Simultaneous determination of aconitine, mesaconitine, hypaconitine, bulleyaconitine and lappaconitine in human urine by liquid chromatography-electrospray ionization-tandem mass spectrometry

Abstract

Aconitine, hypaconitine, mesaconitine, bulleyaconitine and lappaconitine are Aconitum alkaloids that have been proved to be neurotoxic and cardiotoxic. A method with liquid–liquid extraction and liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated for the quantification of the five Aconitum alkaloids in human urine. Upon the successful chromatographic separation, the alkaloids were determined by ion trap tandem mass spectrometry. The precursor and major product ions of aconitine at m/z 646 → 586, mesaconitine at m/z 632 → 572, hypaconitine at m/z 616 → 556, bulleyaconitine at m/z 644 → 586 and lappaconitine at m/z 585 → 535 were monitored in positive ion mode. Linear calibration curves were generated from 0.02–5 μg mL⁻¹ with coefficients of greater than 0.99. The method was validated with the intra-day and inter-day precisions represented by relative standard deviation of less than 7% in urine, and the recoveries from spiked urine samples varied from 89.8% to 96.4% for lappaconitine, 87.4–96.3% for hypaconitine, 81.5–90.6% for mesaconitine, 82.6–90.0% for aconitine and 82.2–88.7% for aconitine. Stability of aconitine was assessed under acidic, neutral and alkaline conditions. Aconitine hydrolysis was not observed during the urine sample pretreatment under neutral and weak acidic conditions.