BioProtocols--Met6

Amino Acids

Amino acid derivative: n-trifluoroacetyl-n-butyl ester (TAB)

Reagents:

1. 0.4N HCl in butanol (Regis, discontinued. BF3-butanol-Supelco )

2. Trifluoroacetic anhydride (TFA Supelco 33165-U)

3. Methylene chloride (Dichloromethane, Fisher D151-4)

Standards: unlabeled amino acids.

Procedure:

1. To a dried amino acid sample, add 200 mL butanolic HCl. Leave on heating block at 100^oC for at least one hour. Dry with nitrogen.

2. Add 100 mL methylene chloride and 25 mL TFA. Leave at room temp for 20 min. Dry under nitrogen. Transfer to ALS vials with methylene chloride.

GC/MS:

n-trifluoroacetyl-n-butyl esters of amino acids used to be separated on a packed TAB-sorb column, made by Regis. It is now discontinued, and the HP-5, or equivalent capillary columns can be used for the same application. A detailed method with masses and retention times is described for glutamate.

Comments:

Ref. 1 describes the general method for TAB derivatives of all amino acids. Ref. 2 is an example of the use of isotopomer analysis to study glutamine oxidation.

References:

1. Leimer K.H., R. H. Rice and C.W. Gehrke. Complete mass spectra of n-trifluoroacetyl-n-butyl esters of amino acids. Journal of chromatography, 141 121-144 (1977)

2. Lee W.N.P., J. Edmond, S. Bassilian and J.W. Morrow, Mass isotopomer study of glutamime oxidation and sythesis in primary culture of astrocytes. Dev Neurosci 18: 469-477 (1996)

Glutamate

Glutamate derivative: n-trifluoroacetyl-n-butyl ester

Reagents:

1. 0.4N HCl in butanol (Regis, discontinued. BF3-butanol-Supelco )

2. Trifluoroacetic anhydride (TFA Supelco 33165-U)

3. Methylene chloride (Dichloromethane, Fisher D151-4)

Standards: unlabeled glutamate.

Procedure:

1. To a dried glutamate sample, add 200 mL butanolic HCl. Leave on heating block at 100^oC for at least one hour. Dry with nitrogen.

2. Add 100 mL methylene chloride and 25 mL TFA. Leave at room temp for 20 min. Dry under nitrogen. Transfer to ALS vials with methylene chloride..