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...
