In this chapter, a wide variety of derivatization reactions used for the GC analysis of amines has been surveyed. Some potential problems with derivatization procedures include the formation of unwanted derivatives, the presence of unchanged derivatization reagents, and the requirement for non-aqueous reaction conditions. Amines in environmental and biological samples are usually found in aqueous solutions except for air samples, and are often at trace levels among complex matrices containing a number of coexisting substances. It is therefore desirable that these amines be derivatized in aqueous solutions and be sensitively detected. Of the derivatization reactions presented in this chapter, silylation and acylation usually require anhydrous reaction conditions because their respective reagents are very sensitive to moisture and hydrolyze. In contrast, alkylation, halogenation and the formation of Schiff base, carbamates, sulfonamide and phosphonamide derivatives can be generally performed in aqueous solutions. GC detectors must be not only sensitive to the minute amounts of analytes, but selective enough to discriminate among coexisting substances. The nitrogen selective detector NPD and GC-MS have been used to obtain results with high sensitivity and selectivity. In addition, the conversions of amines into fluorinated derivatives to introduce ECD responses and into sulfur- and phosphorus-derivatives to introduce FPD responses have been devised for these purposes. The GC methods with selective detectors, described in this chapter, have advantages and disadvantages and to date there is no all-powerful analytical methods. The choice of an analytical method depends on the presence of amines at low parts per billion or less and the variety and complexity of the sample. To achieve the efficient isolation and preconcentration of amines in environmental and biological samples, several methods of sample preparation have been developed using a number of different purification techniques. Among these, SPME is simple and rapid, and may be applied directly to the sample in combination with derivatization. Finally, it is the author's hope that this review will serve as a guide for choosing the most effective techniques for the GC analysis of amines. Derivatization for the protection of amino groups is widely employed to increase volatility, to form diastereomers, and to improve the chromatographic and, if possible, the mass spectral properties of derivatives. In this chapter, we summarize derivatization methods for the determination of amines by gas chromatography (GC), especially with regard to the reactivity, selectivity and sensitivity of these reactions. Among the derivatives used for GC analysis are the silyl, acyl, alkyl, carbamate, sulfonamide, phosphonamide, Shiff base and thiourea derivatives of amines. Some derivatives can be selectively and sensitively detected using a variety of methods, including thermionic, flame photometric, electron capture and mass selective detectors. The recent applications of these derivatization methods to the analysis of individual aliphatic and aromatic amines in food, environmental, clinical chemistry and other areas are also described.
Summary Pre-column derivatization of amino acids with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or 4-dimethylaminoazobenzene-4′-sulfonyl chloride (dabsyl chloride) is described. These pre-column derivatization methods enable a sensitive HPLC analysis of amino acids. Dansyl derivatization has originally been applied to the sequential analysis of peptides and proteins. It finds another use in biochemistry for the fluorogenic labeling of proteins and enzymes. Dansyl chloride is the most widely used for the derivatization of amino acids. Dansyl chloride readily reacts with primary and secondary amino groups of amino acids. The reaction medium is usually an aqueous-organic mixture (e.g., 1:1 acetone-water) adjusted to a pH of 9.5-10. Dansylation reaction is usually carried out at elevated temperatures. Various dansylation reaction conditions are reported, involving 60 °C for 60 min or at 38 °C for 90-120 min. Dansyl amino acids absorb light in the UV region. For example, absorption maxima are observed at 214, 246 and 325 nm for dansyl glycine, and the absorption at 214 nm is the strongest. Dansyl group is also fluorescent and dansyl amino acids can therefore be detected by a fluorimetric detector. The excitation and emission wavelengths for dansyl glycine are 324 and 559 nm, respectively. Dansyl amino acids are mostly separated in the reversed-phase mode on a C8 or C18 column with a linear gradient. Dabsyl chloride has a number of advantages over other derivatization methods, including a simple derivatization procedure, very good stability, good reproducibility and a good limit of detection for the method, complete HPLC separation of all the amino acids, and specific detection at a wavelength in the visible region. Dabsyl chloride also reacts with primary and secondary amino groups of amino acids as does dansyl chloride. Dabsylation reagent solution can be prepared by dissolving dabsyl chloride in acetone or in acetonitrile, followed by mixing with buffer (e.g., carbonate, pH 8.5-9.5). Derivatization of amino acid with dabsyl chloride is also carried out at elevated temperatures as for dansylation. For example, samples were incubated at 70 °C for 15-30 min. There have been a number of literatures dealing with application of dansylation to the determination of amino acids contained in various sample sources, involving biological fluids, tissues, foods, peptide or protein hydrolyzates, etc. Dabsylation also covers nearly the same application areas as for dansylation. Enantiomeric separation of dansyl and dabsyl amino acids can be carried out by ligand-exchange chromatography. The use of native, and derivatized β- and γ-cyclodextrin stationary phases as well as the use of γ-cyclodextrin mobile phase additive are other options for the chiral separation of these derivatized amino acids.
