Rapid Identification and Simultaneous Quantification of Aristolochic Acids by HPLC-DAD and Confirmations by MS in <i>Aristolochia chilensis</i> Using a Limited Biomass

Abstract

Six aristolochic acids were identified in the Chilean species Aristolochia chilensis 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 µ L of the extract that contains the aristolochic acids present in the Aristolochia chilensis 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 −1 . The column InertSustain C18 250 × 4.6 mm and 3 µ m was used, the injection volume was 20 µ L, and the time of run was reduced to 15 min. Calibration curves were constructed with r 2 being 0.9997. The quantification limit for AAI was 0.138 ± 0.010 µ g/mL, and for AAII, it was 0.558 ± 0.042 µ g/mL.