Catecholamines dansylation

V- Much effort has been expended in the development of more sensitive methods for the analysis and detection of catecholamines. They have been analyzed as the dansyl derivatives (376) or after precolumn derivati-zation with o-phthalaldehyde (377, 378). Postcolumn derivatization followed by fluorometric analysis have been described in which the fluoro-phore was formed with o-phthalaldehyde (379) or with 9,10-dimethoxyanthracene-2-sulfonate as the ion-pair (380). Several laboratories have shown the sensitivity and specificity in electrochemical detection methods (381 -383). 

TLC separation of DNS-amines is usually made on layers of silica gel with solvents covering a range of polarity, e.g., chloroform, ethyl acetate, diisopropyl ether and methanol. Seiler and Wiechmann [97] developed 30 solvent systems for the separation of DNS derivatives of over 100 biogenic amines on TLC plates of silica gel. The selective reaction of DNS-C1 with the amino group of catecholamines has been examined [98]. The drugs dopamine, norepinephrine and epinephrine are adsorbed on alumina which protects their hydroxyl groups from dansylation. The N-dansylated compounds are separated with benzene-dioxane-acetic acid (90 25 4) on layers of silica gel. 

A less costly alternative to fluorescamine is o-phthaldehyde (OPT), the derivatives of which are more stable and consequently can be stored overnight if necessary. It is used in a similar manner to fluorescamine the detection limits being about 0.1 ng ca. 4 x g/ml). OPT has been used in the analysis of dopamine, catecholamines and histamines. Other fluorescence reagents that are sometimes used include 4-bromoethyl-7-methoxycoumarin, diphenylindene, sulphonyl chloride, dansyl-hydrazine and a number of fluorescent isocyanates. 

Precolumn derivatization methods include 1,2-diphenylethylenediamine treatment, dansylation of E, NE and DA, derivatization of NE and DA by o-phthalaldehyde and mercaptoethanol and derivatization of catecholamines with 9-fluorenylmethyloxycarbonyl chloride (FMOC-Cl). Derivatization with o-phthalaldehyde increases the sensitivity of NE and DA, but E is not measured because only primary amines are derivatized. Co-analysis of catecholamines, metanephrines and other related compounds by combined electrochemical oxidation and fluorescence derivatization had also been reported. ° This approach involves sequential chromatographic separation, coulometric oxidation and final chemical derivatization with 1,2-diphenylethylenediamine to fluorescent products. 

H. Tsuchiya, M. Tatsumi, N. Takagi, T. Koike, H. Yamaguchi and T. Hayashi, High-performance chromatographic determination of urinary catecholamines hy pre-column solid-phase dansylation on alumina. Anal. Biochem., 15S, 28-33 (1986). 

Peroxyoxalate chemiluminescence reactions are analytically important (particularly in HPLC). The most commonly used reagents are bis-(2,4,6-trichlorophenyl)oxalate (TCPO) and bis-(2.4-din-itrophenyl)oxalate (DNPO). Fluorescent compounds (including anthracene, perylene, aminoanthracenes, and aminopyrenes) and suitably derivatized analytes (such as amines, steroids with dansyl chloride thiols with N-[4-(6-dimethylamino-2-benzofuranyl)-phe-nyljmaleimide and catecholamines with fluoresca-mine) can be sensitively detected. 

Most studies in TLC have been qualitative, and considerable experimentation may be necessary to obtain quantitative results, although excellent commercial layers and new equipment have aided the problem. Phenolic compounds have been analyzed by spectrophotometry off the plate at 725 nm (133) by means of Folin-Ciocalteu reagent, essentially sodium tungstate and molybdate with lithium sulfate (134). Chlorophenols have been quantified after reaction with dansyl chloride and separation (135). Channel TLC (linear relationships between spot lengths and concentration between 1 and 8 pg/spot) has been used (136). The phenols in cashew nut shell liquid have been quantified by off and on the plate methods with a flying-spot scanning procedure and densitometry (137) and the distribution of unsaturateds by TLC/MS (138). Catecholamines and their metabolites in urine have been quantified (12) and determined by on the plate fluorimetry (139). Hindered phenols have been analyzed by densitometry (140). Semiquantitative determinations of the coupling products of 4,4 -... 

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