Disodium hydrogen phosphate dihydrate

An aqueous bulk solution of Interferon-alpha-2a (Roche, Penzberg) was dialysed (Spectra-Por) into 50 mM Pi (di-Sodium hydrogen phosphate dihydrate: VWR Chemicals, Leuven, Sodium dihydrogenphosphate dihydrate: Grüssing GmbH, Filsum) buffer at pH 7.0. The solution was filtered using a 0.22 µm cellulose acetate filter (VWR Chemicals, Leuven), which were shown to be low protein binding (46) . A protein concentration of 1.4 mg/ml was obtained as determined by measuring the light absorption at 280 nm using a NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA).
Excipient stock solutions were prepared by dissolving the excipient in 50 mM Pi buffer at pH 7.0 and adjusting the pH to 7.0 as required either with hydrochloric acid or concentrated sodium hydroxide.
Buffer was then added to obtain a final excipient concentration of 500 mM. The excipient stock solution was then filtered using a 0.22 µm filter (VWR Chemicals, Leuven).

The chromatographic analyses were performed using an Agilent 1100 HPLC system (Santa Clara, CA, USA) with Agilent G1314A UVD and G1321A FLD. The spectral analyses of the UV-detected compounds were made with an Agilent 8453 UV-Vis Spectroscopy System (Santa Clara, CA, USA). The reference compounds TRP, 5-HT, KYN, KYNA, 3NLT; perchloric acid (PCA), zinc acetate (ZnAc) and phosphoric acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetonitrile (ACN) was obtained from Scharlau (Barcelona, Spain) and acetic acid from VWR International (Radnor, PA, USA). The di-sodium-hydrogen phosphate dihydrate was obtained from VWR International (Radnor, PA, USA) and potassium dihydrogen phosphate from Applichem Panreac (Darmstadt, Germany). The IS used for the FLD (HCA) was synthesized at the Department of Pharmaceutical Chemistry, University of Szeged, involving the ring closure of anthranilamide with diethyl oxalate, followed by the hydrolysis of the ester function [33] (link).