Comparison of different chloroformates for the derivatisation of seleno amino acids for gas chromatographic analysis

Abstract

Three chloroformate reagents, ethyl chloroformate (ECF), methyl chloroformate (MCF) and menthyl chloroformate (MenCF), have been used for the derivatisation of seleno amino acids and their performance was compared. Chromatographic parameters and the inertness of the different instrumental configurations used (gas chromatography–atomic emission detection (GC–AED), and GC–MS) were shown to have a significant influence on the detection of various seleno amino acids (selenomethione, selenoethione and selenocysteine) and some sulphur-containing amino acids (methionine, cysteine, cystine and methylcysteine) which were included in the experiments for comparison. Methyl chloroformate was the preferred derivatisation reagent, since it generally performed best in terms of derivatisation yield and reproducibility and also showed less significant conditioning effects than ethyl chloroformate. Methyl and ethyl chloroformate derivatives of selenomethionine, selenoethionine, cysteine and methionine were detectable, while the detection of the menthyl chloroformate derivatives of selenocystine and cystine was not reproducible. Overall efficiencies for the determination of selenomethionine and selenoethionine from aqueous extracts ranged from 40 to 100% for methyl chloroformate, over 30–75% for ethyl chloroformate to 15–70% for menthyl chloroformate for different series measured over a period of months. The relative standard deviation of the method for the methyl and menthyl chloroformate derivatisation ranged from 7 to 13% without internal standard and was improved to 2% for the determination of selenomethionine using selenoethionine as internal standard. This indicates that, despite the limited reproducibility of the method, its repeatability is good enough to allow accurate determination of seleno amino acids, which was also demonstrated by the analysis of selenium supplementation tablets for human diet that contained selenomethionine.