Ultra clear system

The HPLC–HRMS chromatographic system included an Accela 1250 pump, a PAL HTC autosampler, an Accela PDA detector and a Xcalibur data system (ThermoFisher Scientific, Waltham, MA, USA). Water was purified by an Ultra Clear system (Siemens, Berlin, Germany). Chromatographic separation of R-PZQ, S-PZQ and IS mebendazole were obtained on a column of Daicel  CHIRALPAK^® AS-RH (4.6 mm × 150 mm, 5 μm) fitted with a guard column (10 × 4.0 mm, 3 μm) at room temperature (about 25 °C). The autosampler was set at 4 °C during analysis. The mobile phase consisted of methanol and formic acid (90:10, v/v) at the flow rate of 0.55 ml/min. The relative parameters were as follows: ESI ion source, 3.00 kV spray voltage, sheath gas (N₂) and auxiliary gas (N₂) 35 Arb and 10 Arb, respectively, capillary heater temperature 320 °C. The [M -H]⁺ quasi-molecular ion peaks of R-PZQ and S-PZQ were detected at 313.1911 mass-to-charge (m/z) ratio (the two enantiomers have the same molecular weight and molecular structure and differ only with regard to symmetry), while the IS (mebendazole) was detected at 296.1036 m/z. The total run time was 25.0 min.

The following reagents used are high-performance liquid chromatography (HPLC) grade: acetonitrile (Fisher Scientific Los Angeles, CA, USA), methanol (Fisher Scientific Los Angeles, CA, USA), formic acid (Sigma-Aldrich, St Louis, MO, USA) and leucine-enkephalin (Sigma-Aldrich, St Louis, MO, USA). Ultrapure water was prepared using an Ultra Clear System (Siemens Water Technologies, Nuremberg, Germany).