Perchloric acid 70

The collected samples were washed with Milli-Q water, length measured using a ruler, dissected, and the tissues were separated on-site, frozen, and stored at -20 °C in clean polypropylene bottles until acid digestion. The samples were weighted, lyophilized (Adolf Kühner, Birsfelden, Switzerland), and water content was calculated as performed in our previous study (Ngelinkoto et al., 2014 ). The digestion of samples was performed according to the methods described by Rashed (2001) (link), Sivaperumal et al. (2007) and Ngelinkoto et al. (2014) with minor modifications. Briefly, a portion of edible tissue from each sample was freeze-dried (Adolf Kühner, Birsfelden, Switzerland) and ground to obtain a fine powder. Approximately 1 g was digested in 10 mL of a suprapur HNO₃ (Nitric acid 65% Suprapur, Merck KGaA, Darmstadt Germany)-HClO₄ (Perchloric acid 70%, Merck KGaA, Darmstadt Germany) mixture (3:1), in Teflon bombs and heated overnight at 110 °C. The digested samples were cooled at room temperature and centrifuged. Inductively coupled plasma mass spectroscopy (ICP-MS) analysis was performed after dilution with suprapur 1% HNO₃ (Nitric acid 65% Suprapur, Merck KGaA, Darmstadt Germany). Acid dilutions were performed using ultrapure water (Millipore, Milli-Q, 18MW, Merck, Darmstadt, Germany).

All chemicals and solvents used were of analytical or HPLC grade. Ascorbic acid, Folin-Ciocalteu’s phenol reagent, gallic acid, calcium chloride, perchloric acid 70%, ammonium iron (III) sulfate dodecahydrate, ferrous ammonium sulfate hexahydrate and ortho-phosphoric acid 99% were purchased from Merck (Germany). Other chemicals such as 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent, Ferrozine, Trolox, quercetin, Nα-benzoyl-L-arginine ethyl ester (BAEE), DL-Dithiothreitol (DTT), 2,3-butanedione monoxime, L-citrulline, Bradford reagent and chloro-amidine were procured from Sigma-Aldrich (USA).