Determination of selected amino acids in milk using dispersive liquid–liquid microextraction and GC-MS

An ethyl chloroformate derivatisation followed by dispersive liquid–liquid microextraction (DLLME) method combined with GC-MS was developed for the analysis of free amino acids in various milk products. The method was applied to milk samples to investigate any changes in the free amino acid concentration during the shelf life of the product. Both the derivatisation and DLLME procedures were optimised using a design of experiments approach. The optimum derivatisation conditions were determined: 600 μL of both ethyl chloroformate and pyridine for a sample volume of 1 mL. The optimum DLLME conditions were 100 μL of chloroform, 0.89 mL of ACN, and 5 mL of H2O. Under these optimal conditions the method provided linearity in the range of 0.5–9.12 μg mL−1, with R2 values of >0.98. Interday reproducibility ranged from 7.8–10.30% RSD. The LOD ranged from 0.31–0.84 μg mL−1 while the LOQ ranged from 0.95–2.56 μg mL−1. The amino acid concentration was shown to increase significantly for 3 amino acids during the storage period: alanine, glycine, and glutamic acid.