Zorbax stablebond c18 reversed phase column

To evaluate the relative abundance of cardenolides sequestered by caterpillars reared on the different milkweed species, adult wings were dried (at 50 °C) and ground. 21–76 mg of ground tissue, spiked with 20 μg of digitoxin as internal standard, was extracted with 1.8 mL of methanol in a sonicating water bath at 55 °C for 20 min. Wing cardenolides correlate tightly with body cardenolide concentrations^{46 (link),89 (link)}. After evaporating the solvent, the residue was resuspended in 0.5 mL methanol. Samples were analyzed by HPLC using a Zorbax StableBond C18 reversed phase column (5 μm, 150 × 4.6 mm, Agilent Technologies, Santa Clara, CA, USA) and an Agilent 1100 series instrument with diode array detection. The 15 uL injection was eluted at a constant flow of 0.7 mL/min with a gradient of acetonitrile and water as follows: 0–2 min 16% acetonitrile; 25 min 70% acetonitrile; 30 min 95% acetonitrile with a final 8 min hold. Peaks were detected by a diode array detector at 218 nm, and absorbance spectra were recorded from 200–400 nm. Peaks showing a characteristic symmetrical absorption band with a maximum between 217–222 nm were recorded as cardenolides^{90 (link)}. Sample concentrations were quantified by relating abundances to the peak area of the internal standard.

Standard stock solutions (0.1 mg/ml) of four components (i.e. THSG, gallic acid, emodin-8-O-β-d-glucopyranoside, and emodin), and Ibuprofen were prepared individually by dissolving an appropriate amount of each chemical standard in a known volume of methanol. The standard stock solutions prepared were kept at −20 C when not used. The standard working solutions of the four components and Ibuprofen were prepared individually by diluting each stock solution with methanol.
The components of PA were analyzed by HPLC-MS. The samples were separated on a ZORBAX StableBond C₁₈ reversed-phase column (2.1 × 50 mm, 1.8 μm) (Agilent Technologies Co., CA, United States). The autosampler was set at 20 C, and gradient elution was performed with 0.1% formic acid as solvent A and acetonitrile as solvent B. The gradient program was as follows: 0–10 min, 88%–40% A; 10–12 min, 40%–1% A; and 12–15 min, 1% A. The flow rate was set at 0.3 ml min⁻¹, and the injection volume was 4 μL. The total run time was 15 min for each sample.