Ultrasound Bath-Assisted Enzymatic Hydrolysis Procedures as Sample Pretreatment for the Multielement Determination in Mussels by Inductively Coupled Plasma Atomic Emission Spectrometry

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Abstract
Ultrasound energy has been applied to speed up enzymatic hydrolysis processes of mussel tissue in order to determine trace and ultratrace elements (As, Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn). The element releases, by action of three proteases (pepsin, pancreatin, trypsin), lipase, and α-amylase, have been evaluated by inductively coupled plasma atomic emission spectrometry. Different variables such 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−Burman design and central composite design). Results showed that the hydrolysis time was statistically nonsignificant (confidence interval of 95%) for most of the elements and enzymes, meaning that the hydrolysis procedure can be finished within a 30−60-min range. These hydrolysis times are far shorter than those obtained when using thermostatic cameras, between 12 and 24 h. Statistically significant factors were the ultrasound frequency (the highest metals releasing at high-ultrasound frequency), pH, sonication temperature, and ionic strength. All metals can be extracted using the same operating conditions (pH of 1.0 and sodium chloride at 1.0% for pepsin; pH of 7.5, temperature at 37 °C, and 0.4 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for amylase; pH of 8.0 and 0.5 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer for pancreatin; pH of 5.0 and 0.5 M potassium dihydrogen phosphate/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 and quantification, repeatability of the overall procedure, and accuracy, by analyzing DORM-1, DORM-2, and TORT-1 certified reference materials, were finally assessed for each enzyme.