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

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 mu 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 x 4.6 mm and 3 mu m was used, the injection volume was 20 mu 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 mu g/mL, and for AAII, it was 0.558 +/- 0.042 mu g/mL.