Epmotion workstation

OVCAR3 cells were administered with vehicle, niraparib, niraparib plus P4, or P4 for 24 h, and then samples were thawed on ice bath to diminish sample degradation. Twenty microliters of plasma/serum was added to a 96-well plate. Then, the plate was transferred to the Eppendorf epMotion Workstation (Eppendorf Inc., Humburg, Germany). Ice-cold methanol (180 μl) with partial internal standards was automatically added to each sample and vortexed vigorously for 5 min. The plate was centrifuged at 4,000g for 30 min (Allegra X-15R, Beckman Coulter Inc., Indianapolis, IN, USA). Then, the plate was returned back to the workstation. Forty microliters of supernatant was transferred to a clean 96-well plate, and 20 μl of freshly prepared derivative reagents was added to each well. The plate was sealed, and the derivatization was carried out at 30 °C for 60 min. After derivatization, 420 μl of ice-cold acetonitrile was added to dilute the sample. Then, the plate was stored at −20 °C for 20 min and followed by 4,000g centrifugation at 4 °C for 30 min.

Mice feces were used for metabolomics analysis. For this purpose, an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) system (ACQUITY UPLC-Xevo TQ-S, Waters Corp., Milford, MA, USA) was used to quantify metabolites present in feces. Briefly, three to five fecal pellets from each mouse were thawed on ice-bath to diminish degradation, homogenated with zirconium oxide beads and methanol containing internal standard added to extract the metabolites, followed by centrifugation at 18,000 g for 20 min. Subsequent procedures were conducted on the Eppendorf epMotion Workstation (Eppendorf Inc., Humburg, Germany). ACQUITY UPLC BEH C18 1.7 µm VanGuard pre-column (2.1× 5 mm) and ACQUITY UPLC BEH C18 1.7 µm analytical column (2.1 × 100 mm) were employed to determine compounds to be tested. The column temperature was set at 40°C and the flow rate of mobile phase was 0.4 mL/min. The raw data files generated by UPLC-MS/MS were processed using the TMBQ software (v1.0, Metabo-Profile, Shanghai, China), which can perform a collection of data processing, interpretation, and visualization.

The metabolomics progress was performed based on a standard method as described previously [15 , 16 (link)]. In short, standards were diluted at a concentration of 5.0 mg/ml. The Eppendorf epMotion Workstation (Eppendorf Inc., Germany) was operated when the samples were injected into 96-well plates mixed with methanol for 5 min and followed by centrifugation (4000 × g, 30 min). Samples were further diluted by the 50% methanol. Then, 135 μl supernatant was transferred into a clean plate followed by LC-MS procedures. ACQUITY UPLC BEH 1.7 μM VanGuard precolumn (2.1 × 5 mm) and ACQUITY UPLC BEH C18 1.7 μM analytical column (2.1 × 100 mm) were used in this procedure. 0.1% formic acid water was used as mobile phase A, and 0.1% acetonitrile isopropanol was used as mobile phase B. The elution procedure was as follows: 0-1 min, 5% B; 1-5 min, 5-30% B; 5-9 min, 30-50% B; 9-11 min, 50-78% B; 11-13.5 min, 78-95% B; 13.5-14 min, 95-100% B; 14-16 min, 100% B; 16-16.1 min, 100-5% B; and 16.1-18 min, 5% B. The flow rate was 0.4 ml/min. For the mass spectrometer settlement, 1.5 (ESI+), 2.0 (ESI-) was used for capillary voltage, 150°C for source temperature, and 550°C for desolvation temperature. Data process and analysis were consistent with previous study [17 (link)].