Fluorescamine catecholamines

Method for catecholamines by HPLC. A few microlitres of a 10 3-Af solution of dopamine, norepinephrine or similar compounds are transferred to a small test-tube and diluted to 20 jul with 0.05 M sodium phosphate (pH 8) at 4 °C [ 102]. 10 jul of a 0.2% solution of fluorescamine in acetone are then added with vigorous shaking. An aliquot portion of this mixture is applied directly to the HPLC column. The derivatives are separated on Hitachi 3011 or 3010-OH gels (column, 50 cm X 3 mm) with methanol-0.05 M Tris-hydrochloric acid buffer of pH 8 (7 3) at room temperature at a flow-rate of 0.72 ml/min. The separation of fluorescamine derivatives of dopamine and norepinephrine with this system is shown in Fig.4.52. 

Nakamura, H. and Pisano, J.J., Specific detection of primary catecholamines and their 3-O-methyl derivatives on thin-layer plates using a fluorigenic reaction with fluorescamine, J. Chromatogr., 154, 51, 1978 Chem. Abs., 89, 117958x, 1978. 

Fluorescamine (4-phenylspiro(furan-2-(3H),r-phthalan)3,3 -dione) is also a commonly used fluorescence reagent. It reacts almost instantly and selectively with primary amines, while the excess of the reagent is hydrolyzed to a non-fluorescent product. The reagent itself is non-fluorescent. The reaction is carried out in aqueous acetone at a pH of about 8-9 and the derivatives can be chromatographed directly. The excitation and emission wavelengths are 390 nm and 475 nm respectively. Two disadvantages of the reagent are its cost and the fact the products are less stable, cannot be stored and should be injected onto the column immediately after formation. Fluorescamine has been employed in the analysis of polyamines, catecholamines and amino acids. 

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. 

Fluorescence detection is often used where no other property of the solute (e.g. UV of RI detection) is convenient and can be either an intrinsic property of the solute itself or a derivatised form of the solute. Solution studies have indicated that the sensitivity of detection can be increased by up to three orders of magnitude over UV. This has increased the popularity of post-column fluorescence detection methods for may compounds, including physiological fluids, catecholamines, and other polyamines. A popular use of fluorescence detection is in peptide chemistry where no convenient intrinsic chro-mophore is present. Derivatising agents such as orthophthalaldehyde and fluorescamine are used extensively in both pre- and post-column systems allowing detection of low picomole quantities (Chapter 11). In addition, detection can be performed using the intrinsic fluorescence of many compounds such as steroids, vitamins, and nucleotides. 

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