Feasibility of Pressurization To Speed Up Enzymatic Hydrolysis of Biological Materials for Multielement Determinations

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• 作者：Antonio Moreda-Piñ ; eiro ; Adela Bermejo-Barrera ; Pilar Bermejo-Barrera ; Jorge Moreda-Piñ ; eiro ; Elia Alonso-Rodrí ; guez ; Soledad Muniategui-Lorenzo ; Purificaci&oacute ; n L&oacute ; pez-Mah
• 刊名：Analytical Chemistry
• 出版年：2007
• 出版时间：March 1, 2007
• 年：2007
• 卷：79
• 期：5
• 页码：1797 - 1805
• 全文大小：245K
• 年卷期：v.79,no.5(March 1, 2007)
• ISSN：1520-6882

文摘

The feasibility of pressurized solvents (liquids at a highpressure and/or high temperature without the subcriticalpoint being reached) has been newly investigated toaccelerate 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, andZn) were released from dried mussel tissue by action oftwo proteases (pepsin and pancreatin), and they havebeen evaluated by inductively coupled plasma opticalemission spectrometry (ICP-OES). Variables inherent tothe 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 statisticallysignificant factors (confidence interval of 95%) underpressurized conditions for pepsin, while pH and ionicstrength affected pancreatin activity. This means thatmetal extraction is mostly attributed to enzymatic activity.The static time (enzymatic hydrolysis time) was foundstatistically nonsignificant for most of the elements, meaning that the hydrolysis procedure can be finished withina 2-15 min range. For pepsin, optimized conditions (pH1.0, temperature 40 ges/entities/deg.gif">C, pressure 1500 psi, static time 2min, and number of cycles 3) gave quantitative extractionsfor As, Cd, Co, Cu, Hg, Li, Mn, Pb, Se, Sr, V, and Zn. Thepepsin mass was 0.05 g, and the solution was Milli-Qwater at pH 1.0 (adjusted with hydrochloric acid). Forpancreatin, quantitative recoveries were only reached forAs, Cd, Cu, Li, Pb, and Sr at room temperature, at apressure of 1500 psi, for a static time of 2 min and anumber of cycles of 3. The extraction solution was a 0.3M potassium dihydrogen phosphate/potassium hydrogenphosphate buffer at a pH of 7.5 working at room temperature. Around 0.5 g of diatomaceous earth was used asdispersing agent for hydrolyses with either enzyme. Analytical performances, such as limits of detection andquantification and repeatability of the overall procedure,have been established. Finally, accuracy of the methodswas assessed by analyzing seafood certified referencematerials (GBW-08571, DORM-2, DOLT-3, TORT-2),fatty tissues certified reference materials (BCR 185, NIST1577b), and fibrous certified reference materials (BCR62, GBW-08501).