Derivative Development

Amino acids are not sufficiently volatile or stable at the temperatures required for analysis by GC. Thus, they must be converted to derivatives having the desired characteristics. It was to be no simple task to derivatize or mask the several functional groups in even the 20 proteic amino acids. Carboxy, amino, hydroxy and sulfhydryl groups all need to be converted to eliminate internal zwitterionic charges and hydrogen bonding, and thus increase the volatility of the derivatives. It was thought in those early years that the molecular mass also required to be reduced but it was later realized that this was not an absolute requirement. As new reagents became available, it was found that volatility could be significantly increased while increasing the derivative mass. Apart from the multiplicity of functional groups, it is also necessary that each group should be quantitatively converted.

The first report of amino acid analysis by gas liquid chromatography was published in 1956. Hunter, Dimick and Corse oxidized isoleucine and leucine with ninhydrin to form volatile aldehydes. These were resolved using a 10 ft long silicone oil - celite column operated isothermally at 69°C. Peaks were detected at about 44 and 48 min (Figure 1). The aldehydes were generated using 2-5 mg of each amino acid. Either of the leucines could be assayed in the presence of 10-fold quantities of the other. However, only about eight simple amino acids yield volatile aldehydes.

From this simple but momentous beginning, there followed, in the next two decades, a proliferation of reaction schemes to prepare stable, volatile amino acid derivatives. Various oxidation, hydrocracking, pyrolysis and reduction reactions were explored but significant progress was to evolve from those procedures which focused on substituting the exchangeable protons of the reactive groups. In 1957, Bayer, Reuther and Born separated glutamic acid, leucine, methionine, norleucine, norvaline, phenylalanine, sarcosine and valine methyl esters on a silicone oil-sodium caproate packing. The use of an acyl ester constituted the first report of a key component in

Table 1 Advances in gas chromatography of amino acids

1956 First GC analysis (Hunter, Dimick and Corse) 1959 Acyl amino acid alkyl esters separated (Youngs) 1965 Resolution of alanine, leucine and valine enantiomers

(Gil-Av, Charles and Fischer) 1962-79 Development of derivatization and separation procedures for the proteic amino acids (Gehrke) 1971 First single column separation of all proteic amino acids (Moss, Lambert and Diaz) 1971-76 Further improvements in resolution 1977 Development of Chirasil Val® (Frank, Nicholson and Bayer)

1989 Use of cyclodextrins for enantiomer resolution (Konig,

Krebber and Mischnick) 1991 4 min analysis of proteic amino acids (Hussek)

a derivatization strategy which would eventually prove to be successful. One year later, Bayer reported that good resolution could be achieved using N-tri-fiuoroacetyl (TFA) amino acid esters. This work represented the first use of N-TFA derivatives, representatives of a class of compounds which would feature strongly in later developments.

In the next decade, N-formyl and -acetyl derivatives were combined with a variety of alkyl esters such as methyl, ethyl, propyl, isopropyl, isobutyl, amyl and isoamyl. The work of Youngs in 1959 was the first in which N-acyl derivatives were combined with alkyl amino acid esters. N-acetyl ethyl and butyl esters of six simple amino acids were separated on

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Figure 1 Separation of 3-methylbutanal and 2-methylbutanal using a 10ft column filled with a silicone-celite mixture. (Reproduced with permission from Hunter IR, Dimick KP and JW Corse (1956) Determination of amino acids by ninhydrin oxidation and gas chromatography. Chemistry and Industry 294-295.)

hydrogenated vegetable oil. This approach was to provide the foundation for developments leading, over the next several years, to the quantitative resolution of all the amino acids in a protein hydrolysate. In 1964 Karmen and Saroff showed that excellent yields of N-TFA amino acid methyl esters were obtained when the esters were first prepared and then acylated. This general protocol remains in use.

The use of N-TFA derivatives in combination with amino acid alkyl esters was first reported by Ettre in 1962. Starting in the same year, Gehrke and his colleagues systematically studied the derivatization and chromatography of the N-TFA «-butyl amino acid esters. TFA derivatives were used in amino acid chemistry by Weygand as early as 1952 but were first applied in the context of GC analysis in 1960. In the first report, 22 naturally occurring amino acid derivatives were resolved in less than 45 min using a 2 m column packed with Gas Chrom A coated with 1% neopentyl succinate. Subsequently, the esterification reaction was simplified by using direct esterification instead of methylation followed by interesterification. Direct on-column injection and an all-glass system were demonstrated to avoid degradation of some derivatives. Rigorous exclusion of water is necessary both for complete derivatization and to prevent hydrolysis of derivatives once formed. These and other procedures developed by Gehrke formed a solid quantitative foundation for subsequent studies by others.

Continued refinement of both the reaction chemistry and the columns culminated in the complete separation of the 20 proteic amino acids in 1971. Seventeen amino acids were resolved using a 4 mm i.d. x 1.5 m glass column packed with 0.65% ethylene glycol adipate (EGA) on 80-100 mesh Chromo-sorb W-AW. The derivatives of arginine, histidine, tryptophan and cystine were separated from those of the other amino acids on a 4 mm i.d. x 1.5 m glass column packed with a mixed stationary phase of 2% OV-17 and 1% OV-210 coated on 100-200 mesh Supelcoport. In particular, histidine could be directly assayed. The two columns were operated simultaneously, resulting in an analysis time of 15-30 min. In 1979, the same derivatives were separated on a single EGA liquid phase but no significant improvement over other available procedures was obtained.

Gehrke also conducted a thorough assessment of possible sources of contamination. As detection sensitivity increased, contamination became a significant problem. At the nanogram level, contamination was shown to derive from laboratory reagents such as butanol, methylene chloride and water, and from human sources such as dandruff, fingerprints, hair, saliva and skin fragments.

