Sil 20a ht autosampler

All analyses were performed using a Shimadzu Prominence series HPLC system (Shimadzu Corporation, Kyoto, Japan), equipped with an LC-20AB binary pump (Serial: L20124200883), SIL-20A HT autosampler (Serial: L20345256104), CTO-20AC temperature-controlled column oven (Serial: L2021525077), SPD-M20A photodiode array detector and CBM-20A communications bus (Serial: L20235154327). All equipment were controlled by Shimadzu Lab Solutions Lite software version 5.71 SP2. For separation, an Ultra C18 column, 3 μm, 50 × 4.6 mm (RESTEK Corporation, Bellefonte, PA) was used. Dynantin samples were analyzed at a constant solvent flow rate of 0.7 mL/min at 35 °C using a binary gradient (Table 1). Solvent A consisted of a 25% solution of acetonitrile (HPLC grade, Fisher Scientific, Fairlawn, NJ, USA) in ddH₂O (0.2 μm filtered) and solvent B consisted of acetonitrile with each solvent containing 0.1% trifluoroacetic acid (v/v, protein sequencing grade, Sigma Aldrich, Fairlawn, NJ, USA).

HPLC analysis was performed using a Shimadzu HPLC system, which consisted of SIL-20A HT autosampler, LC-20 AT multisolvent delivery system, DGU-20A degasser, CTO-20 AC cooler, and SPD-M20A UV/Visible detector, and Chromolith RP-18 endcapped column (100 × 4.6 mm) was associated with the LC solution software. HPLC-grade organic solvents, i.e., acetonitrile, methanol, and phosphoric acid (BDH), were used. The mobile phase consisted of a mixture of ultrapure water with 0.1% phosphoric acid (A) and acetonitrile (B). Analyses of zerumbone and α-humulene were carried out by the following elution gradient: 0.01 min, 50% A, 50% B; 2.00 min, 15% A, 85% B; 7.00 min, 15% A, 85% B; 7.50 min, 50% A, 50% B; 11.00 min, 60% A, 40% B. The total running time was 20 min at a flow rate of 1.0 mL/min. The temperature of the column was set at 35 °C at UV detection wavelength 243 nm. All samples were filtered through 0.25 µm membrane filters prior to injection into the HPLC. The zerumbone and α-humulene compounds were identified by matching the retention times and spectral characteristics of a sample to a commercially available standard (Sigma Aldrich, St. Louis, Missouri USA). Quantitative analysis was performed using the peak area calibrated to external standard concentration. Three replicates were prepared for each standard concentration. The injection was also performed in triplicate.

The quantification of TTT-28 in plasma and tumors was conducted using an isocratic Shimadzu LC-20AB HPLC equipped with a Shimadzu SIL-20A HT autosampler and LC-20AB pump connected to a Dgu-20A3 degasser (Shimadzu, OR). A reverse-phase, Phenomenex Luna C18 column (250 × 4.6 mm i.d., 5 μm; Phenomenex, CA) with an ODS guard column (4 mm × 3 mm; Phenomenex, CA), was used. The injection volume was 20 μl, and the mobile phase used for the separation of TTT-28 in plasma and tissue homogenate samples consisted of acetonitrile and water (90:10, v/v) delivered at a flow rate of 1.0 ml/min. For TTT-28 detection, the Shimadzu UV SPD-20A (Shimadzu, OR) detector set was at 210 nm. Data acquisition and analysis was achieved using LC Solution software version 1.22 SP1 (Shimadzu, OR). All samples were analyzed in duplicate. Under these chromatographic conditions, the total run time was 10 min with a retention time of 5.6 min for TTT-28. Standard curves for TTT-28 in plasma and tissue homogenates were prepared in the ranges of 10–10,000 ng/ml. The preparation and storage of samples and quantification of paclitaxel in tumor and plasma were performed as previously described36 (link).

A Multi-Mode Microplate Reader (BioTek Instruments, Winooski, VT, USA) was employed to carry out the conventional optical analytical methods, such as TPC, TFC, FRAP and DPPH.
The HPLC analyses were carried out in a Shimadzu HPLC Prominence system (Kyoto, Japan) equipped with a LC-20AD pump, a DGU-20AS degasser, a CTO-10AS VP column oven, a SIL-20A HT autosampler, and an SPD-M20A photodiode array detector.
SWV was performed in an Autolab PSTAT 10 controlled by GPES software, version 4.8 (EcoChemie, Utrecht, The Netherlands). A conventional three-electrode cell was used, and included a homemade CPE (2 mm in diameter; electroactive area of 0.047 cm²) as a working electrode, a platinum wire-counter electrode and an Ag|AgCl|KCl sat reference electrode to which all potentials are referred. The CPE was prepared by mixing 1.8 g of paraffin oil as pasting liquid with 5 g of spectroscopic grade graphite powder. The unmodified carbon paste (CP) was introduced into the well of a Teflon electrode body provided by a stainless-steel piston. The surface was smoothed against a plain white paper while a slight manual pressure was applied to the piston. Since CP oxidizes when exposed to air, for each analysis a new CPE surface was freshly prepared [6 (link)].