Validation of a microscale extraction and high-throughput UHPLC-QTOF-MS analysis method for huperzine A in Huperzia

Abstract

Herein we report on an improved method for the microscale extraction of huperzine A (HupA), an acetylcholinesterase-inhibiting alkaloid, from as little as 3 mg of tissue homogenate from the clubmoss Huperzia squarrosa (G. Forst.) Trevis with 99.95% recovery. We also validated a novel UHPLC-QTOF-MS method for the high-throughput analysis of H. squarrosa extracts in only 6 min, which, in combination with the very low limit of detection (20 pg on column) and the wide linear range for quantification (20-10,000 pg on column), allow for a highly efficient screening of extracts containing varying amounts of HupA. Utilization of this methodology has the potential to conserve valuable plant resources.

Fig. 1

Identification of HupA in H. squarrosa extracts. (A) Total Ion Current (TIC) and (B) Extracted Ion Chromatograms (EIC) obtained by UHPLC-QTOF-MS in APCI (+) mode for the HupA standard (red trace) and the 1 % acetic acid extract (black trace). HupA elutes at 0.62 min. (C) MS and (D) MS/MS spectra of the H. squarrosa extract at 0.62 min. (E) MS and (F) MS/MS spectra of the authentic HupA standard at 0.62 min. The following fragments were identified in the MS/MS spectra of the extract: (1) 243.1477 = [M+H]⁺, (2) 226.1225 (loss of NH₃), and (3) 210.0890 (unannotated).

Fig. 2

Evaluation of matrix effects on HupA detection by UHPLC-QTOF-MS. The blue line indicates the calibration curve with neat HupA standard (N = 6), while the red line indicates measured peak areas for known quantities of HupA spiked into an extract obtained from H. lucidula, a close relative of H. squarrosa that does not contain detectable levels of HupA (N = 6). Standard deviations are indicated, but are mostly smaller than the symbols used to plot the mean of the measurements.