HORT640 - Metabolic Plant Physiology
Amino Acid Derivatization to Heptafluorobutyryl Isobutyl (HFBI) Derivatives
Derivatization is an important part of sample preparation in GC-MS applications. Derivatization serves to convert a non-volatile compound to a volatile derivative which can be more easily
transferred to the gaseous phase by heating. Amino acids are derivatized to their N(O,S)-heptafluorobutyryl isobutyl esters as follows:
1. Transfer ion exchange purified amino sample (redissolved in 0.6 mL 60% methanol) to a 1 mL microreaction vessel. Evaporate to dryness under a stream of air.
2. Add 0.2 mL methylene chloride. Evaporate to dryness under a stream of air.
3. Prepare a 5 : 1 v/v mixture of isobutanol : acetyl chloride (e.g. add 0.2 mL acetyl chloride to 1 mL of ice-cold isobutanol; caution, this reaction is exothermic .... add acetyl chloride drop-wise to pre-chilled isobutanol in the fume hood)! This
reagent is moisture sensitive and should be prepared freshly each day.
4. Add 0.2 mL of freshly prepared isobutanol:acetyl chloride (3. above) to dried amino acid sample. Cap with cap and teflon coated septum. Vortex. Heat at 120oC for 20 min in a pre-heated heating block.
5. After 20 min, remove vials from heating block. Allow to cool to room temperature, remove caps and septa, and evaporate to dryness under a stream of air.
6. Add 0.1 mL heptafluorobutyric anhydride in the fume hood. Cap vials with caps and teflon-coated septa. Mix and heat at 120oC for 10 min.
7. After 10 min, remove vials from heating block. Allow to cool to room temperature, remove caps and septa, and evaporate to the point of dryness under a stream of air (do not allow to dry beyond incipient dryness .... the more volatile amino acid
derivatives will be lost!).
8. Remove from air stream, add 0.05 to 0.2 mL of ethyl acetate : acetic anhydride (1 : 1 v/v). Cap vials with caps and teflon-coated septa. Mix, and analyze 1 uL to 2 uL samples by GC and/or GC-MS. Samples can be stored at room temperature for several
days. The methionine derivative is the most labile, and it is recommended that samples be analyzed within 24 h after derivatization when methionine is to be quantified.
Brunk DG, Rhodes D 1988 Amino acid metabolism of Lemna minor L. III. Responses to aminooxyacetate. Plant Physiol. 87: 447-453.
Golan-Goldhirsh A, Hogg AM, Wolfe FH 1982 Gas chromatographic analyses of the free amino acid pool of the potato and gas chromatography-mass spectrometry identification of gamma-aminobutyrate and ornithine. J. Agric. Food Chem. 30: 320-323.
Hall NT, Nagy S 1979 Response amplification of histidine in gas-liquid chromatography analysis of amino acid mixtures. J. Chromatog. 171: 392-397.
Husek P 1982 Gas chromatography of cyclic amino acid derivatives: A useful alternative to esterification procedures. J. Chromatog. 234: 381-393.
Huang Z-H, Wang J, Gage DA, Watson JT, Sweeley CC, Husek P 1993 Characterization of N-ethoxycarbonyl ethyl esters of amino acids by mass spectrometry. J. Chromatog. 635: 271-281.
Lapidot A, Nissim I 1980 Application of nitrogen-15 GCMS in metabolic studies of amino acids in man. Advances in Mass Spectrometry 8B: 1142-1154.
MacKenzie SL, Tenaschuk D 1974 Gas-liquid chromatography of N-heptafluorobutyryl isobutyl esters of amino acids. J. Chromatog. 97: 19-24.
MacKenzie SL, Tenaschuk D 1979 Quantitative formation of N(O,S)-heptafluorobutyryl isobutyl amino acids for gas chromatographic analysis. I. Esterification. J. Chromatog. 171: 195-208.
Makita M, Yamamoto S, Kiyama S 1982 Improved gas-liquid chromatographic method for the determination of protein amino acids. J. Chromatog. 237: 279-284.
Mayer RR, Cherry JH, Rhodes D 1990 Effects of heat shock on amino acid metabolism of cowpea cells. Plant Physiol. 94: 796-810.
Moodie IM 1981 Gas-liquid chromatography of amino acids: The heptafluorobutyryl-isobutyl ester of tryptophan. J. Chromatog. 208: 60-66.
Moodie IM, Burger J 1981 Gas-liquid chromatography of amino acids: Columns and methodology as a basis for routine amino acid analysis using capillary gas chromatography. Journal of High Resolution Chromatography & Chromatography Communications 4: 218-212.
Rhodes D, Deal L, Haworth P, Jamieson GC, Reuter CC, Ericson MC 1986 Amino acid metabolism of Lemna minor L. I. Responses to methionine sulfoximine. Plant Physiol. 82: 1057-1062.
Rhodes D, Handa S 1989 Amino acid metabolism in relation to osmotic adjustment in plant cells. In (JH Cherry, ed) "Environmental Stress in Plants: Biochemical and Physiological Mechanisms." NATO ASI Series G: Ecological Sciences, Vol. 19, Springer-Verlag, Berlin, pp. 41-62.
Rhodes D, Handa S, Bressan RA 1986 Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol. 82: 890-903.
Rhodes D, Hogan AL, Deal L, Jamieson GC, Haworth P 1987 Amino acid metabolism of Lemna minor L. II. Responses to chlorsulfuron. Plant Physiol. 84: 775-780.
Rhodes D, Myers AC, Jamieson GC 1981 Gas chromatography-mass spectrometry of N-heptafluorobutyryl isobutyl esters of amino acids in the analysis of the kinetics of [15N]H4+ assimilation in Lemna minor L. Plant Physiol. 68: 1197-1205.
Rhodes D, Rich PJ, Brunk DG 1989 Amino acid metabolism of Lemna minor L. IV. 15N-Labeling of the amide and amino groups of glutamine and asparagine. Plant Physiol. 89: 1161-1171.
Siezen RJ, Mague TH 1977 Gas-liquid chromatography of the N-heptafluorobutyryl isobutyl esters of fifty biologically interesting amino acids. J. Chromatog. 130: 151-160.