Ultrasound energy has been applied to speed up enzymatic hydrolysis processes of mussel tissue in order todetermine trace and ultratrace elements (As, Al, Cd, Cr,Cu, Fe, Mn, Ni, Pb, Zn). The element releases, by actionof three proteases (pepsin, pancreatin, trypsin), lipase,and
-amylase, have been evaluated by inductively coupledplasma atomic emission spectrometry. Different variablessuch as pH, sonication temperature, ionic strength, hydrolysis time, ultrasound frequency, extracting volume,and enzyme mass were simultaneously studied by applying an experimental design approach (Plackett-Burmandesign and central composite design). Results showedthat the hydrolysis time was statistically nonsignificant(confidence interval of 95%) for most of the elements andenzymes, meaning that the hydrolysis procedure can befinished within a 30-60-min range. These hydrolysistimes are far shorter than those obtained when usingthermostatic cameras, between 12 and 24 h. Statisticallysignificant factors were the ultrasound frequency (thehighest metals releasing at high-ultrasound frequency),pH, sonication temperature, and ionic strength. All metalscan be extracted using the same operating conditions (pHof 1.0 and sodium chloride at 1.0% for pepsin; pH of 7.5,temperature at 37
C, and 0.4 M potassium dihydrogenphosphate/potassium hydrogen phosphate buffer for amylase; pH of 8.0 and 0.5 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer forpancreatin; pH of 5.0 and 0.5 M potassium dihydrogenphosphate/potassium hydrogen phosphate buffer for lipase; pH of 8.0 and 0.2 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for trypsin).Analytical performances, such as limits of detection andquantification, repeatability of the overall procedure, andaccuracy, by analyzing DORM-1, DORM-2, and TORT-1certified reference materials, were finally assessed foreach enzyme.