Amino acid derivatization and analysis in five minutes

Abstract

M~rch 1991Peer Hu,~ek Re.Y¢clreh In~titltte ¢~f l~ndt~crin¢~hJ~v. Nr)rndni ttid, ,~. CS.I i6 ~4 Pr~tx,e I. C:¢chn.~lnvakla Recei red 14 December 1990 A conv©nient method i~rmits amino acid ~par~ttion t~:t capillary Ba~ chroraalogr~aphy in mim~t¢~ =~ the ~uilable anal:clio form~ are preps, reel in tt¢~ond~,Amino ~tcid ~cparatio0; Oa~ chrom;llollr~ph?Among present methods of amino acid determination by both gas.liquid chromatography (GC) and reversedphase liquid chromatography (LC) after a precolumn derivatization, there is no one method which combines rapid sample pretreatment with rapid analysis of the derivatized forms. Because in LC analysis not all the active hydrogen-containing groups need to be treated and condensation of amino group is by far the prevailing approach, the average time for sample pretreatment is shorter than in GC.Derivatization methods have been reported with time requirements ranging from nearly instantaneous derivative formation with chloroformates with a bulky fluorene moiety [1,2], over one to two minutes requirement for treatment with the popular o.phthalaldehyde [3,4], to several minutes for derivatization with, e.g. phenylisothiocyanate [5,6]. With the last mentioned reagent the most rapid analysis of a complete mixture of protein amino acids was achieved, in 12 rain, which is the shortest time for determination of amino acids by LC. However, 60 min were necessary for physiological samples of amino acids [5,71 using the same method and the one hour analysis time can be considered as an average for LC performed amino acid analyses. Unlike GC analysis, use of capillary columns in LC lengthens the total time of a chromatographic run [8,9]. When considering GC in connection with amino acid analysis rather the opposite is to be expected; rapid analysis, but slow derivatization [10]. The widely used esterification-acylation procedures require on average one hour When summarizing criteria aiming to the establishment of a hypothetic, ideal procedure of amino acid determination, the following are required: (a) simple sample handling with possibly one reagent only, (b) ability to derivatize in aqueous medium, (c) an instate. taneous or very rapid reaction course proceeding at room temperature, (d) few-minute analysis with a good resolution and (e) low reagent and instrumental costs. Reviewing the nearly 40 years spent on amino acid methodology it would be rather unrealistic to expect fulfilment of all these requirements in one single procedure.But miracles happen. With the procedure reported here we are close to that ideal. The derivatization may no longer be the Achilles heel of GC. The chemical pretreatment of the sample determines the rapidity of analysis. This method takes about one minute and requires only five microliters of an inexpensive reagent. And the chemical conversion tolerates water, even dilute hydrochloric acid.The procedure is based on the treatment of aqueous amino acid solutions with ethyl chloroformate (ECF). An approach using isobutyl ehloroformate to the same purpo...

Probing in Vivo Metabolism by Stable Isotope Labeling of Storage Lipids and Proteins in Developing Brassica napusEmbryos

“…Acidic spots were visualized with bromphenol blue. An acidic fraction of endosperm liquid was derivatized with ethyl chloroformate as O-ethoxycarbonyl ethyl esters (Husek, 1991) and separated by GC/MS (see below). The content of malic acid in endosperm liquid was measured with malic enzyme (EC 1.1.1.40, Sigma).…”

Development and Evaluation of a GC/FID Method for the Analysis of Free Amino Acids in Quince Fruit and Jam

“…[3][4][5][6] Each dried residue was dissolved in 60 µL of water and 40 µL of ethanol/pyridine (4:1); 5 µL ECF were added and the sample was vortex mixed (3 -5 s). Gas evolution (carbon dioxide) usually occurs.…”

“…This problem was solved by Husek, [3][4][5][6] who developed rapid derivatization procedures based on the treatment of amino acids with chloroformates.…”

Identification and Synthesis of a Novel Selenium−Sulfur Amino Acid Found in Selenized Yeast: Rapid Indirect Detection NMR Methods for Characterizing Low-Level Organoselenium Compounds in Complex Matrices

“…with ethyl pyrocarbonate (23), giving N-ethylcarbamate S-(methylseleno)cysteine ethyl ester (3a) and N-ethylcarbamate Se-(methylthio)selenocysteine ethyl ester (4a), respectively, which could be rigorously purified and fully characterized by spectroscopic methods. These two compounds were used as standards for GC-AED and NMR studies.…”

“…The derivatization procedure of Husek was followed (23). Diethyl pyrocarbonate (700 µL, 4.76 mmol) was added to a suspension of 6 (130 mg, 0.50 mmol) in ethanol/water/pyridine (32:60:8 v/v/v; 1.5 mL).…”