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Abstract

This paper introduces a newly developed method of applying liquid chromatography- tandem mass spectrometry (LC/MS/MS) to the analysis of aristolochic acids (AAs) in Chinese herbal (medicinal) preparations. Sensitive and highly accurate readings were obtained for this study using both a photodiode array (PDA) detector and tandem mass spectrometer. The following optimized conditions for LC/MS/MS were set for this study: Separation was accomplished using a reverse phase C18 column (2.1 × 150 mm, 5 μm). The mobile phase comprised 35% acetonitrile and a 65% aqueous solution containing 0.1% formic acid and 0.1% ammonium acetate at a flow rate of 0.3 mL/min with a split ratio of 1:1 into the PDA detector and the tandem mass spectrometer. MS/MS qualitative analysis was performed at 3.0 kV capillary voltage, with a collision energy level for aristolochic acid I (AA-1) of 10 eV and for aristolochic acid II (AA-II) of 12eV. The electrospray ionization source was operated in the positive mode. AA-I [M + NH4]+ ions at m/z 359 and AA-II [M + NH4]+ ions at m/z 329 were selected as precursor ions for daughter ion scanning. The characteristic daughter ion mass spectra for AA-I and AA-II were generated and studied carefully. Quantification of results was done using the multiple reaction monitoring (MRM) method. The [M + NH4]+ and [(M + NH4)- NH3-CO2]+ ions of both AA-I and AA-II were selected as precursor and daughter ions for the MRM analysis. MS/MS detection limits were defined as 2.0 ng/mL of AA-I, and 2.8 ng/mL of AA-II. The linear regression correlation coefficients of the calibration cure were 0.9992 of AA-I within the range of 0.02-16.00 μg/mL and 0.9988 of AA-II within the range of 0.028-22.40 μg/mL. Relative standard deviations of 0.73% and 10.44% for AA-I, and 1.38% and 6.10% for AA-II were determined for the intraday and interday tests. Recoveries for five differing concentrations of AA-I ranged from 99.0% to 106.9% and, for five differing concentrations of AA-II, ranged from 92.0% to 104.5%. Levels of AA-I and AA-II detected in the 12 commercial Chinese medicinal preparation samples ranged from 11.1 to 3376.0 μg/g and from 5.4 to 725.4 μg/g, respectively.

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