Reversed phase high performance liquid chromatography rp hplc

All amino acids and resins were purchased from AAPPTec LLC (Louisville, KY, USA), Chem-Impex International Inc. (Wood Dale, IL, USA), and CEM Corporation (Matthews, NC, USA). All organic solvents were purchased from Millipore Sigma Corporation (St. Louis, MO, USA), Fischer Scientific (Pittsburgh, PA, USA), and Gyros Protein Technologies, Inc (Tucson, AZ, USA). Anticancer agents Doxorubicin (Dox) and Docetaxel (Doce) were purchased from LC laboratories (Woburn, MA, USA). Masses of intermediate and final products were confirmed by high-resolution matrix-assisted laser desorption/ ionization time-of-flight (MALDI-TOF) mass spectrometer from Bruker Inc. (GT 0264, Billerica, MA, USA) or Applied Biosystems (4800 MALDI TOF/TOF Analyzer, Foster City, CA, USA). Intermediate and final compounds were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) from Shimadzu (Prominence, Columbia, MD, USA) using a gradient system of acetonitrile and water with 0.1% trifluoroacetic acid using reverse phase C18 column (XBridge BEH130 Prep C18), from Waters Corporation (Milford, MA, USA). 5(6)-Carboxyfluorescein diisobutyrate (CFDI) was used to synthesize fluorescently-label peptide (USBiological Life Science, Swampscott, MA, USA).

All amino acids and resins were purchased from AAPPTec, LLC (Louisville, KY, USA). All organic solvents and reagents were purchased from Millipore Sigma Corporation (St. Louis, MO, USA) and Fisher Scientific (Pittsburgh, PA, USA). Molecular weights of intermediate and final products were confirmed by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometer from Bruker Inc. (GT 0264, Billerica, MA, USA). Intermediate and final compounds were purified by reversed-phase high-performance liquid chromatography (RP-HPLC) from Shimadzu (Prominence, Columbia, MD, USA) using a gradient system of acetonitrile and water with 0.1% trifluoroacetic acid (TFA) using a reverse-phase column (XBridge BEH C18 OBD Prep Column), from Waters Corporation (Milford, MA, USA).

The amount of CQA covalently and non-covalently bound to the proteins and hydrolysates was measured according to the method described in Ali (2019a (link)). Four mg of sample was dispersed in 1 mL of 8 M urea solution, and after that the protein was precipitated by addition of 20% trichloroacetic acid (50:50). Then, the precipitate was dissolved again in 1 mL of 8 M urea. Shimadzu Reversed-phase high-performance liquid chromatography (RP-HPLC, Kyoto, Japan) with a Perfectsil column (C8 300 ODS, 150 × 4.6 mm, 5 μm) at 37 °C was used to evaluate the content of covalently and non-covalently bound CQA. 0.1% Trifluoroacetic acid (v/v) and acetonitrile were (A) and (B) eluents, respectively with the gradient: B (10−18%), 22 min; B (18−80%), 8 min; B (80%), 3 min; B (80−10%), 2 min; and B (10%), 7 min. The injection volume was 50 μL and the total run time was 42 min. Calibration for 5-CQA was performed at 325 nm.