Agilent 1200 series hplc system

Xylooligomers (0.1 mg each) and birchwood, beechwood, and oat-spelt xylan (1 mg) were reacted using purified wild-type and mutant xylanases (10 U) in 1 mL of 50 mM sodium acetate-acetate buffer solution (pH 5.5) for 12 h at 50 °C. Then the hydrolytic reaction was stopped by heating the reaction mixture at 100 °C for 10 min. The degradation products were identified by high performance liquid chromatography (HPLC) analysis using an Agilent 1200 Series HPLC system (Agilent Co., Santa Clara, CA, USA) equipped with a Sugar-D column (4.6 Inner Diameter (ID) × 250 mm, COSMOSIL, Nacalai tesque, Kyoto, Japan). The mobile phase contained acetonitrile and 30% water at a flow rate of 0.8 mL·min⁻¹, oven temperature 30 °C, refractive index detector temperature 30 °C, and injection volume of 20 μL after using a 0.22 μm filter.

Optical rotations were measured on Jasco P-1020 automatic digital polarimeter. CD spectra were recorded on a Chirascan spectropolarimeter (Applied Photophysics, Leatherhead, Surrey, UK). UV data were obtained from HPLC online analysis. IR spectra were obtained on a Bruker Tensor-27 infrared spectrophotometer with KBr pellets. NMR spectra were carried out on a Bruker Avance III 600 or DRX-500 spectrometer with deuterated solvent signals used as internal standards. ESIMS and HRESIMS were measured using Agilent G6230 time-of-flight mass spectrometer. Preparative MPLC was performed on a Büchi apparatus equipped with Büchi fraction collector C-660, Büchi pump module C-605 and manager C-615. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China), MCI gel CHP-20P (75–150 μm, Mitsubishi Chemical Corporation, Japan), Chromatorex C-18 (40–75 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) were used for column chromatography. Fractions were monitored and analyzed using TLC, in combination with an Agilent 1200 series HPLC system equipped by an Extend-C18 column (5 μm, 4.6 × 150 mm).

Ilomastat was quantified using HLPC (Agilent 1200 series HPLC system, Agilent Technologies, London, UK) fitted with a Synergi RP Phenomenex 4-μm, 15-cm C18 column and equipped with an autosampler, a degasser, and two SL bin-pumps. A flow rate of 1 mL/min was used with 0.1% trifluoroacetic acid in water and acetonitrile as eluents A and B, respectively, with a linear gradient from 80% A to 70% B in 17 minutes. The detection wavelength was 280 nm.

The linear range of the method was tested for each of the melanin oxidation products with a 9-point calibration curve at concentrations ranging from 0.01 to 10 μg/mL by multiple injections. Limit of detection (LOD) and limit of quantitation (LOQ) were determined with the signal-to-noise ratio method for each of the standards based on HPLC measurements with UV detection. LOD was set at 3:1 and LOQ at 10:1 signal-to-noise, respectively.
Recoveries after sample preparation by SPE were tested with a mixture of all four melanin oxidation products in eluent A. Additionally, total method recovery was investigated for all three natural matrices (feather, hair, shell) by a 3-point standard addition (2 times, 5 times and 10 times) of all oxidation products. Standards were added following oxidation of matrices and prior to SPE. SPE recoveries without matrices were measured on an Agilent 1200 Series HPLC system with diode array detector using the same chromatographic conditions as described above.
Additional experiments on the oxidation protocol itself verified the linearity of PDCA and PTCA formation from a synthetic eumelanin standard in the range of 0.05–0.4 mg. A test with an elongated oxidation time (40 h) did not result in significantly higher amounts of oxidation products and even yielded slightly less eumelanin markers in the case of shell samples.

The SK-OV-3, MDA-MB-435, 786-O, MDA-MB-231, and MCF-7 cells were seeded at 500,000 cells per well in 12-well plates. The cells were treated with 10 μM of biguanides for 30 min. The cells in each well were washed with cold PBS, and the amount of biguanides were measured using liquid chromatography tandem mass spectrometry (LC-MS/MS) (Agilent 1200 series HPLC system; Agilent 6430 MS/MS system; Agilent), and the data were analyzed using MassHunter B 01.03. For the AMP-activated protein kinase (AMPK) activation assay, 500,000 MCF-7 cells/well were plated in six-well plates. The cells were treated with concentrations ranging from 3 μM to 10 mM of biguanides and were incubated for 12 h. The cells were treated with 1% Triton X-100 cell lysis buffer, and the supernatant was collected after centrifugation at 12,000 rpm for 20 min. The protein samples (25 μg each) were added to the p-AMPK ELISA plates (Thermo Fisher Scientific), and the absorbances of samples were measured at 450 nm.