Because of its unstable nature, glutamine is normally analyzed after being deamidated to the more stable glutamic acid. However, both glutamine and glutamic acid could be easily converted to pyroglutamic acid (5-oxoproline, 2-pyrrolidone-5carboxylic acid) in solution or in the dry state1-3. The formation of pyroglutamic acid causes more complications to the already difficult task of recovering glutamine from protein hydrolysis and estimating glutamine and glutamic acid in protein hydrolysates. Conventional methods for protein composition analysis, which use acid hydrolysis, analyze glutamine, glutamic acid and pyroglutamic acid in terms of Glx (the sum of the three amino acids). Special methods, which use enzymatic hydrolysis, analyze glutamine and glutamic acid individually but fail to recognize the possible discrepancy in glutamine and glutamic acid due to the formation of pyroglutamic acid. In order to deal with these problems, it is pertinent that methods be developed so that glutamine, glutamic acid and pyroglutamic acid in hydrolysates can be measured accurately. Reports on methods for amino acid analysis are numerous; however, few of them specialize in the analysis of glutamine and glutamic acid, and fewer still are those dealing with pyroglutamic acid. The analysis of glutamine (and asparagine) can be achieved in a modification of the conventional amino acid analysis by the use of lithium citrate buffer on ion-exchange resin4vs, and the pyroglutamic acid in cosmetic products has been analyzed by the elution with sodium sulfate bulfer on a TSK gel co1umn6. In an earlier publication, we reported the use of high-performance liquid chromatography (HPLC) in the analysis of glutamine and asparagine'. In this study we (a) modified our earlier method for the analysis of glutamine and glutamic acid and developed a method using reversed-phase HPLC for the analysis of pyroglutamic acid, (b) analyzed protein hydrolysates in the presence of glutamine, glutamic acid and pyroglutamic acid before and after the deamidation of amide groups, and (c) investigated the formation of pyroglutamic acid and the stability of glutamine, glutamic acid and pyroglutamic acid.
EXPERIMENTAL
Apparatus
Analyses were performed on a Beckman Model 324 gradient liquid chromatograph equipped with a Beckman Model 421 microprocessor-controller, a Beckman