Poroshell c18 column

Eight isoforms of CYPs (CYP1B1, CYP2C29, CYP2D22, CYP2E1, CYP3A11, CYP3A25, CYP7A1 and CYP27A1), ten isoforms of UGTs (UGT1A1, UGT1A2, UGT1A5, UGT1A6a, UGT1A9, UGT2A3, UGT2B1, UGT2B5, UGT2B34 and UGT2B36) and three isoforms of SULTs (SULT1A1, SULT1B1 and SULT1D1) were analyzed. The methods of sample preparation and quantifying DMEs amounts by UHPLC/MS-MS were consistent with our previous study and dynamic MRM chromatograms of 21 subtypes were displayed in Fig. S1 (Chen et al., 2017 (link)). Samples were analyzed by using an Agilent 6490 triple quadruple mass spectrometer coupled with 1290 Infinite UHPLC system. A Poroshell C 18 column (2.1 mm ×100 nm, 2.7 µm; Agilent Technologies) was used for separation. In this study, the protein amounts of DMEs were represented in the form of pmol protein per S9 fraction protein (pmol/mg). The quantification of protein levels was performed two independent experiments. All samples were performed in triplicate in each independent experiment and data were presented as mean ± SD.

Each guayule fraction was dissolved in acetonitrile at a concentration of 1 mg ml⁻¹. Then, a 0.5 mL aliquot was filtered through a 0.22 µm nylon filter. The analysis of the compounds that were present in the guayule extracts was performed using an Agilent 1260 Infinity II quaternary liquid chromatograph (Hewlett Packard, Wilmington, NC, USA) with a multiple wavelength detector and a G6420A triple quad LC–MS detector that was equipped with technology electrospray ion source, which was also from Agilent. The chromatographic separations were performed on an Agilent Poroshell C18 column (150 × 2.1 mm, 2.7 μm particle size). The column temperature was 30 °C and the flow rate was 0.8 mL min⁻¹. Eluents A and B were used for gradient elution. Solvent A was acetonitrile and solvent B was water. The following gradients were used: 60% A (0–10 min), 80% A (10–20 min), 80% A (20–25 min), 100% A (25–32 min), 100% A (32–37 min), 60% A (37–42 min) and 60% A (42–45 min.). The MS analyses were conducted in positive ion mode and the operating parameters were optimized as follows: ESI–MS/MS in scan mode (m/z = 50–600) with a scan time of 500 ms and a fragmentor of 135 V. The gas flow and temperature were established at 12 L min^-1 and 300 °C, respectively. The pressure of the nebulizer was 50 psi and the capillary voltage was established at 4000 V.

To determine the encapsulation efficiency, high-performance liquid chromatography (HPLC) analysis was used. The GSG-modified polymer membrane was disrupted by the use of Triton-X (Merck, Zug, Switzerland) in water and acetonitrile (VWR International, Dietikon, Switzerland). In detail, 25 µL of 1 mg/mL nanoparticle (theoretically containing 0.2 mg/mL paclitaxel), 25 µL of 0.4% Triton-X in water and 50 µL acetonitrile were combined, vortexed and centrifuged for 3 min at 4000 × g. The amount of paclitaxel encapsulated in the nanoparticles was detected with an Agilent 1100-series equipped with diode array and evaporating light scattering detector (Agilent Technologies, Basel, Switzerland). The mobile phase consisted of ddH₂O (buffer A) and acetonitrile (buffer B, VWR International, Dietikon, Switzerland). Separation was achieved with a Poroshell C18 column (3.0 × 100 mm 2.7-micron, Agilent Technologies). Starting at 50% A, 50% B changing from minute 1 to minute 11 to 0% A, 100% B, with a flow rate of 0.45 mL/min. The paclitaxel peak had a retention time of 4.8/4.9 min. To calculate the concentration of encapsulated paclitaxel a paclitaxel standard curve (Cathepsin B Table S3A,B; AUC versus concentration) was recorded.