4.3.2. HPLC Analysis
Metabolites were quantified using a 1260 Infinity Quaternary LC System liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) coupled with a quaternary pump, an autosampler, and a DAD. The column used was a 5 Prodigy ODS3 column (250 × 4.60 mm) (Phenomenex Cheshire, UK) with a 1 mL/min flow rate and a column temperature of 25 °C. The mobile phase consisted of two solvents: (A) 0.5% formic acid in water and (B) 0.5% formic acid in acetonitrile. The gradient used was 10% (B) at the beginning, 10% at 5 min, 20% at 15 min, 25% at 25 min, 35% at 33 min, 50% at 38 min, 90% at 43–44 min and 10% at 45 min. Anthocyanins and other phenolic compounds were detected by monitoring elution at 525 and 270 nm, respectively. Quantification was based on the calibration curves of the PCA, VA, iVA, HA, HBA, and HPA standards. The concentrations were expressed as μmol/mmol creatinine for urine samples, and the elimination values were expressed in μmol. Urinary creatinine was evaluated using a Labtest kit (Labtest Diagnóstica S.A., Lagoa Santa, Minas Gerais, Brazil). The area under the curve (AUC) was calculated using the trapezoidal rule (data expressed as μmol/h), and the maximum concentration (Cmax) was calculated using the Origin 6.0 software (OriginLab Corporation, Northampton, MA, USA) and expressed as the mean and standard error of the mean (SEM). The samples collected from 0 to 3 h (after the juçara juice intake) were indicated as 3 h urine and the samples collected from 3 to 6 h were designated as 6 h urine to indicate the period of excretion. The standards’ purity varied from 95 to 98%.