2695 hplc instrument

DZD of 0.9 g/mL was concentrated at 60°C in a rotary evaporator under reduced pressure. The yield of final extract was 25.07%. 0.2261 g of the sample was dissolved in 80% methanol, ultrasonicated, and then filtered on a 0.45 μm filter for HPLC analysis. The extract of DZD was analyzed by Waters 2695 HPLC instrument (2998PDA detector). The sample was separated on a Waters Symmetry C18 column (4.6 × 250 mm, 5 μm) and the linear gradient was 5–95% B (A = 0.1% formic acid water, B = acetonitrile) for 70 min. The flow rate was 1.0 mL/min and the column temperature was maintained at 30°C. Sample of 10 μL was detected at 280 nm.
Reference compounds consisted of mulberroside A, phillyrin (purchased from Chengdu Pulse Biological Technology Co., Ltd.), tetrahydropalmatine, ferulic acid, and tanshinone IIA (purchased from National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). The method of HPLC was the same as above.

OP9 cells were cultured for 24 hours with media supplemented with 3 μM DMBA. The media were collected and treated with β-glucuronidase (Sigma) at 2000 U/mL to optimize recovery of the lipid-soluble residual PAHs. Samples were spiked with an extraction control standard (dibenzo[a,l]pyrene) (NCI Chemical Carcinogen Reference Standard Repositories, Midwest Research Institute, Kansas City, MO) prior to extraction using a 2 : 1 ethyl acetate : acetone (Sigma) organic phase, with repeated extraction centrifugation. The organic phase was dried under liquid nitrogen and the extracted compounds were stored at −80°C until analysis. Dried samples were resuspended in 100% methanol immediately prior to injection onto the HPLC column. Individual PAHs were separated using a Waters (Milford, MA) 2695 HPLC instrument with a C₁₈ column, employing both a UV detector (wavelength 254 nm) and a Waters 470 scanning florescence detector. Data was collected with Waters Empower 3 software. Peaks were separated under a 50–100% methanol gradient over a period of 55 minutes [23 (link)].