Aspartame derivatization

That derivatization may increase rather than decrease peptidase-catalyzed degradation is illustrated with aspartame (6.79, R = MeO), the C-terminal methyl ester of the dipeptide Asp-Phe. The metabolism of this artificial sweetener was compared to that of the underivatized dipeptide (6.79, R = H) and of the corresponding amide Asp-Phe-NH2 (6.79, R = NH2) in microvillar membranes obtained from human duodenum, jejunum, and ileum [189]. The activities monitored were clearly those of peptidases as shown by the effects of inhibitors. Whereas the peptide bond in Asp-Phe and Asp-Phe-NH2 was hydrolyzed at a comparable rate, that in aspartame was hydrolyzed approximately twice as fast. This is an interesting and favorable situation, given that aspartame is expected to be degraded once it has elicited its effect in the buccal cavity. 

CE has been applied extensively for the separation of chiral compounds in chemical and pharmaceutical analysis.First chiral separations were reported by Gozel et al. who separated the enantiomers of some dansylated amino acids by using diastereomeric complex formation with Cu " -aspartame. Later, Tran et al. demonstrated that such a separation was also possible by derivatization of amino acids with L-Marfey s reagent. Nishi et al. were able to separate some chiral pharmaceutical compounds by using bile salts as chiral selectors and as micellar surfactants. However, it was not until Fanali first showed the utilization of cyclodextrins as chiral selectors that a boom in the number of applications was noted. Cyclodextrins are added to the buffer electrolyte and a chiral recognition may... 

Aspartame has been quantified by UV detection at 254 nm and at 200-217 nm. However, because aspartame has a relatively low extinction at 254 nm, quantification at lower wavelengths provides increased response. Detection can also be performed with increased specificity by fluorescence after postcolumn derivatization with o-phthaldehyde (76). 

K Hayakawa, T Schilpp, K Imai, T Higuchi, OS Wong. Determination of aspartic acid, phenylalanine and aspartylphenylalanine in aspartame-containing samples using a precolumn derivatization HPLC method. J Agric Food Chem 38(5) 1256-1260, 1990. 

Additives that specifically interact with an analyte component are also very useful in altering the electrophoretic mobility of that component. For example, the addition of copper(II)-L-histidine (12) or copper(II)-aspartame (54) complexes to the buffer system allows racemic mixtures of derivatized amino acids to resolve into its component enantiomers. Similarly, cyclodextrins have proven to be useful additives for improving selectivity. Cyclodextrins are non-ionic cyclic polysaccharides of glucose with a shape like a hollow truncated torus. The cavity is relatively hydrophobic while the external faces are hydrophilic, with one edge of the torus containing chiral secondary hydroxyl groups (55). These substances form inclusion complexes with guest compounds that fit well into their cavity. The use of cyclodextrins has been successfully applied to the separation of isomeric compounds (56), and to the optical resolution of racemic amino acid derivatives (57). 

Different mechanisms have been devised to achieve chiral resolution by electromigration, but often we have to deal with mixed-mode separations rather than pure processes. In any case, chiral resolution results from stereospecific interactions of a chiral selector, with the enantiomers of the compound giving rise to a difference in migration velocity between the two entities. Chirally selective ligands, such as Cu(II)-L-histidine and Cu(II)-aspartame, have been used for derivatized amino acid mixtures. 

Gas chromatography Acesulfame-K, aspartame, cyclamate, saccharin, and stevioside are determined by gas chromatography, but the main drawback of this technique is that a derivatization is required. Acesulfame-K is methylated with ethereal diazomethane, aspartame is converted into its N- 2-methylpropoxycarbonyl) methyl ester derivative, menthol and isobutyl chloroformate are used to convert aspartame to 3-[(isobutoxycarbonyl)amino]-4-[[a-(methoxycarbonyl)phenethyl]amino]-4-oxobutyric acid, cyclamate is determined as cyclohexene resulting from the reaction with nitrite, saccharin is converted to N-methylsaccharin, and stevioside is hydrolyzed. Detection is carried out utilizing flame-ionization, flame-photometric electron-capture detectors or nitrogen-phosphorus detection. 

A new chiral reagent, NSP-Cl was synthesized and used to derivatize amino acids and the resulting diastereomers were resolved by TLC (166), Chiralplate with MeCN-MeOH-HjO (4 1 1) as mobile phase was used to evaluate reaction products in the synthesis of modified phe and tyr derivatives (167), and also to separate aspartame and its precursor stereoisomers (168). Tryptophans and substituted tryptophans were separated on cellulose layers developed with copper sulphate solutions (169) when excess of Cu " ions decreased the chiral discrimination of the system, and with aq a-cyclodextrin (1 to 10%) plus NaCl solutions (O.IM, 0.5M, l.OM) when the best results were... 

NaOH aqueous solution. The use of copper electrodes permitted the direct detection of amino acid species in CE at constant applied potentials and without derivatization. The detection limits they obtained varied from 1 to 10 fmol for most amino acids. The feasibility and the application of the approaches they developed have been demonstrated included the determination of amino acids in human urine, of the dipeptide aspartame in diet soft drinks, and of pentapeptide products of a solid-phase synthesis procedure. 

Cabero et al. [80] developed a method based on the conversion of cyclamate to cyclo-hexylamine and the subsequent reaction with l,2-naphthoquinone-4-sulfonate, yielding a spectrophotometrically active derivative, which is detected at 480 nm thus, other sweeteners, such as saccharin or aspartame, do not interfere in these determinations. The hydrolysis step is performed batchwise by treatment of cyclamate with hydrogen peroxide and hydrochloric acid, while the cyclohexylamine derivatization is carried out in the flow injection system (Figure 24.9). Rocha et al. [81] reported a flow system based on multicommutation for fast and clean determination of cyclamate. The procedure exploits the reaction of cyclamate with nitrite in an acidic medium and the spectrophotometric determination of the excess of nitrite by iodometry. The flow system was designed with a set of solenoid micropumps to minimize reagent consumption and waste generation (Figure 24.10). 

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