Fluorescamine, postcolumn fluorescence

Fluorescamine was developed by Weigele et al. in 1972 [8], based on the fact that strongly fluorescent pyrrolinones were formed by the reaction of ninhydrin, phenylacetaldehyde, and primary amines. The reagent, 4-phenylspiro[furan-2(3H),l -phthalan]-3,3 -dione (fluorescamine), is nonfluorescent, and it reacts with primary amines, amino acids, and peptides under aqueous conditions in a few minutes at room temperature to form intensely fluorescent substances (Figure 6.1). On the other hand, nonfluorescent derivatives are formed by the reaction of fluorescamine and secondary amino compoimds. Therefore, fluorescamine can be used for the selective determination of primary amino compounds, and the fluorophore produced by the reaction is the expected pyrrolinone. Because the reaction is sufficiently rapid and the hydrolysis products are nonfluorescent, the fluorescamine reaction is applicable for the postcolumn fluorescence derivatization of primary amino compounds [9]. The amino acids are separated by a cation-exchange column similar to the ninhydrin method, and the column effluent is mixed with an alkaline-buffered solution and fluorescamine reagent. The fluorescent derivatives are detected at 480 nm with excitation at 390 nm. 

Wetai Ion Analysis. We have reported a sensitive trace-metal analysis based upon HPLC separation of p-aminophenyl EDTA chelates and fluorescence detection by postcolumn reaction with fluorescamine (23). An application of the pyridone chemistry already discussed leads to a fluorescent-labeled EDTA (VIII). 

Fluorescence is not widely used as a general detection technique for polypeptides because only tyrosine and tryptophan residues possess native fluorescence. However, fluorescence can be used to detect the presence of these residues in peptides and to obtain information on their location in proteins. Fluorescence detectors are occasionally used in combination with postcolumn reaction systems to increase detection sensitivity for polypeptides. Fluorescamine, o-phthalaldehyde, and napthalenedialdehyde all react with primary amine groups to produce highly fluorescent derivatives.33,34 These reagents can be delivered by a secondary HPLC pump and mixed with the column effluent using a low-volume tee. The derivatization reaction is carried out in a packed bed or open-tube reactor. 

Fluorometric detection has also been employed for the determination of sulfonamides in edible animal products, because it confers the advantages of selectivity and sensitivity. Although sulfonamides possess weak native fluorescence, their sensitive lluorometric detection necessitates use of precolumn or postcolumn derivatization producing the corresponding fluorescent derivatives. The most commonly used derivatizing reagent for precolumn derivatization is fluorescamine (217, 228, 230, 238, 239), while for postcolumn derivatization fluorescamine (231), and o-phthalaldehyde (OPA), and -mercaptoethanol (219) are most often used. 

Fluorescamine reacts instantaneously with the primary amino groups of peptides, yielding a fluorescent product with an excitation peak at 390 nm and an emission band at 475 nm. It is insoluble in water and is usually prepared in acetone. Neither the reagent nor the degradation products of the excess reagent in an aqueous medium are fluorescent, which is a great advantage, particularly when postcolumn derivatization is used. 

This approach is based on the reaction of SAs with fluorescamine, yielding both highly fluorescent and relatively stable derivatives. The reaction conditions enable one to apply this technique in both pre- and postcolumn operation modes. 

Since BAs occurring in food do not exhibit satisfactory absorbance or fluorescence in the visible or ultraviolet range, chemical derivatization, either pre- (35-37) or postcolumn (38), is usually used for their detection in HPLC. The most frequently employed reagents for precolumn derivatization are fluorescamine, aminoquinolyl-lV-hydroxysuccinimidyl carbamate (AQC) (39, 40), 9-fluorenylmethyl chloroformate (FMOC) (41-43), 4-dimethylaminoazobenzene-4 -sul-fonyl chloride (dabsylchloride, DBS) (44), N-acetylcysteine (NAC) (45,46), and 5-dimethyl-amino-1-naphthalene-1-sulfonyl chloride (dansylchloride, DNS) (47,48), phthalaldehyde (PA), and orf/to-phthaldialdehyde (OPA) (49-51), together with thiols such as 3-mercaptopropionic acid (MPA) (37) and 2-mercaptoethanol (ME) (35,49). 

Fluorescamine (4-phenyl-spiro[furan-2(3Fl), l -phthalan]-3,3 -dione) Fluorescent (ex. 390 nm, em. 475 nm) 20-100 pmol 30-90 min postcolumn Secondary amine pretreatment, critical concentration, may give background interference Rapid reaction, stable product, good reagent separation... 

