Nexera xr hplc system

Microsomal incubations were performed three times in 0.1 M potassium phosphate buffer (pH 7.4) in 1.5 mL tube. NADPH-dependent metabolism was evaluated by PF-543, derivatives, or Verapamil (positive control) into pooled liver microsomes of human (HLM), dog (DLM), rat (RLM), and mouse (MLM) in the presence of NADPH regenerating system. The final mixtures contained 0.5 mg/mL HLM, 0.1 M potassium phosphate buffer (pH 7.4), and NADPH regenerating system (10 mM MgCl₂, 1 mM NADPH). HLM was added and the mixture was preincubated at 37 °C in a shaking incubator at approximately 350 rpm for 5 min under Thermo mixer (Eppendorf., Hamburg, Germany). The reaction was started by adding 1 mM NADPH and was then terminated by adding 40 µL of ice-cold acetonitrile containing 10 µM chlorproamide as an internal standard at 0, 30 min. The incubation mixtures were centrifuged at 15,000× g for 5 min at 4 °C. A 2 µL of the supernatant was taken and directly injected into the LC-MS/MS system. The LC-MS/MS system is the Nexera XR HPLC system (Shimadzu Co., Kyoto, Japan) coupled to the TSQ Vantage triple quadrupole mass spectrometer equipped with Xcalibur version 1.1.1 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

About 50 mg of freeze-dried FPH was weighed into glass tubes, 1 ml 6 M HCl was added. The glass tubes placed into a heating cupboard for 24 h, at 105 °C. Samples were diluted 50 times using distilled water before filtering through 0.22 μm.
For the derivatization, 300 μl of the sample were transferred to a glass tube, containing 600 μl 0.4 M borate buffer (pH 9). 600 μl FMOC (9-fluorenylmethoxycarbonyl chloride, 15 mM in acetonitrile) was added, mixed, and then allowed to stand at room temperature for 10 min on an orbital shaker. After amino acid derivatization with FMOC, 600 μl ADAM (12 mM in acetone:water 1:1) was added.
Amino acids were analyzed using a Shimadzu Nexera XR HPLC system, equipped with a PDA detector (Shimadzu, USA). Separation of amino acids were carried out on a Restec ARC-18 column (10 mm × 2.1 mm) at 30 °C. The mobile phase was 0.1% formic acid with 20 mM ammonium formate and 0.1% formic acid with 10 mM ammonium formate in 90:10 acetonitrile water in gradient mode, with a flowrate of 0.35 ml min⁻¹.

ICR mice (n = 3) were dosed with CA140 dissolved in DMSO/Tween-80/saline (10:5:85%) via a single intravenous administration (10 mg/kg). Blood was collected by cardiac puncture at 5 min and then centrifuged to isolate plasma. The brain was collected at 5 min and homogenized in PBS after washing with fresh PBS. The concentrations of CA140 in the plasma and brain were determined by LC-MS/MS. The LC-MS/MS system comprised a Nexera XR HPLC system (Shimadzu Co., Kyoto, Japan) coupled to a TSQ Vantage triple quadrupole mass spectrometer equipped with Xcalibur version 1.1.1 (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Acetone:methanol lipophilic extracts were analyzed via reverse-phase HPLC using a Kinetex 2.6 μm PS C18 100 Å (150 × 2.1 mm) column (Phenomenex; Torrance, CA) attached to a Shimadzu Nexera XR HPLC system. The mobile phase was a binary gradient of Solvent A (70% acetonitrile/30% water) and Solvent B (70% acetonitrile/30% isopropanol) flowing at 0.45 mL/min. Absorbance was measured between 200 and 600 nm using a Shimadzu SPD-M20A photodiode array detector. An astaxanthin commercial standard (Sigma-Aldrich) was used to identify and quantify astaxanthin in the extracts. astaxanthin eluted at a retention time of 12.6 min.

A Nexera XR HPLC system (Shimadzu, Tokyo, Japan) coupled to a 4500 Qtrap mass spectrometer (Sciex, Toronto, ON, Canada) equipped with a heated ESI source (V source) was used for the analysis, following Oliva et al. [47 (link)]. The different PCs were separated using an Excel 2 C18-PFP (10 cm × 2.1 mm ID) column from Advanced Chromatography Technologies (Aberdeen, UK) with 2 μm particles and safety protection. H₂O with 1% acetic acid was used as mobile phase (A) and ACN as phase (B). The injection volume was set to 6 μL and the flow rate was set to 0.300 mL min⁻¹. All analytes were detected in negative ionization with a capillary voltage of −4500, nebulizer gas (air) at 40 psi, and turbogas (nitrogen) at 40 psi and 500 °C. Data collection and processing were performed with Analyst 1.7.2 software and quantification with Multiquant 3.0 software (Sciex).