Amino compounds derivatization

The principal directions of amino compound derivatization for GC analysis include the following types of chemical reactions ... 

While amino compounds can be derivatized with acids and acid chlorides, it is possible to separate racemic acids (vice versa) with unichiral amino compounds... 

Both -NH2 and -COOH groups are hydrophilic esterifying the -COOH group under acidic conditions would render surfactant properties to the final derivative (Figure 3). If both the -COOH and -NH2 functionalities of the amino acid are derivatized, making it a secondary amino compound, then it can be used as fuel stabilizer, although the exact mechanism of -NH- as free radical stabilizer is still uncertain. 

ALL Duchateau, H Knuts, JMM Boesten, JJ Guns. Enantioseparation of amino compounds by derivatization with o-phthalaldehyde and D-3-mercapto-2-methylpropionic acid. J Chromatogr 623 237-245, 1992. 

Table 2 Physicochemical and GC Properties of Some Carbonyl Reagents and Their Analogs for Derivatization of Amino Compounds...

The selection of derivatization methods for amides and imides is not as great as for other classes of amino compounds. The active hydrogen atom in the structural fragments —CO — NH— or SO2 — NH— is highly acidic and, hence, sometimes recommended TMS, acetyl, or TEA derivatives of these compounds are unstable during hydrolysis. The best derivatization method is their exhaustive alkylation (preferably methylation), because permethylated amides and imides are volatile enough for their GC analysis. This general statement can be illustrated by retention data... 

The second group of hydroxy compound derivatization reactions includes acylation of OH groups with the formation of esters. The most important are listed below (for the table of physicochemical and gas chromatographic constants of acylation reagents refer to the entry Derivatization of Amines, Amino Acids, Amides, and Imides for GC Analysis). 

As mentioned in Section 3.1, the derivatization of luminol results in a much reduced quantum yield. On the other hand, isoluminol compounds (derivatized at the 6-amino position) exhibit higher quantum yields than the parent compound. This fact dictates the way in which each compound is utilized as an analytical agent. Compounds such as ABEl (Fig. 14) can be covalently conjugated to a variety of substances, ranging in size and complexity from steroid hormones to immunoglobulins. These chemiluminescent labels can be subsequently utilized in immunoassays according to the general scheme outlined in Section 1.3.1. 

For a review on the preparation of derivatives prior to TLC, see Ref. 41. Derivatization is frequently used in the TLC of amino acids where certain amino acids are easily converted into the so-called DANS forms (amino acid derivatives on silica gel. Derivatives are also used when a volatile compound can be changed into a nonvolatile compound, or when the detection sensitivity of the derivative is greater than that of the original compound. Derivatization is advantageous as a clean-up procedure when it is easier to clean up the derivative than the original compound. 

The coupling of the CZE step to detection systems other than UV has required the development of separation conditions compatible to the detection system used. For instance, the presence of primary amines, such as DAB, in buffers needed to be avoided for compatibility with laser-induced fluorescence (LIE) of compounds derivatized with fluorogenic substrates through their amino groups [90]. Baseline resolution of eight peaks in approximately the same time was achieved by substituting DAB by morpholine and tricine by boric acid (to avoid potential traces of primary amines in the tricine buffer) and by adjusting the concentration of other buffer components to compensate for the increase in electrical current. In the same work, modifications were also required to achieve compatibility with MS detection where nonvolatile salts, urea, and amines should be usually avoided. A physically adsorbed polyethylenimine-coated capillary was used to overcome protein adsorption to the capillary walls in the absence of cationic additives and the use of an acetate buffer at pH 5.05 allowed the partial resolution of at least five bands of rhEPO. Other types of coated capillaries have been used for the analysis of EPO by CE-MS as detailed in Section 22.4.3.3 [30,37,42,62,96]. 

The number of reagents for derivatization of amino compounds is larger than for any other class of compounds. Two of the most useful reagents for precolumn fluorescence labeling m liquid chromatography are dansyl chloride and o-phthaldialdehyde (OPA). 

Gentamicin, a polyfiinctional amino compound, was determined by using RP-HPLC after labelling with ben-zenesulphonyl chloride [20]. The derivatization reaction was complete in 10 min at 75 °C. The HPLC separation was carried out on a Cjg column with acetonitrile/ methylene dichloride/water/methanol as the mobile phase. Separation of gentamidn isomers was not achieved. 

For urinary amino adds [22], 0.5 ml of urine was mixed with 0.5 ml of 0.5 M sodium bicarbonate and 2 ml of DABSCl (2 g P in acetone). The reaction mixture was maintained at pH 8.5-9.0 and 25-26 "C for 30 min. The filtrates were directly applied to a Cjg column for HPLC separation. Interfering amino compounds present in the urine also form derivatives with DABSQ, but may be removed by extraction with chloroform. Ammonia reacts readily with DABSCl, resulting in a derivative coeluted with DABS-methionine. Furthermore, ammonia may consume so much DABSCl that it seriously interferes with the derivatization of amino acids. Therefore the ammonia should be removed by lyophilizing the samples at pH 8.9 before derivatization. This method allowed the detection of urinary amino acid down to concentrations of about 16mgl ... 

Nitrobenzenes are most commonly used for the derivatization of amino compounds. The 4-nitrobenzoyl group has been shown to impart high UV absorptivity to amines. l-Fluoro-2,4-dinitrobenzene has been used as a label for aminoglycosides (Figure 5) such as neomycin [25, 26], fortimicin A [27], amikacin [28, 29], tobramycin [30-33], gentamicin and sissomicin [31-33]. 

The derivatization of 6-amino-2-methyl-2-heptanol (heptaminol) with 4-N,N -dimethylaminoazobenzene-4 -isothiocyanate was used for the HPLC determination of this amino compound in pharmaceutical preparations [63,64]. Labelling with p-phenylbenzoyl isothiocyanate [65] may also be a good technique for the HPLC determination of amines. 

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. 

Tris(2,2 -bipyridyl)ruthenium(n) (Rulbyp) ) is used as the electrogenerated CL (ECL) reagent for the amino compounds in the LC-CL detection system. The CL reaction is supposed to proceed as follows initially, the oxidation of Rulbypls to Ru(byp) is performed at the electrode surface, and then Ru(byp) reacts with amines to emit light at 620 nm. The ECL intensity is proportional to the amount of amines, and the order of increasing CL intensities is tertiary > secondary > primary amines. Dansyl derivatization of primary, secondary, and tertiary amines are used to increase the sensitivity. DNS-Glu can be determined by ECL with the detection limit of 2 pmol. 

Hydroxy compoimds such as alcohols, carbohydrates, steroids, and phenols are known to play important physiological roles. Although many analytical reagents have been developed for determination of hydroxy compounds, some of them can be also applied to amines as described in the previous section (e.g., isocyanates, sulfonyl chlorides, acyl chlorides, halogenobenzofurazans, acyl amides, and chloroformates). The derivatization conditions for hydroxy compounds are relatively more drastic than those for amines. By using fairly different conditions from those for amino compounds. 

Table 3 Physicochemical and gas chromatographic properties of some derivatization reagents for acylation of amino compounds.

The derivatization methods available for amides and imides is not so vast as for other classes of amino compounds (remember that numerous amides themselves are used as the target analytical derivatives of amino compounds). The active hydrogen atoms in the stmctural fragments -CO-NH- or SO2-NH- are rather acidic and, hence, sometimes the recommended acetyl or TFA derivatives of these compounds (with additional acidic protection groups) are unstable with respect to hydrolysis. 

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