The stationary phases used during the early years of development fell into three main classes: silicones, polyglycols and polyesters. Because it was difficult to separate even the proteic amino acids on a single phase, mixed phases were common. Eventually, however, the silicone phases, in nonpolar or slightly polar forms, became favoured and were essential for quantitative elution of arginine, cystine and histidine derivatives. Dual- and triple-column procedures were to give way in the search for a single column separation of the proteic amino acids. The first such resolution was achieved in 1971 by Moss, who prepared the N-heptafluorobutyryl (HFB) «-propyl esters. These were resolved on a 10 x 1/4 in glass column packed with 3% OV-1 coated on 80-100 mesh HP Chromo-sorb W (Figure 2). No quantitative data were provided. There followed other variations on the same theme. The N-HFB isoamyl (1973), isobutyl (1974) and isopropyl (1979) esters provided similar resolutions but with subtle separatory advantages depending on the relative proportions of specific amino acids present. Resolution was primarily a function of the ester, while the acyl group mainly moderated the volatility.

The search for a single-column resolution of the proteic amino acids was paralleled by a search for a single reaction which would derivatize all the functional groups present in amino acids. Trimethyl-silylation was introduced as early as 1961 by Riihl-man and Giesecke who reacted trimethylchlorosilane with amino acid salts. Six amino acids were separated in less than 30 min. A fuller account in 1963 reported that tyrosine and histidine derivatives tended to decompose in the presence of moisture or oxygen. The early reagents were generally silylated amines or monosubstituted amides and double derivative formation was a significant problem. However, newer reagents, for example bzs-(trimethylsilyl)trifiuoroacet-amide, were considerably more potent and derivatiz-ation became quantitative. In more recent work (1993), all 22 proteic amino acids, including glutamine and asparagine, which would not be present in protein hydrolysates, have been quantitatively resolved as the N(0)-£ert-butyldimethylsilyl derivatives in 41 min on a DB-1 column. The derivatives are formed in 30 min at 75°C.

Other approaches have also been used in the search for the simplest derivatization commensurate with reproducibility and stability, and with good chromatographic characteristics. Reaction with di-chlorotetrafluoroacetone forms stable 2,2-bis(chloro-difluoromethyl)-4-subst-1,3-oxazolidine-5-one derivatives. All the proteic amino acids and more than 30 other a-amino acids have been studied. However, a second reaction with HFB anhydride is required and analysis of the diaminodicarboxylic acids histidine and tryptophan required a second column.

Alkoxycarbonyl alkyl esters, specifically the iso-butoxycarbonyl methyl esters, were first prepared by Makita in 1976. Twenty proteic amino acid derivatives were separated using a dual-column system but the derivatization procedure involves multiple extraction. Arginine was first converted to ornithine. At that time, this procedure offered no significant advantage over the other protocols available. However, the method was subsequently improved so that, in 1996, all the proteic amino acid derivatives were resolved as single peaks in 9 min using a DB-17 capillary column. Serum amino acids could be assayed without any prior clean-up except for deproteiniz-ation. The isobutoxycarbonyl derivatives have also been effectively combined with tert-butyldimethyl-silyl esters.

Figure 2 Separation of N(O, S)-heptafluorobutyryl n-propyl amino acids. (Reproduced with permission from Moss CW, Lambert MA and Diaz FJ (1971) Gas-liquid chromatography of twenty protein amino acids on a single column. Journal of Chromatography 60: 134-136.)

Figure 3 Analysis of N(O, S)-ethoxycarbonyl amino acid ethyl esters on a 10 m x 0.25 mm i.d. capillary column coated with OV1701. (Reproduced with permission from Hussek P and Sweeley CC (1991) Gas chromatographic separation of protein amino acids in four minutes. Journal ofHigh Resolution Chromatography 14: 751-753.)

Figure 3 Analysis of N(O, S)-ethoxycarbonyl amino acid ethyl esters on a 10 m x 0.25 mm i.d. capillary column coated with OV1701. (Reproduced with permission from Hussek P and Sweeley CC (1991) Gas chromatographic separation of protein amino acids in four minutes. Journal ofHigh Resolution Chromatography 14: 751-753.)

In 1991, Husek prepared derivatives in the same general class but using ethyl chloroformate which reacts with all the functional groups found in amino acids. The N(O, S)-ethoxycarbonyl ethyl esters are formed in seconds in an aqueous medium. The derivatives were resolved in less than 5 min using a moderately polar OV1701 capillary column (Figure 3).

A variety of derivatization options are now available. The N-HFB isoamyl, isobutyl or isopropyl esters are equally effective for the relatively simple task of assaying the standard proteic amino acids. However, procedures requiring only a single derivatization step are more convenient and are preferred. Either the isobutoxycarbonyl methyl esters or the ethoxycar-bonyl ethyl esters can be quickly prepared and resolved in less than 10 min using moderately polar capillary columns.

The several hundred amino acids which are present in physiological fluids cannot be resolved by any single method. Each method has advantages in a specific context. Frequently, however, the target is a single or a few structurally related amino acids. In such a context, any of the methods cited above may be appropriate, depending on the specific separations required. However, methods based on alkoxycar-bonyl alkyl esters are more convenient to implement. Furthermore, some physiological samples, such as sera, can be assayed directly after deproteinization.

Very few amino acids are not amenable to being analysed by GC. Furthermore, the resolving power of capillary column chromatography cannot be matched by any other separatory medium. GC remains the method of choice for assaying amino acids in complex physiological samples.

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