Dionex rf fluorescence detector

Briefly, 50 mg sample was hydrolyzed in 1 mL 6 M HCl at 105 °C for 22 h (n = 2). Prior to quantification, the samples were neutralized by NaOH and HCl to pH 7.0 ± 0.5 and filtered through a Whatman glass microfiber filter (GF/C) using suction. The samples were diluted 1:100 with distilled water followed by a 0.22 μm filtration. Then the amino acids were quantified by High-Performance Liquid Chromatography (HPLC) (Dionex UltiMate^® 3000 HPLC+ focused, Dionex UltiMate^® 3000 Autosampler, Dionex RF Fluorescence Detector, Thermo Scientific, USA) including precolumn derivatization of the amino acids with o-phtaldialdehyde and Nova-Pak^® column (C18, 4 μm). Tryptophan was destroyed in acid hydrolysis, thus not detected. The chromatographic peaks for glycine and arginine gathered in one, therefore an average of their molar masses was used to calculate their content.
The total protein content was calculated by summing the total moles of amino acids as recommended by Angell et al. (2016) [18 (link)] and FAO (2003) [19 ] and then subtracting the water mass (18 g H₂O mol⁻¹ amino acid), which was integrated during disruption of peptide bonds in the acid hydrolysis [20 (link)].

The moisture content was analyzed by drying the carcass components (skin, meat, and frame) (2.0 g) at 104 °C for 20 h [50 ]. The ash content was determined by combusting the pre-weighed samples in a muffle furnace at 540 °C for 16 h [51 ]. Crude protein was determined using the Kjeldahl titration method [52 ] and the lipid content was determined by the Bligh and Dyer method [53 (link)]. The determination of the total amino acid composition of the freeze-dried skin sample was performed as described by Blackburn [54 ], using a reversed-phase Ultra High-Performance Liquid Chromatography (RP-HPLC) (Dionex UltiMate^® 3000 UHPLC+ Focused, Dionex UltiMate^® 3000 Autosampler, Dionex RF Fluorescence Detector, Thermo Scientific, Waltham, MA, USA) and NovaPak column (Nova-Pak C18 4 μm, 3.9·150 mm, Waters tech. Ltd, Wexford, Ireland). The method cannot detect proline, hydroxyproline, hydroxylysine, or cysteine. Glycine and arginine are determined together as their peaks merge.