Amino acids fluorimetric methods

One method that combines the good chromatographic properties with improved limit of detection is the separation of isoindole derivatives of amino acids that may be detected fluorimetrically. This method may be applied to protein hydrolysates, and used in automated format in routine analyses [22]. 

Consider one small molecule, phenylalanine. It is an essential amino acid in our diet and is important in protein synthesis (a component of protein), as well as a precursor to tyrosine and neurotransmitters. Phenylalanine is one of several amino acids that are measured in a variety of clinical methods, which include immunoassay, fluorometry, high performance liquid chromatography (HPLC see Section 4.1.2) and most recently MS/MS (see Chapter 3). Historically, screening labs utilized immunoassays or fluorimetric analysis. Diagnostic metabolic labs used the amino acid analyzer, which was a form of HPLC. Most recently, the tandem mass spectrometer has been used extensively in screening labs to analyze amino acids or in diagnostic labs as a universal detector for GC and LC techniques. Why did MS/MS replace older technological systems The answer to this question lies in the power of mass spectrometer. 

There are several compounds that will react with amino acids to give coloured or fluorescent products and as a result can be used in qualitative or quantitative methods. Fluorimetric methods are gaining in popularity and offer some important advantages over absorption spectrophotometry for amino acid analysis. 

The fluorimetric methods often offer improved specificity and sensitivity over colorimetric procedures and the quantitative assays for the aromatic amino acids tyrosine and phenylalanine illustrate this point. 

Derivatization of primary amino acids with o-phthalaldehyde (OPA) is simple and the poor reproducibility due to the instability of the reaction product can be improved by automation and the use of alternative thiols, e.g. ethanthiol in place of the 2-mercaptoethanol originally used. An alternative fluorimetric method using 9-fluoroenylmethylchloroformate (FMOC-CL) requires the removal of excess unreacted reagent prior to column chromatography. This procedure is more difficult to automate fully and results are less reproducible. However, sensitivity is comparable with the OPA method with detection at the low picomole or femtomole level, and it has the added advantage that both primary and secondary amino acids can be determined. 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

There are numerous references in the literature to the preparation of quinoxalinones as characterizing derivatives for a-keto acids. Paper chromatographic, " gas-liquid chromatographic, and fluorimetric methods have been used for the microestimation of quinoxalinones. Quinoxalinone formation is also the basis of a method for amino acid identification, as shown in Scheme 18. It has found application in... 

In a recent publication, Lindroth and Mopper (1979) have presented a method for precolumn derivatisation followed by separation on reversed phase packing materials with subsequent fluorimetric detection of the amino acid-derivatives. The method has been applied for the analysis of small volumes (100 jul or less) of seawater with separation of up to 25 amino acids within 25 min at sensitivities in the femtomolar (lO M) range. The speed of analysis together with the sensitivities attainable on a routine basis and the simplicity of the analytical instrumentation and procedure, makes the technique eminently suitable for real-time analysis in the field (Garrasi et al., 1979). It is certain that this approach is an exciting development in marine analytical organic chemistry and sets the standard for discovery of equivalent techniques to detect and quantify other biochemical compounds. 

Hydrolysis method After alkaline hydrolysis, amino acids produced are reacted with ninhydrin or with ophthalaldehyde and then measured spectrometrically or fluorimetrically (ninhydrin) 20-500 pg ml" (o-phthalaldehyde) 10-1000ngml" ... 

Proteins in foods such as milk have been the subject of much fluorimetric study involving measurement of the intrinsic fluorescence of tryptophan and tyrosine however, since proteins vary in their content of these amino acids, the fluorescence intensity also varies markedly from protein to protein and, for a single protein, with the experimental conditions. Also, the natural fluorescence of peptides is limited to peptide-containing tryptophans and tyrosines. However, the joint use of LC and derivatizing fluorogenic agents such as dansyl chloride, ninhydrin, fluorescamine, and o-phthaldialdehyde (OPA) has allowed the development of a number of methods for the determination of proteins, peptides, amino acids, and amines in food samples. 

Some fluorimetric methods for the individual determination of amino acids in foods have also been reported. Thus, the native fluorescence of tryptophan has been used for its determination in food and feed hydrolysates using ion-exchange chromatography. Also, 3-methylhistidine has been determined in meat and meat products using LC and precolumn derivatization with fluorescamine or postcolumn derivatization with OPA and 2-ME. 

The method was further extended for analysis of d- and L-amino acids in mouse kidney in a similar manner. HPLC methods, for the resolution of amino acid enantiomers that utilized a chiral stationary phase or a chiral mobile phase additive were considered expensive and less satisfactory due to the high retention results in broad peaks. The separation of FDAA derivatives followed by HPLC, as described above, was found to be much more successful as there was neither needed a post column derivatization nor fluorimetric analysis and moreover a subnanomolar sensitivity was attained [33], d- and L-enantiomers of glutamate, aspartate, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine, and histidine were derivatized with FDAA and the diastereomers were separated by 2D TLC. Each was separated except for the two spots comprising Tyr and Val, and He, Leu, and Phe. Only histidine was separated further into D- and L-diastereomers by TLC. The excess hydrolyzed FDAA moved to the front... 

More-specific methods are available for identifying and quantitating the typical, amino sugar component of heparin (and some heparan sulfate species), namely, 2-deoxy-2-sulfoamino-D-glucose. Most of these methods are based on conversion of these residues into 2,5-anhydro-D-mannose by deamination with nitrous acid (see Section VIII,2). The 2,5-anhydro-D-mannose residues may be determined either colorimetrically,52-54 or fluorimetrically.55... 

Tsanaclis et al. [33] described a reversed-phase HPLC method for determination of vigabatrin in plasma after derivatization with phthalal-dehyde. Serum was mixed with y-amino-y-phenylbutyric acid and methanol. Derivatization was carried out with phthaldehyde in borate buffer (pH 9.5) containing 2-mercaptoethanol. The resulting mixture was separated on a column (15 cm x 4.6 mm) of C18 Microsorb (5 /an) with 10 M H3P04-acetonitrile-methanol (6 3 1) as mobile phase with a flow rate of 2 ml/min and fluorimetric detection between 418 and 700 nm (excitation at 370 nm). The detection limit was 0.08 fig/ml of vigabatrin. The CVs were 9%, 5%, and 5% at 2.14, 20.1, and 83.61 fig/ml/min, respectively. Common anticonvulsant did not interfere. 

A sensitive fluorimetric assay for 2-amino-2-deoxy-D-hexoses with increased sensitivity over the Elson-Morgan and Dische-Borenfreund indole methods has been reported. The 2,5-anhydrohexoses produced by nitrous acid deamination of heparin, heparan sulphate, and 2-amino-2-deoxy-D-hexoses have been treated with 3,5-diaminobenzoic acid to give stable derivatives. Neither neutral sugars nor acetamido sugars interfere in the reaction. In a modification of an earlier procedure, equivalent colour complexes were formed by the interaction of 3-methyl 2-benzothiazoline and the 2,5-anhydrohexoses produced by deamination of 2-amino-2-deoxy-D-glucose and 2-amino-2-deoxy-D-galactose. ... 

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