Pressure-assisted electrokinetic injection stacking for verteporfin drug to achieve highly sensitive enantioseparation and detection in artificial urine by capillary electrophoresis

Pressure-assisted electrokinetic injection (PAEKI) was applied for negatively charged verteporfin (VER) overloading and inline stacking, which targeted highly sensitive enantioseparation by CE. The essential step of PAEKI is a constant pressure used to counterbalance the electroosmotic flow (EOF), consequently, the large amount of analyte could be permitted into capillary and concentrated at the motionless boundary of the sample zone and background electrolyte (BGE). Aiming to know the balance, the velocity of the whole BGE in capillary by the impetus of pressure (0.2–2.0 psi), and the velocity of EOF depending on the length of sample plug and voltage (5.0–20 kV) was investigated, respectively. The velocity of bulk flow in capillary has good linearity with the pressure or applied voltage. Through the pattern of EOF marked peak and analyte peaks (dissolved in pure water), the constant pressure (0.8 psi) vs. the added voltage (−10.3 kV) during PAEKI was confirmed to immobilize the bulk flow of BGE, thus the sample injection time could sustain 2.0 min without compromising separation efficiency. The obtained LOD (S/N = 3) of each isomer at UV detection (428 nm) was around 10.3 μg/L, which was improved to 116 and 39-fold in comparison with normal hydrodynamic injection (HDI) and electrokinetic injection (EKI). The LOD is far below the reported value with LIF detection of VER. The RSD (n = 5) of migration time and peak area was, respectively, around 3.5% and 5.7% for the proposed PAEKI method. Finally, PAEKI was used for the detection of VER in artificial urine to investigate the matrix interference.