717 plus autosampler system

The tissues were extracted and homogenized in ice-cold 0.1 M perchloric acid (v/w = 30:1) and immediately centrifuged at 15, 000 g for 15 min at 4 °C. The supernatant was collected and prepared for HPLC analysis or stored at -80 °C for later HPLC analysis with fluorometric detection. The column used was a REPROSIL-PUR 120 C18 5 μm (4.6 × 250.0 mm) from ThermoFisher. The mobile phase consisted of 0.05 M citric acid, 0.05 M sodium acetate and 0.5 mM sodium EDTA, 13% methanol, pH 3.1; the flow rate was 0.4 ml/min. The excitation wavelength used was 280 nm and the emission wavelength was 330 nm; the sample injection volume was 20 μL. The equipment included a Waters 717 Plus Autosampler System, a Waters 1525 Binary HPLC Pump and a Waters 2475 Multi λ Fluorescence Detector.

The amount of drug in the donor and receptor solutions was quantified using a Waters HPLC apparatus fitted with a 717 Plus Autosampler system and a UV detector. A SunFire C18 4.6 × 150 mm column with 4.5 µm pores (Waters, Ireland) was selected. The analysis was performed at room temperature (25 °C) with an 80 µL injection volume and a flow rate of 1 mL/min. The mobile phase consisted in a mixture of 0.01 M phosphate buffer (pH 6.0) [26 ] and acetonitrile in the ratio of 72:28 v/v. Dexamethasone sodium and bromfenac sodium were quantified at 242 nm and 265 nm, respectively. The retention time of dexamethasone sodium and bromfenac sodium was 4.20 min and 14.65 min, respectively. Two calibration curves were prepared to quantify the drugs in different concentration ranges in PBS (i.e., 0.05–10 µg/mL and 25–500 µg/mL). The curves were prepared in triplicate and validated with respect to linearity, accuracy and precision. All samples were filtered (0.22 µm) before testing. The quantification limits of the method were 7 ng/mL and 27 ng/mL for dexamethasone sodium and bromfenac sodium, respectively.

The quality control was performed using a 717_plus Autosampler system, a 1525 binary pump, a 2996 photodiode array detector (Waters, USA), and a Flowstar LB 513 (Berthold, France) gamma detector. The system was operated with the Empower^® 3 (Waters) software. Analytical HPLC was performed using a normal phase Chiralpak-AD 250 × 4.6 mm 5 µm column (Chiral Technologies, France) and a mixture of heptane/ⁱPrOH/MeOH/N,N-diisopropylamine (40/30/30/0.5 v/v/v/v, 1 mL/min) as the eluent. UV detection was performed at 333 nm. Molar activity was calculated as the ratio of the activity of the collected peak of [¹⁸F](R)-crizotinib measured in an ionization chamber (Capintec^®, Berthold) over the molar quantity of crizotinib determined using a calibration curve. MA is calculated as the mean value of three consecutive runs.