Primary amino group, analysis

Abou-Ouf et al. [16] described a spectrophotometric method for the determination of primaquine phosphate in pharmaceutical preparation. Two color reactions for the analysis of primaquine phosphate dosage form, which are based on 2,6-dichlor-oquinone chlorimide and l,2-naphthoquinone-4-sulfonate, were described. The reactions depend on the presence of active centers in the primaquine molecule. These are the hydrogen atoms at position 5 of the quinoline nucleus and the primary amino group of the side chain. The method was applied to tablets of primaquine phosphate and a combination of primaquine phosphate and amodiaquine hydrochloride. 

Acylation of a / -configurated axially chiral diamine 1 containing a primary amino group with equimolar amounts of monocyclic anhydrides 2 and 4 in toluene or dichloromethane at low temperatures gives the amides 3 or 5 with good diastereoselectivity 10°. Diastereomeric ratios were determined by HPLC analysis after conversion to the methyl esters with diazomethane absolute configurations are based on chemical correlation. 

Since fluorescamine reacts only with primary amino groups, secondary amino acids do not give a fluorescent product with this reaction. A method for converting secondary amino acids into primary amines has been described for analysis using fluorescamine [87], and is based on treatment of the amino acid with N-chlorosuccinimide. The reaction involves an oxidative decarboxylation of the amino acids. This method has been incorporated into the automatic analysis of amino acids with fluorescamine [88]. The fluorescence spectra and the sensitivities are similar to those of the derivatives of the primary amino acids. 

Sanger s reagent, l-fluoro-2,4-dinitrobenzene (FDNB), which was used in the earlier days for the quantitation of primary amino groups by colorimetric determination, can also be used in the identification of amino-terminal residue, but not for sequencing. At the present time, N-terminal analysis is performed on a protein sequencer. 

Fonnation of Isoindoks. In 1971 Roth described a sensitive analytical method for amino adds based on their reaction with o-phthaldia dehyde (OPA) and a thiol, 2-mercaptoethanol (117). The reaction produces an intensely fluorescent isoindole derivative of the amino acid and is specific for the primary amino group (118,119). Subsequently, the OPA method was extended to the enantiospedfic LC analysis of amino adds by substituting an optically active (single enantiomer) thiol for 2-mercaptoethanol in the derivatization reaction (120-122). This results in the formation of two... 

This is a traditional titration that is still often used to determine compounds containing primary amino groups on the aromatic ring, with the endpoint being monitored with a Pt indicator electrode. This method, applied to the determination of pharmaceuticals (sulfonamide-based compounds, benzocaine, procaine, etc.), is rapid, and the results are in good agreement with official methods of analysis. ... 

The esterification of carboxylic acids can be provided, also, by synthetic equivalents of alcanols acetals RCH(0R )2 (with acid catalysis), ortho-esters RC(0R )3 (acid catalysis), and dialkylcarbonates C0(0R)2 (base catalysis). The series of bifunctional reagents of this type [dimethylformamide dialkylac-etals (CH3)2N-CH(0R)2] is commercially available. Besides the esterification of carboxyl groups, these compounds react with primary amino groups and, thus, are used for GC analysis of amino acids (see the entry Derivatization of Amines, Amino Acids, and Related Compounds for GC Analysis ) ... 

Reactive xenobiotic Intermediates may be covalently bound to amino acids, free or In proteins. For example the reactivity of epoxides have been studied In relation to formation of covalent bound products with the hemoglobin amino acids histidine, valine and to some extent also cysteine In dose monitor experiments (249,250). The hemoglobin must be hydrolyzed prior to analysis of histidine adducts while that Is not neccessary In order to characterize adducts to the N-termlnal valine (251). A few reference cysteine, histidine and valine xenobiotic adducts have been synthesized (250,252-254). The primary amino group of histidine Is occupied In the peptide bond, therefore this group must be protected In order, to favor tjeactlon at the other nucleo-... 

Most GC determinations of primary amino groups in organic compounds (see, for instance, refs. 103-107) are based on a widely known deamination reaction used first for the determination of amines by Van Slyke [108]. A study of the kinetics and stoichiometry of the reaction showed that for quantitative analysis the reaction conditions must be chosen in accordance with the type of organic compounds to be analysed and the correctness of the procedure must be checked with the aid of model compounds [15]. 

The development of high performance stationary phases for amino acid analysis has been vigorously pursued since the introduction of the first amino acid analyser (Spackman et al., 1950). The low UV extinction coefficient of most amino acids means that current detection methods depend upon the reaction of the primary amino group of the amino acid to yield a coloured or fluorescent derivative. The most popular reagents, referred to by their most common nomenclature, are the following ... 

The chromatographic analysis of amino acids with spectrophotometric detection usually requires the formation of derivatives, because of httle absorption of UV light above 210 run. Precolumn deiivatization is usually preferable. o-Phthalaldehyde (OPA) is the deiivatization reagent that probably has the best characteristics. It reacts with primary amino groups in the presence of a thiol at pH 9.5 and room temperature to form l-alkylthio-2-alkyl substituted isoindoles (Fig. 10.5). The derivatives show maximum absorption at 335 nm and are highly fluorescent, with excitation wavelength at 340 nm and emission at 445 nm. Mercaptoethanol has been more extensively used than other thiols for the derivatization, but the OPA-mercaptoethanol isoindoles are unstable. The stability of isoindoles is improved when A-acetyl-L-cysteine (NAC) is used instead of mercaptoethanol [12]. 

This reaction is commonly used in both qualitative and quantitative analysis of amino acids. Nineteen of the 20 protein-derived a-amino acids have primary amino groups and give the same purple-colored ninhydrin-derived anion. Proline, a secondary amine, gives a different, orange-colored compound. 

Only the nitrogen atom of the violet dye comes from the amino acid the rest of the amino acid is converted to an aldehyde and carbon dioxide. Therefore, the same violet dye is produced from all a-amino acids with a primary amino group, and the intensity of its color is directly proportional to the concentration of the amino acid present. Only proline, which has a secondary amino group, reacts differently to give a yellow dye, but this, too, can be used for analysis. 

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