Optimisation of microwave-assisted acid hydrolysis for the determination of seleno-amino acids bound to proteins in powdered milk, lyophilized milk and infant formula

Selenium (Se) is an essential element present in milk as seleno-amino acids and seleno-proteins. To perform an accurate seleno-amino acid analysis, a complete hydrolysis of proteins is necessary. Microwave-assisted acid hydrolysis (MAAH) can be used; however, some variables must be optimised to avoid incomplete hydrolysis and oxidation. Multivariate calibration showed that the volume of the reducing agent, the concentration of the acid used, and the microwave power were statistically significant (95% confidence), and since these are the variables controlling the system, a final optimisation was performed to increase the method efficiency. This was performed using a 2(4−1) central composite design. A robustness study was performed employing the Taguchi design, which demonstrated that the optimised MAAH technique was not dependent on the protein concentration of the milk samples. Following MAAH optimisation, a 30-fold recovery improvement was obtained for seleno-amino acids, reaching a limit of detection of 0.053 µg 100g-1 for selenocysteine. Selenocysteine, selenomethionine, and seleno-methyl-selenocysteine were determined at concentration ranges of 5.06 ± 0.20 – 22.5 ± 0.90, 0.75 ± 0.03 – 19.7 ± 1.2 and 1.25 ± 0.05 – 16.3 ± 0.85 respectively, in commercial powder milk samples and infant formula, lyophilized selenium-enriched ewe milk, lyophilized human milk and reference material ERM-BD150 skimmed milk powder.