Agilent chemstation software rev a 10.02

An Agilent 1100 Series HPLC instrument equipped with a G1311A quaternary pump, a G1379A degasser, a G1392A autosampler, a G1315B diode-array detector, and a computer with the Agilent Chemstation software (Rev. A 10.02), all from Agilent Technologies (Waldbronn, Germany), was employed to obtain the HPLC-UV chromatographic fingerprints for the PCA and PLS studies. Chromatographic separation was carried out in reversed-phase mode by using a Zorbax Eclipse XDB-C8 column (150 × 4.6 mm i.d., 5 µm particle size) also provided by Agilent Technologies. Formic acid (0.1%, v/v) aqueous solution (solvent A) and methanol (solvent B) were used as mobile phase to stablish the gradient elution as follows: 0–2 min at 10% B (initial conditions); 2–4.5 min linear gradient from 10% B to 25% B; 4.5–7 min at 25% B; 7–22 min linear gradient from 25% B to 90% B; 22–24 min at 90% B; 24–25 min back to initial conditions at 10% B; and 25–30 min at 10% B for column equilibration. A mobile phase flow-rate of 1 mL min⁻¹ and an injection volume of 10 µL were employed. Photodiode array (PDA) acquisition from 190 to 600 nm was performed to register UV spectra and to guarantee peak purity when necessary. HPLC-UV fingerprints for PCA and PLS analysis were obtained by direct UV absorption detection at 257, 280, and 316 nm.