Kromasil c18 column

The Kromasil C₁₈ column was chosen for HPLC (Agilent Technology, Palo Alto, USA). The mobile phases were acetonitrile (A) and 0.2% acetic acid aqueous solution (B) [29 ]. Gradient elution was adopted, and the gradient was: 0–5 min, 10–90%; 5–50 min, 52–48%; 50–55 min, 10–90%. The flow rate was set at 0.8 mL/min. Injection volume was 20 μL. Column temperature was set at 30 °C. Detection wavelength was 327 nm. The conditions of mass spectrometry were electrospray ionization source (ESI) of positive and negative ion modes were used for analysis. Spray voltage was set to 3.5 kV. Interface temperature was set to 300 °C. Temperature of desolventizing was 526 °C. Flow rate of heating gas was 10 L/min. Flow rate of atomizing gas was set to 3 L/min. Scanning range is 50–500 m/z.

Grapes at 8, 12, and 16 WAA of 5 cultivars were used to determine individual anthocyanins. Specific extraction steps have been described previously [20 (link)]. Three independent samples were extracted for each skin, and were stored at −40 °C until analysis. Before injection, the extracts were filtered through 0.45 µm filters (cellulose acetate and nitrocellulose, CAN).
Individual anthocyanins of extracts were analyzed using an Agilent 1100 series LC-MSD trap VL equipped with a diode array detector and a Kromasil C18 column (250 × 4.6 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA) utilizing a binary solvent gradient, where mobile phase A was 2% formic acid in water and B was 2% formic acid in acetonitrile. The detailed LC procedures and MS conditions have been described previously report [20 (link)].
The elution order and retention time of the anthocyanins were compared with those of the standard, and the weights of molecular ions and fragment ions were compared with the weights of standard and reference products, allowing different anthocyanins to be identified [25 (link),26 (link)]. Anthocyanins were quantified using malvidin-3-O glucoside as an external standard.

Detection of treprostinil in samples was conducted using an HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a Kromasil C18 column (4.6 × 250 mm, 5 μm). A solvent combination of acetonitrile and water (40:60 v/v) with a pH of 4 adjusted using trichloroacetic acid was used as the mobile phase. The analytical condition for separation of the drug was; flow rate (1.5 mL/min), column temperature (25 °C), run time (15 min), internal standard (kanamycin sulfate), injection volume (50 μL), and detection wavelength (220 nm). Specimens collected were filtered using a hydrophilic polyvinylidene difluoride membrane with a pore size of 0.45 µm and sonicated for 5 min before injecting in the HPLC system.