AF4-UV-ICP-MS for detection and quantification of silver nanoparticles in seafood after enzymatic hydrolysis

A method based on asymmetric flow field-flow fractionation (AF4) coupled to ultraviolet-visible (UV-vis) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) has been developed for silver nanoparticles (Ag NPs) detection and quantification in bivalve molluscs. Samples were pre-treated using a conventional enzymatic (pancreatin and lipase) hydrolysis procedure (37 degrees C, 12 h). AF4 was performed using a regenerated cellulose (RC) membrane (10 kDa, 350 mu m spacer) and aqueous 5 mM Tris-HCl pH = 7.4 as carrier. AF4 separation was achieved with a program that included a focusing step with tip and focus flows of 0.20 and 3.0 mL min(-1), respectively, and an injection time of 4.0 min. Elution of different size fractions was performed using a cross flow of 3.0 mL min(-1) for 15 min, followed by linear cross flow decrease for 7.5 min, and a washing step for 9.4 min with no cross flow. Several bivalve molluscs (clams, oysters and variegated scallops) were analysed for total Ag content (ICP-MS after microwave assisted acid digestion), and for Ag NPs by the method presented here. Results show that Ag NPs are detected at the same elution time than proteins (UV monitoring at 280 and 405 nm), which suggests a certain interaction occurred between Ag NPs with proteins in the enzymatic extracts. AF4-UV-ICP-MS fractograms also suggest different Ag NPs size distributions for selected samples. Membrane recoveries, determined by peak area comparison of fractograms with and without application of cross flow, were within the 49-121% range. Confirmation of the presence Ag NPs in the investigated enzymatic extracts was demonstrated by SEM after an oxidative pre-treatment based on hydrogen peroxide and microwave irradiation.