Amino acids have an important role in the field of medicine, pharmacy, food, and feed industries and many more. Development of routine and accurate analysis of both L- and D-enantiomers is crucial for research in medicine and pharmacy and for quality control of foods and feed and determination their authenticity. The analysis is difficult due to the large differences in concentrations of enantiomers in biological samples, the need for chiral separation and additional step of protein and peptide hydrolysis. A partial racemisation occurs during the hydrolysis conditions, which leads to inaccurate determination of enantiomeric ratios. Hydrolysis using deuterated hydrochloric acid along with HPLC-MS/MS analysis offers a possible solution to the partial racemisation where the racemised amino acids are not included in the determination. This research consists of the development of such a method. I have prepared diastereoisomeric derivatives of amino acids using S-NIFE chiral reagent. Using reverse-phase column, I have managed to separate 35 amino acids. I have optimised the parameters of mass spectrometry detection and performed a basic validation of the method by determining reproducibility, linearity, lower limits of detection, accuracy, and the recovery of amino acids after hydrolysis. I have used the method to analyse a sample of insulin medication and a sample of protein dietary supplement. I have determined the degree of racemisation in the use of both deuterated and nondeuterated hydrochloric acid.
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