HPLC-Based Protein Binding Estimation

Retention times of the analytes were measured with Shimadzu HPLC system on the CHIRALPAK®HAS stationary phase (50 × 3 mm, 5 μm, Chiral Technologies, DAICEL Group, Europe SAS, France). The mobile phase A consisted of 50 mM aqueous ammonium acetate buffer (pH 7.4) and phase B of 2-propanol according to Valko et al.^{65 (link)} Analysis was performed at prolonged 1 mL min⁻¹ flow rate in the linear gradient. Retention capacity factors (k′) were calculated by using DMSO or a substance with 0% HAS binding for systems' dead time (R_t0). The system was calibrated by injecting the reference compounds: acetylsalicylic acid (CAS 69-72-7), betamethasone (CAS 378-44-9), budesonide (CAS 5133-22-3), carbamazepine (CAS 298-46-4), cimetidine (CAS 51481-61-9), ciprofloxacin (CAS 85721-33-1), indomethacin (CAS 53-86-1), isoniazid (CAS 54-85-3), metronidazole (CAS 443-48-1), nicardipine (CAS 55985-32-5), nizatidine (CAS 76963-41-2) and warfarin (CAS 81-81-2) obtained from Sigma-Aldrich, diclofenac (CAS 15307-86-5) from EMD Chemicals Inc., flumazenil (CAS 78755-81-4) from ABX and ketoprofen (CAS 22071-15-4) from LKT Labs. The logarithmic capacity factors of the references' Rt (log(k′)) on the HSA column were plotted against the %PPB values from literature. The slope and the intercept were used to convert the log(k′) of the compounds (6a, c, f, h, m–o) to %PPB using the regression equation.^{66 (link)}