Temperature controlled ionic liquid aqueous two phase system combined with affinity capillary electrophoresis for rapid and precise pharmaceutical-protein binding measurements.

Temperature controlled ionic liquid aqueous two phase system (ILATPS) was used to improve the precision of pharmaceutical-AGP (human alpha (α1)-acid glycoprotein) binding measurements by affinity capillary electrophoresis (ACE). The effect of different types of short-chain alkyl imidazolium ILs within the concentration range of 10.0-1000.0 μmol L on a propranolol (PRO)-AGP model was firstly investigated by ILATPS/ACE system. Use of 100.0 μmol L 1-butyl-3-methylimidazolium chloride (BMImCl) in 67.0 mmol L potassium phosphate buffer (pH 7.4) containing low concentrations of AGP (5.0-100.0 µmol L) gave the highest precision value (2.98 ± 0.14 × 10 L/mol) which is in concord with the reported one (3.00 × 10) under similar experimental conditions. The proposed BMImCl/phosphate solution was a unique temperature controlled system to preserve AGP activity during the pre-analysis and within ACE measurements under lab conditions for about 30 days. This period could be prolonged by converting the one-phase solution into biphasic solution at 4 °C storage temperature and again it could get rapidly back into one-phase by raising the temperature with gentle shaking. This behavior could be attributed to the electrostatic interaction and π-π interaction between BMIm cations and negatively charged AGP ions (pI = 2.7). Moreover, the compatibility of ILATPS with ACE has been the critical factor to avoid precipitation of salts formed by anion exchange in the running buffer. The current ILATPS/ACE system was further used to rapidly and precisely estimate the binding constants of anticancer drugs methotrexate (MTX) and vinblastine (VBL) with human AGP. The obtained binding values have been in good agreements with their findings by high performance affinity chromatography (HPAC). This ILATP/ACE system could similarly be applied to improve the precision of other proteins binding measurements with consuming a small amount of protein and with shortening ACE analysis time.