Hplc dad system

The chromatographic method was adapted from Tang et al. [32 (link)], with some modifications. The extracts were injected into a Jasco (Tokyo, Japan) HPLC-DAD system, which consisted of a PU-4180 pump, an MD-4015 PDA detector, and an AS-4050 autosampler. The stationary phase was an Agilent (Santa Clara, CA, USA) Zorbax Eclipse Plus C18 reverse-phase column (100 × 3 mm I.D., 3.5 μm). The mobile phase was a mixture of solvent A (water/formic acid 95/5, v/v) and solvent B (methanol/acetonitrile 95/5, v/v), with a composition gradient ranging from 95% to 5% of solvent A and flowing at 0.7 mL/min. Injection volume was 20 µL for all determinations, and analyte detection was carried out with a diode array detector (DAD) by monitoring at 280, 329, and 360 nm. Quantification was performed with pure standards using calibration curves ranging between 1.25 and 30 μg mL⁻¹ (r² ≥ 0.9634).

The caffeine content of the extracts was analyzed using an HPLC-DAD system (Jasco, Tokyo, Japan). This system consisted of an LC-NetII/ADC hardware interface, an automatic sampler (Jasco AS-2057 Plus), a pump (Jasco PU-2089 Plus), a multi-wavelength diode array detector (DAD, Jasco MD-2018 Plus), and a column oven (Jasco CO-2060 Plus). The gradient elution used was the following: 0 min, 5% B; 40 min, 25% B; 55 min, 45% B; 60 min, 60% B; 65 min, 5% B (solvent A: 0.5% acetic acid; solvent B: 100% methanol), with a flow rate of 1.1 mL/min. The chromatographic column was a Zorbax-SB-C18 (5 μm, 250 mm × 4.6; Agilent Technologies, Santa Clara, CA, USA), at 28 °C. The DAD recorded data from 200 to 600 nm were monitored at 274 nm. For HPLC analyses, the lyophilized extracts were dissolved in H₂O (10 mg/mL) and the injected volume was 20 µL. Caffeine was used as the standard for HPLC analyses validation. The calibration curve (y = 36,096x − 227,800; R² = 0.9996) was constructed in the linear range of 1.5–800 µg/mL. The detection limit of the method was 1.24 µg/mL.