Hplc gradient grade acetonitrile

Materials used in this study were corn grits, and hazelnut, camelina, pumpkin, and hemp seed press cakes. Press cakes were obtained by screw pressing in a screw expeller (Model SPU 20; Senta, Serbia) and were defatted via supercritical CO₂ extraction (custom-made extractor described by Jokić et al. [41 ]). These processes are described in detail in our previous article, with the same preparation for usage in the extrusion process, along with the approximate composition [18 (link)].
Gallic acid, Folin–Ciocalteu, DPPH, high-purity acrylamide, ¹³C₃-acrylamide, and 5-hydroxymethylfurfural were purchased from Sigma-Aldrich (Diesenhofen, Germany). HPLC gradient grade acetonitrile and methanol were purchased from J.T. Baker (Deventer, The Netherlands).

High purity acrylamide, ¹³C₃-acrylamide and 5-hydroxymethylfurfural were purchased from Sigma-Aldrich (Diesenhofen, Germany). HPLC gradient grade acetonitrile and methanol were purchased from J.T. Baker (Deventer, Holland). All other chemicals used in this study were of analytical grade. Zorbax Extend-C18 column (250 × 4.6 mm, 5 μm) was purchased from Agilent Technologies (Santa Clara, CA, USA), and 0.45 μm PVDF (polyvinyl difluoride) filters Chromafil Xtra PVDF-45/25 from Macherey-Nagel (Düren, Germany). Ultra-pure water was used throughout the experiments (MilliQ system, Millipore, Bedford, MA, USA).
Stock solutions of acrylamide (1 mg/mL), ¹³C₃-acrylamide (1 mg/mL) and HMF (1 mg/mL) were prepared by dissolving in distilled water:acetonitrile (50:50 v/v). Working solutions were prepared by diluting the stock solution with water:acetonitrile (50:50 v/v) to concentrations of 0.75, 1, 2.5, 5, 10, and 25 ng/mL for acrylamide and ¹³C₃-acrylamide; 50, 100, 250, 500, 1000 and 2500 ng/mL for HMF. Stock solutions and working standards were kept at 4 °C. Carrez I solution was prepared by dissolving 15 g of potassium hexacyanoferrate in 100 mL of water, and Carrez II solution by dissolving 30 g of zinc sulfate in 100 mL of water.

Secretoneurin (SN) and Alexa Fluor® 647 labeled SN were synthesized and purified by piChem (Graz, Austria) and provided as freeze-dried product. Full length SN sequence was used as reported by Fischer-Colbire et al: TNE IVE EQY TPQ SLA TLE SVF QEL GKL TGP NNQ [30 (link)]. Single stranded DNA oligonucleotides (ODN 1826 control—5’-TCC ATG AGC TTC CTG AGC TT-3’) were obtained from InvivoGen (San Diego, USA) and contain full phosphorothioate backbone. ODN 1826 control does not cause any immunostimulatory effect and was therefore used as non-active ODN component. Protamine free base (Grade IV, Histone free) was purchased from Sigma Aldrich (Vienna, Austria). For our in-vivo biodistribution study oligonucleotides were synthesized by Biospring (Frankfurt am Main, Germany) with the same sequence and backbone properties as mentioned above.
Stock solutions for each component were prepared in Milli-Q (MQ) water and stored at -24°C. MQ water (ultra-pure water) was provided by a Milli-Q-Gradient system (Merck-Millipore, Darmstadt, Germany). In addition, the water was sterilized before usage at 121°C for 20 min.
For HPLC measurements, acetonitrile (HPLC gradient grade) was obtained from VWR (Leuven, Belgium) and trifluoroacetic acid from Merck (Darmstadt, Germany). Mannitol was purchased from Carl Roth (Vienna, Austria) and was used as cryoprotectant for lyophilization.

Citric acid monohydrate (≥ 99.8%) and hydrochloric acid (37%) were purchased from Fisher Scientific UK and VWR BDH chemicals, respectively. EDTA standard (9.56% nitrogen) from LECO Corp., USA, was used for LECO analyses. The amino acid calibration standard mix (analytical standard grade) was obtained from Sigma Aldrich (Copenhagen, Denmark). Acetonitrile (HPLC gradient grade) and methanol (HPLC gradient grade) were obtained from VWR International (Søborg, Denmark). Deionised water was used for all the extraction experiments and was obtained from Millipore Milli-Q Direct 8 Water Purification System (Southern Scientific Instruments, USA).