| Summary: | A rapid, specific, and sensitive ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) method to examine the chemical differences between <em>Aconitum</em> herbs and processed products has been developed and validated. Combined with chemometrics analysis of principal component analysis (PCA) and orthogonal projection to latent structural discriminate analysis, diester-diterpenoid and monoester-type alkaloids, especially the five alkaloids which contributed to the chemical distinction between <em>Aconitum</em> herbs and processed products, namely mesaconitine (MA), aconitine (AC), hypaconitine (HA), benzoylmesaconitine (BMA), and benzoylhypaconitine (BHA), were picked out. Further, the five alkaloids and benzoylaconitine (BAC) have been simultaneously determined in the <em>Xiaohuoluo pill</em>. Chromatographic separations were achieved on a C<sub>18</sub> column and peaks were detected by mass spectrometry in positive ion mode and selected ion recording (SIR) mode. In quantitative analysis, the six alkaloids showed good regression, (<em>r</em>) > 0.9984, within the test ranges. The lower limit quantifications (LLOQs) for MA, AC, HA, BMA, BAC, and BHA were 1.41, 1.20, 1.92, 4.28, 1.99 and 2.02 ng·mL<sup>−1</sup>, respectively. Recoveries ranged from 99.7% to 101.7%. The validated method was applied successfully in the analysis of the six alkaloids from different samples, in which significant variations were revealed. Results indicated that the developed assay can be used as an appropriate quality control assay for <em>Xiaohuoluo pill</em> and other herbal preparations containing <em>Aconitum</em> roots.
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