Agilent 1260 infinity quaternary pump

The HPLC system consisted of the Agilent 1260 infinity Quaternary Pump (G1311B), Agilent 1260 infinity Standard Auto Sampler (G1329B), Agilent 1260 Infinity Column Thermostat Compartment (G1316A), and Agilent 1260 Infinity Variable Wavelength Detector (G1314F) as instrumental system, and ZORBAX Eclipse Plus C18 column (250 × 4.6 mm i.d., 5 μm particle size) (Milford, MA, United States) was used as the stationary phase. For simultaneous detection of the four compounds, gradient elution composition was as follows: (0–10 min, 5%–9% channel B; 10–30 min, 9%–9% channel B; 30–60 min, 9%–30% channel B; 60–62 min, 30%–50% channel B; 62–65 min, 50%–5% channel B; and 65–70 min, 5%–5% channel B). Channel A contained 0.4% phosphoric acid in HPLC-grade water, and channel B contained acetonitrile. The wavelength was 327 nm, with a column temperature of 35°C and an injection volume of 5 μL. The four analytical standards and DM plant extract under observation HPLC generated peaks are merged and provided in Supplementary Figure S1.

The reference substances, benzoic acid, toluene, anthraquinone and benzyl benzoate were dissolved in HPLC grade methanol. Their retention time on RP-C18 column was then determined. The HPLC analysis employed reversed-phase C18 column (Agilent HC-C18(2), 5 μm, 150 mm × 4.6 mm, Agilent Technologies, Inc., Santa Clara, CA, US) using photodiode detector (Agilent 1,260 Infinity DAD, Agilent Technologies, Inc., Santa Clara, CA, US). For analysis, an isocratic mobile phase of 3:1 (v/v) methanol and water was employed. A volume of 20 μL (Agilent 1,260 Infinity High S61 performance autosampler) was injected with a flow rate of 0.6 mL/min (Agilent 1,260 Infinity Quaternary Pump). Temperature of the column oven was kept at 25°C (Agilent 1,260 Infinity Thermostatic Column Compartment). A calibration curve of Log k versus LogP of the reference compounds was constructed to obtain its regression equation (Hansch and Leo, 1980 ). The most active nanocomplex (DE-FeO NPs) was dissolved in the mobile phase that was injected into the column. The partition coefficient of the tested nanocomplex was calculated from interpolating its capacity factor value in the calibration curve regression equation. LogP of this nanocomplex was calculated by extrapolating its t ratio using regression equation.

Quenched and reduced samples were analyzed with a nanoACQUITY UPLC reversed-phased chromatographic system equipped with HDX technology (Waters, Milford, MA) coupled to a Synapt G2 electrospray ionization mass spectrometer (Waters). Desalting was performed by applying a flow of 300 µL/min buffer A (0.23% [v/v] formic acid [FA]) to an ACQUITY UPLC BEH C18 1.7-μm, 2.1 × 5 mm Vanguard Pre-Column by an Agilent 1260 Infinity Quaternary pump (Agilent Technologies, Santa Clara, CA). Peptides were separated on a 1.0 × 100 mm ACQUITY UPLC BEH C18 1.7-μm analytical column by a 12-min gradient from 95% buffer A to 50% buffer B (0.23% [v/v] FA in acetonitrile) at a flow of 40 µl/min. Proteins were digested online with an Upchurch guard column (1.0 × 20 mm, IDEX, Oak Harbor, WA) packed with agarose-immobilized pepsin (Thermo Scientific Pierce, Rockford, IL). Peptides from peptic digests were identified from DDA MS/MS runs using ProteinLynx Global Server v2.4 (Waters) and MassAI v1.07 (MassAI Bioinformatics, Stenstrup, DK, http://www.massai.dk). Deuterium incorporation for intact proteins and peptides was quantified with DynamX V2.0 (Waters).