Amino acid analysis high-performance liquid chromatography

Modern tHcy methods include enzyme immunoassays and chromatographic-based methods. In practice, immunoassays " are most often used for routine purposes (e.g., fluorescence polarization immunoassay IFPIA] as run on Abbott s IMx and AxSYM platforms) Chromatographic assays include amino acid analysis high-performance liquid chromatography (HPLC) with ultraviolet, fluorescence, or electrochemical detection ° " capillary electrophoresis with fluorescence detection gas chromatography-mass spectrometry (GC-MS) and liquid chromatography with tandem MS (MS-MS). 

S Weinstein, MH Engel, PE Hare. The enantiomeric analysis of a mixture of all common protein amino acids by high-performance liquid chromatography using a new chiral mobile phase. Anal Biochem 121, 370, 1982. 

SA Cohen, DP Michaud. Synthesis of a fluorescent derivatizing reagent, 6-aminoquinolyl-A -hydroxysuccinimidyl carbamate, and its application for the analysis of hydrolysate amino acids via high-performance liquid chromatography. Anal Biochem 211 279-287, 1993. 

Larsen, B. R. and West, F. G., A method for quantitative amino acid analysis using precolumn o-phthaladehyde derivatization and high performance liquid chromatography, /. Chromatogr. Sci., 19, 259, 1981. 

Heinrikson, R. L. and Meredith, S. C., Amino acid analysis by reverse-phase high-performance liquid chromatography precolumn derivatization with phenylisothiocyanate, Anal. Biochem., 136, 65, 1984. 

Desai,M.J., Armstrong, D.W. (2004). Analysis of native amino acid and peptide enantiomers by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. J. Mass. Spec. 39, 177-187. 

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. 

Because of these ever-widening interests, the measurement of plasma tHcy is undertaken in many clinical chemistry and routine laboratories. Various methods are employed, including high-performance liquid chromatography (HPLC) assays, conventional amino acid analysis, capillary electrophoresis, gas chromatography with or without mass spectrometry, liquid chromatography with tandem mass spectrometry, and in many routine clinical chemistry laboratories immunoassays. In this chapter, those methods that are often available in laboratories involved in the investigation of inborn errors of metabolism are described, namely HPLC and tandem mass spectrometry. 

Even more versatile than the dansyl method is the Edman method (Figure E2.4). The NH2-terminal amino acid is removed as its phenylthiohydan-toin (PTH) derivative under anhydrous acid conditions, while all other amide bonds in the peptide remain intact. The derivatized amino acid is then extracted from the reaction mixture and identified by paper, thin-layer, gas, or high-performance liquid chromatography. The intact peptide (minus the original NH2-terminal amino acid) may be isolated and recycled by reaction with phenylisothiocyanate. Since this method is nondestructive to the remaining peptide (aqueous acid hydrolysis is not required) and results in good yield, it can be used for stepwise sequential analysis of peptides. The method is now automated. 

Evaporate a 5- ll aliquot to dryness under a vacuum. Redissolve the residue in a suitable buffer prior to analysis by an amino acid analyzer or into the appropriate eluant if high performance liquid chromatography (HPLC) analysis is used. 

MF Maimer, LA Schroeder. Amino acid analysis by high-performance liquid chromatography with methanesulfonic acid hydrolysis and 9-fluorenylmethylchloroformate derivatization. J Chromatogr 514 227-239, 1990. 

JF Davey, RS Ersser. Amino acid analysis of physiological fluids by high-performance liquid chromatography with phenylisothiocyanate derivatization and comparison with ion-exchange chromatography. J Chromatogr Biomed Appl 528 9-23, 1990. 

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