Selenium speciation in bay scallops by high performance liquid chromatography separation and inductively coupled plasma mass spectrometry detection after complete enzymatic extraction.

Selenium (Se) species, Se-methyl-seleno-cysteine (MeSeCys), seleno-cystine (SeCys2), seleno-methionine (SeMet), selenite (SeO3(2-)) and selenate (SeO4(2-)), in the three main anatomical tissues of bay scallops (Argopecten irradians), the adductor muscle, the mantle and the visceral mass, were completely released by enzymatic hydrolysis and detected by high performance liquid chromatography (HPLC) in combination with inductively coupled plasma mass spectrometry (ICP-MS). For the thorough hydrolysis of the proteins to free the Se species, bay scallop tissues were pre-treated (pre-hydrolyzed) with papain in a 1molL(-1) sodium bicarbonate solution containing 5mmolL(-1) sodium thiosulfate at 30-40°C for 24h, then hydrolyzed by the combination of Flavourzyme(®) 500 L, carboxypeptidase Y and trypsin (3+1+1) at 45°C, at a constant pH of 8.00 for 6h. Under the optimized conditions, the quantification limits of MeSeCys, SeCys2, SeMet, SeO3(2-) and SeO4(2-) were 0.69, 0.48, 0.93 0.53 and 1.22μgL(-1), respectively (equivalent to 0.14, 0.097, 0.19, 0.11 and 0.24μgg(-1) for real samples). The working curves in the concentration ranges of 2 to 500μgL(-1) were linear with all the RSD (n=5) smaller than 15% and regression coefficients greater than 0.999. The recoveries of the species for spiked samples at 4μgg(-1) (equivalent to 20μgL(-1) in the final hydrolyzates) levels all exceeded 90%. The developed method was validated by the determination of SeMet in SELM-1, a Se enriched yeast certified reference material (CRM). Selenate was the only absent species, whereas the other four species did exist in bay scallops.