU), histamine (300 jig), or 0.9 normal saline (0.2 ml). Throughout this paper all doses are expressed as weight units of free base. Ten minutes after the injection they were decapitated and the plasma separated ffom the pooled heparinized blood, was frozen and stored at -12 C until assayed for its ACTH content. The results as shown in Table 1, indicate that norepinephrine is more potent than epinephrine which is in turn more potent than isoproterenol in stimulating ACTH secretion, and that dopamine, histamine, and vasopressin in larger amounts have a similar effect. Since the potency of the catecholamines in this respect appeared to be... 

---