Rapid Identification and Simultaneous Quantification of Aristolochic Acids by HPLC-DAD and Confirmations by MS in Using a Limited Biomass.

Six aristolochic acids were identified in the Chilean species using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) and subsequent confirmation with mass spectrometry (MS). The fractions of each signal were collected and injected directly into an Orbitrap mass detector model Q Exactive Focus (Thermo Scientific). The acids extraction was done with 0.10-0.50 g of lyophilized and pulverized sample and concentrated in Soxhlet extraction equipment. The liquid-liquid separations and a subsequent solid phase extraction (SPE) C18 were performed using 100 L of the extract that contains the aristolochic acids present in the plant. The HPLC conditions used a single mobile phase acetonitrile : water (1 : 1) acidified with 0.1% acetic acid and an isocratic elution to 1 mL·min. The column InertSustain C18 250 × 4.6 mm and 3 m was used, the injection volume was 20 L, and the time of run was reduced to 15 min. Calibration curves were constructed with  being 0.9997. The quantification limit for AAI was 0.138 ± 0.010 g/mL, and for AAII, it was 0.558 ± 0.042 g/mL.