Summary The recent advances in evaporative light scattering (ELS), chemiluminescent nitrogen (CLN) and mass spectrometry (MS) techniques provide new opportunities in native amino acid determination. Seven chromatographic detectors (low-wavelength UV, refractive index, conductivity detector, nuclear magnetic resonance, ELSD, CLND, MS/MS) are compatible with a direct detection of amino acids. The triple quadrupole ESI-MS/MS was found to be the most promising detector due to its high sensitivity. However, in spite of its high specificity, a high number of amino acids have to be separated due to the intrinsic limitations of MS when the simultaneous analysis of closely related chemical structures is required. By using perfluorinated carboxylic acids of longer n-alkyl chain than trifluoroacetic acid, as ion-pairing reagents in the mobile phase, high amino acid selectivities can be obtained on C8, C18 silica or porous graphitic carbon. They are efficient reagents for the separation of underivatized amino acids and are volatile enough for their use in MS/MS. LC coupled with MS has been shown to be a viable alternative to traditional detection techniques for the separation of underivatized amino acids and their determination in biological extracts without any further treatment. The use of stable isotopically labeled internal standards ensured the accuracy of quantification and eliminated matrix effects. Quantification limits for each molecule were found compatible with their measurement in plasma and urine.
Taking to heart the meaning of the proverb “Simple is best”, we have been aiming to develop a method for the determinations of biogenic amines that is so simple that almost anyone could perform it. The present platform, oncolumn derivatization CEC, has the following advantages: 1) No additional apparatus or devices are needed. 2) Biogenic amines can be separated and detected, regardless of the presence of other substances in the sample, such as amino acids. This selectivity makes unnecessary the need for any sort of pre-treatment of the sample to remove unwanted components, thereby resulting in a streamlining of the analytical procedure. 3) A high-sensitivity detection can be easily obtained with an on-line preconcentration technique. 4) Downsizing the format onto a μ-chip is possible. Although oncolumn derivatization CEC still has some problems requiring solution or improvement, I believe this will be one of the best platforms for the high-throughput determinations of biogenic amines in food or biological sample in the near future
This chapter summarizes the advances in the enantioseparation of Dns-DL-AAs by CE and CEC. The basic principle of chiral recognition with the different chiral selectors is discussed by combining the typical examples for resolving the Dns-AAs. Cu(II) complexes with Lamino acids, L-amino acylamides or other complex-forming agents, CDs or CD derivatives, chiral surfactants and other chiral selectors have been well used as the chiral selectors added in the CE background electrolytes for chirally resolving Dns-DL-AAs. With the introduction of micellar phase of anionic surfactants in the CE electrolytes, two separation modes, LE-MEKC and CD-MEKC, have been developed for improving the separation selectivity. In addition, the combination of different chiral selectors, the addition of organic modifiers, and the adjustment of separation conditions like pH, ionic strength, buffer composition were also used to manipulate the selectivity. In CEC, with the advent of monolithic column technology, chiral stationary phases have been immobilized on the monolithic matrix by in situ co-polymerization and/or post-modifications or grafting of chiral selectors. Monolithic column technology makes CEC a very powerful technique for chiral separation with high separation selectivity and efficiency. New chiral selectors and column technology are expected to develop for the chiral separation of Dns-AAs in the future
