4.3. Quantification of Artemisinin by HPLC-MS/MS

The HPLC system used including columns and the mass spectrometer are described above. For sample preparation, 30 mg of Momundo capsule content were dissolved in 1.5 mL acetonitrile and extracted for 1 h at RT with continuous stirring. After centrifugation (16,000 g, 10 min), 1 mL supernatant was added to 1 mL water and filtered through regenerated cellulose (0.45 µm). The resulting solution with a sample concentration of 10 mg/mL was analyzed in triplicates. For chromatographic separation, the flow rate was set to 600 µL/min and the injection volume to 70 µL. The mobile phase consisted of eluent A (deionized, ultrapure water + 0.1% acetic acid and 10 mM ammonium acetate) and eluent B (acetonitrile + 0.1% acetic acid). Initial conditions were 60% eluent A and 40% eluent B followed by a linear gradient to 90% eluent B over 10 min, then 90% eluent B until 13 min. Thereafter, a linear gradient continued to initial conditions until 15 min and reequilibration until 20 min. The column was kept constantly at 28 °C.

MS/MS analysis was performed in the positive atmospheric pressure ESI and multiple-reaction monitoring (MRM) detection modes. For quantification, the precursor ion at m/z 300.2 ([M + NH₄]⁺) and the product ion of highest intensity at m/z 151.2 were selected. To achieve linearity and to determine the limit of detection (LOD) and the limit of quantification (LOQ), standard solutions in the range from 7.5 ng/mL to 100 ng/mL (6 levels) were analyzed, each in triplicates, yielding a LOD of 2 ng/mL and a LOQ of 8 ng/mL. To evaluate accuracy, the recovery was determined by using the standard addition method. Hence, a real sample was spiked at six levels, extracted as described above in sample preparation and analyzed, each in triplicates, yielding a recovery of 94.8% (± 9.6% SD). Precision was determined by analysis of a reference sample with six replicates at four different days yielding the intraday variation of 1.5% (RSD) and the interday variation of 1.8% (RSD). Sample analysis showed that the artemisinin concentration of the Momundo extract was below the LOD. The method was also used for analysis of three additional commercial Artemisia annua herbal preparations. The analysis revealed artemisinin contents below the LOQ in ‘Artemisinin′ (Euro Nutrador B.V., Landgraaf, Netherlands) and ‘Artemisia Extrakt 400 mg′ (Vita-World, Taunusstein, Germany), whereas ‘Artemisia annua intense^® 600 mg′ (Novofrom Pharma GmbH, Gaggenau, Germany) contained 38 µg/mg artemisinin, which complies with the manufacturers specification of 4% artemisinin.