title: Feasibility of pressurization to speed up enzymatic hydrolysis of biological materials for multielement determinations.
abstract: The feasibility of pressurized solvents (liquids at a high pressure and/or high temperature without the subcritical point being reached) has been newly investigated to accelerate enzymatic hydrolysis processes of mussel tissue for multielement determinations. The target elements (Al, As, Cd, Co, Cu, Fe, Hg, Li, Mn, Pb, Se, Sr, V, and Zn) were released from dried mussel tissue by action of two proteases (pepsin and pancreatin), and they have been evaluated by inductively coupled plasma optical emission spectrometry (ICP-OES). Variables inherent to the enzymatic activity (pH, ionic strength, temperature, and enzyme mass) and factors affecting pressurization (static time, pressure, and number of cycles) were simultaneously studied by applying a Plackett-Burman design (PBD) as the screening method. Results showed that pH, ionic strength, and temperature were the most statistically significant factors (confidence interval of 95%) under pressurized conditions for pepsin, while pH and ionic strength affected pancreatin activity. This means that metal extraction is mostly attributed to enzymatic activity. The static time (enzymatic hydrolysis time) was found statistically nonsignificant for most of the elements, meaning that the hydrolysis procedure can be finished within a 2-15 min range. For pepsin, optimized conditions (pH 1.0, temperature 40 degrees C, pressure 1500 psi, static time 2 min, and number of cycles 3) gave quantitative extractions for As, Cd, Co, Cu, Hg, Li, Mn, Pb, Se, Sr, V, and Zn. The pepsin mass was 0.05 g, and the solution was Milli-Q water at pH 1.0 (adjusted with hydrochloric acid). For pancreatin, quantitative recoveries were only reached for As, Cd, Cu, Li, Pb, and Sr at room temperature, at a pressure of 1500 psi, for a static time of 2 min and a number of cycles of 3. The extraction solution was a 0.3 M potassium dihydrogen phosphate/potassium hydrogen phosphate buffer at a pH of 7.5 working at room temperature. Around 0.5 g of diatomaceous earth was used as dispersing agent for hydrolyses with either enzyme. Analytical performances, such as limits of detection and quantification and repeatability of the overall procedure, have been established. Finally, accuracy of the methods was assessed by analyzing seafood certified reference materials (GBW-08571, DORM-2, DOLT-3, TORT-2), fatty tissues certified reference materials (BCR 185, NIST 1577b), and fibrous certified reference materials (BCR 62, GBW-08501).
authors.affiliation: Department of Analytical Chemistry, Nutrition and Bromatology, Faculty of Chemistry, University of Santiago de Compostela, Avenida das Ciencias, s/n, 15782 Santiago de Compostela, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Moreda-Piñeiro
authors.forename: Adela
authors.initials: A
authors.lastname: Bermejo-Barrera
authors.forename: Pilar
authors.initials: P
authors.lastname: Bermejo-Barrera
authors.forename: Jorge
authors.initials: J
authors.lastname: Moreda-Piñeiro
authors.forename: Elia
authors.initials: E
authors.lastname: Alonso-Rodríguez
authors.forename: Soledad
authors.initials: S
authors.lastname: Muniategui-Lorenzo
authors.forename: Purificación
authors.initials: P
authors.lastname: López-Mahía
authors.forename: Darío
authors.initials: D
authors.lastname: Prada-Rodríguez
mesh_terms: D000818:Animals; D049872:Bivalvia / Q000737:chemistry; D002021:Buffers; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008670:Metals / Q000032:analysis* / Q000302:isolation & purification*; D010194:Pancreatin / Q000737:chemistry*; D010434:Pepsin A / Q000737:chemistry*; D011312:Pressure; D012997:Solvents / Q000737:chemistry; D013696:Temperature
chemical_list: D002021:Buffers; D008670:Metals; D012997:Solvents; D010194:Pancreatin; D010434:Pepsin A