Chromatogram catecholamines

Note The pre- and post-treatment of the chromatograms with the basic tri-ethylamine solution, which can be replaced by an alcoholic solution of sodium hydroxide [1,4] or a phosphate buffer solution pH = 8.0 (c = 0.2 mol/1) [5], serves to stabilize the fluorescence of the amino derivatives [2]. A final spraying with methanolic hydrochloric acid (chci = 5 mol/1) or 70% perchloric acid renders the detection reaction highly specific for histamine [4] and for catecholamines and indolamines [5]. 

Figure 16. Chromatogram of cyanobenz[f]isoindole derivatives of catecholamines.

Fig. I Chromatogram of catecholamines, serotonin and some metabolites together with creatinine A) examination at X = 365 nm, B) examination at A. = 254 nm.

The detection limits for catecholamines are 10 to 50 ng substance per chromatogram zone [11] and 50 ng substance per chromatogram zone for carbaryl and a series of other... 

Figure 15 Chromatograms of catecholamines obtained from (a) human and (b) Sprague-Dawley rat plasma. Peaks NE = norepinephrine E = epinephrine I = 3,4-dihydroxybenzylamine DA = dopamine. (From Ref. 88.)...

Figure 1-1 shows an example of the detection limits that can be reached with EC detection. The chromatogram shows the separation and detection of the catecholamines noradrenaline (nor), adrenaline (adr), dihydroxybenzylamine (dhba), and dopamine (dopa), using a glassy carbon electrode at a working potential of +0.6 V. The minimum detectable quantity is less than 3 Pg-... 

Fig. 3.11. HPLC of the hydroxylated products of salicylate (from Halliwell et al., 1988) 2,3-DHB, 2,3-dihydroxybenzoate 2,5-DHB, 2,5-dihydroxybenzoate RS, resorcinol SA, salicylate SU, salicylurate (a major metabolite of aspirin) CA, catecholamines, (a) Separation of a standard mixture of 2,3-dihydroxybenzoate, 2,5-dihydroxybenzoate and resorcinol (internal standard) (b) Separation of an extract of a plasma sample from a healthy individual not consuming aspirin (c) Separation of an extract of a plasma sample from a healthy individual consuming aspirin (the large peaks to the left of the chromatogram are probably catecholamines - marked CA).

Fig. 4 Some applications of pH-zone-refining CCC. (a) Separation of eight CBZ dipeptides (see Table 1) [4,8] (b) separation of amaryllis alkaloids using both the lower phase (upper chromatogram) and upper phase (lower chromatogram) as the mobile phase (see Table 1) [4,9] (c) separation of catecholamines using a ligand (see Table 2) [4,12] (d) separation of two groups of dipeptide each using an affinity ligand [4,13] (see Table 2). Continued)...

Color complexes with catecholamines are generally the least specific and least accurate methods for determining concentrations. However, highly colored complexes are often useful in the visualization of chromatograms. 

Catecholamines and amines of the histamine and tryptophan group are highly polar compounds and hence have low volatility. It is preferable, as is the case with amino acids, q.v., therefore, to form derivatives to obtain satisfactory gas-liquid chromatograms. The main problem in the analysis of urine for these amines lies not in the gas chromatographic aspect of the procedure, but rather in their isolation from the urine prior to GLC, where there may be as little as 1 /tg of amine per 100 ml of urine. It appears that this problem of extracting the amines into a sufficiently small volume of suitable solvent prior to GLC has not yet been solved satisfactorily. The fact that GLC can be used successfully to separate catecholamines through their derivatives when starting with pure substances should provide a stimulus for increased research activity in this field. 

Fig. 2 Total ion chromatograms for the separation of a catecholamine mixture on (A) a hydride-based bidentate Cig column (15 cm X 4.6 mm) and (B) a hydride-based cholesterol column (7.5 cm X 4.6 mm). Mobile phase 5 95 acetonitrile/ 25 mM ammonium formate. Solutes 1 = 1-methyl-dopa (10 xg/ml) 2 = norepinephrine (3 p,g/nil) 3 = epinephrine (10 xg/ml) 4 = dopamine (10 p-g/ml).

O-methylation of catecholamines was suggested by the reported presence of 3,4-dimethyoxyphenylethylamine (which is structurally very similar to mescaline) in schizophrenic urine [388]. The relationship between this substance, dietary factors and the so-called pink spot detected on urinary chromatograms [389] is somewhat confused. The latter is probably a complex mixture which may or may not contain 3,4-dime thoxyphenylethylamine [163, 390, 391]. Furthermore clear evidence that this substance is either a product of endogenous metabolism or that its excretion is elevated in schizophrenia is lacking [392]. 

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