Acetonitrile ch3cn

Levels of total 25-hydroxyvitamin D3 were measured by HPLC (Agilent 1100 series, Chem Station software) using RECIPE’s HPLC complete kit. Deionized water, HPLC-grade methanol (Merck Millipore, Cat.No.1.06007) and Acetonitrile (CH3CN) (Carlo Erba, Cat.No.412372000) were used in all the performed experiments. Serum calibrators and controls (low and high levels) for 25(OH) D3 were also obtained from RECIPE. The chromatographic column was a C18, 150 × 4.6 mm, with a 5 μm particle size (Hector-M). The mobile phase was considered as 90% acetonitrile and 10% methanol in isocratic elution mode with a flow rate of 1.0 ml/min. Additionally, the detection wavelength was 264 nm.
In this study, serum levels of VDBP were determined using polyclonal ELIZA kit (CUSABIO) in terms of the manufacturer’s instructions, with intra-assay precision: CV < 8%, and inter-assay precision: CV < 10%. Moreover, serum albumin levels were determined by enzymatic colorimetric assay [Pars-Asmun kits, Iran] using an auto analyzer [Hitachi 902, Japan]. Finally, the vitamin D bioavailability was estimated based on the ratio of 25 (OH) D3/VDBP.

Protected amino acids, coupling agents (HATU, Oxyma), and Fmoc-Rink Amide AM resin used for peptide synthesis were purchased from IRIS Biotech GmbH (Marktrewitz, DE). Solvents, including acetonitrile (CH3CN) and dimethylformamide (DMF) were purchased from Carlo Erba reagents (Milan, Italy). Other products such as trifluoroacetic acid (TFA), sym-collidine, diisopropylethylamine (DIPEA), piperidine, were from Sigma-Aldrich (Milan, Italy). HPLC analyses for peptides characterization were performed on an Alliance HT WATERS 2795 system, equipped with a PDA WATERS detector 2996, whereas preparative purifications were carried out on a WATERS 2545 preparative system (Waters, Milan, Italy) fitted out with a WATERS 2489 UV/Visible detector.