Not many compounds fluoresce naturally. However, some compounds, when added to another compound, cause that compound to fluoresce. These compounds are called fluorophores. Dansyl chloride and fluorescamine are two that are used to react with primary amines. o-Phthalaldehyde is used as a postcolumn reactant to produce fluorescent compounds. Other compounds are naphthalenedialdehyde (NDA), fluorescein isothiocyanate (FITC), and phenylthiohydantoin (PTH). See Chapter 19, p. 203, for more detail. 

Many reported methods for BA and AA involve pre- or postcolumn (or capillary) derivatization of these compounds before detection using 3-(4-carboxybenzoyl)-2-quinoline-carboxaldehyde," 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein, l,2-naphthoquinone-4-sulfonate," < -Phthalaldehyde (OPA), fluorescamine, and many other procedures reported in Table 30.2. OPA is disadvantageous in that it reacts only with primary amines, and the fluorescent derivatives are associated with significant instability. Dabsyl- and dansylchloride are better in this respect as they react with both primary and secondary amino groups, and provide stable derivatives. Indirect UV, indirect fluorescence detection, conductivity," and electrochemical detection have been utilized after CE separation as well as mass spectrometry Kvasnicka et al. " developed a direct,... 

For increased sensitivity, reagents that yield fluorescent products are employed. These include OPA, fluorescamine, and 4-fluoro-7-nitro-benzo-2-oxa-l, 3-diazole (NBD-F). Derivatization with OPA provides the best limits of detection for most amino acids. Although the products of OPA derivatization are unstable and decompose fairly rapidly to produce nonfluorescent 2,3-dihydro-lH-isoindol-l-ones, this is not a problem with postcolumn derivatization since the products are detected almost instantaneously. 

Fluorescamine has also been employed for postcolumn derivatization with fluorescence detection. As discussed previously, a fluorescent product is generated in the presence of primary amines within a few seconds. Fluorescamine undergoes hydrolysis, but the hydrolysis reaction is much slower than the reaction with primary amines and the product of that reaction does not fluoresce. For postcolumn addition, the reagent is added as a solution in acetone, acetonitrile, or methanol. As with OPA, proline can be detected through conversion to aminobutyraldehyde by oxidation with N-chlorosuccinimide. Unlike OPA, fluorescamine reacts poorly with ammonia. Therefore, problems with baseline drift are not as prominent. Since the reaction of fluorescamine with primary amines produces an equilibrated mixture of two products, it is used most frequently as a postcolumn reagent. 

In these systems, a high-energy intermediate excites a suitable fluorophore, which then emits its characteristic fluorescence spectrum consequently, they are termed indirect or sensitized chemiluminescence. The most common analytical application has been as a postcolumn reaction detector for liquid chromatography. Various fluorescent analytes (polycyclic aromatic hydrocarbons and polycyclic aromatic amines) and compounds derivatized using dansyl chloride, fluorescamine, or o-phthalaldehyde have been determined with sub-femtomole detection limits. 

Some fluorimetric methods for the individual determination of amino acids in foods have also been reported. Thus, the native fluorescence of tryptophan has been used for its determination in food and feed hydrolysates using ion-exchange chromatography. Also, 3-methylhistidine has been determined in meat and meat products using LC and precolumn derivatization with fluorescamine or postcolumn derivatization with OPA and 2-ME. 

Fluorescamine under the alkaline conditions rapidly reacts with primary amines and amino acids to give fluorescent derivatives at room temperature. The advantageous features of this reaction are as follows (1) fluorescamine is nonfluorescent (2) fluorescamine can be hydrolyzed to the nonfluorescent product (3) the reaction with secondary amines can form nonfluorescent derivatives, which allows selectivity to primary amines. For those reasons, fluorescamine can be applied to pre- and postcolumn derivatization of primary amino compounds with LC-FL or CZE-LIF detection. 

Tryptophan and tyrosine are intrinsic fluorophores that are present in many peptides, which then can be identified with fluorescence detection. However, most peptides have no native fluorescence, thus making derivatization a prerequisite for fluorescence detection. Derivatization has been described with naphthalene-2,3-dicarboxaldehyde-(S-mercaptoethanol for determination of substance P and its metabolites, fluorescamine, and 5-carboxyfluorescein succinimidyl ester for luteinizing hormone-releasing hormone (LHRH), neuropeptide Y, and 3-endorphin. Kostel and Lunte compared various postcolumn reactor designs, whereas Advis et al. employed precolumn derivatization, among others. In order to improve sensitivity, laser light is frequently employed for exciting the fluorescent molecules referred to as laserinduced fluorescence (LIE) and provides a 500-1000 times improved sensitivity compared to UV detection. 

---