Fig. 1. Picture of a glandular trichome on a leaf of A. annua L. before (A and B) and after (C and D) chloroform extraction. The cuticle is crumpled after chloroform extraction. The epidermal cells are unaffected by this treatment. Black bar is 10 μm.

Fig. 2. Chromatogram with retention times and chemical structures of (1) arteannuin B, (2) artemisitene, (3) artemisinin, (4) artemisinic acid and (5) the internal standard artemether. This chromatogram is the result of the analysis by electrospray QTOF-MS/MS of an analytical standard containing 1.2 μg/ml of each analyte and 0.4 μg/ml IS.

Fig. 3. This MS/MS spectrum shows the fragmentation of artemisinin (m/z 283) with the optimal collision energy of 7 eV. The MS/MS signal is calculated as the sum of the fragments with m/z 219, 229, 247 and 265. The molecular formulas show that the fragments are mainly formed by dehydration of their parent ion.

Recovery from spiked samples

^aFifteen equal samples of one gram fresh A. annua leaves were prepared. Ten samples were not spiked and analyzed. ^bFive samples were spiked with each analyte. ^cThe total quantity of the analytes present in the spiked samples was calculated as the sum of the spiked quantity and the mean quantity of the analytes in the 10 unspiked samples. ^dThe spiked samples were analyzed and the individual. ^eMean absolute recoveries (% recovery between brackets) were calculated. Quantities are presented as the concentration after sample preparation (multiply by 600 to obtain quantities in μg analyte/g fresh plant material).

Recovery with chloroform extraction of Artemisia annua leaves

^aSix equal samples of one gram fresh leaf material were prepared. Three of them were extracted following a previously described extraction method [19] which uses extraction with toluene after lyophilisation and pulverization of the plant material. ^bThe other three samples were extracted in exactly the same way but after they were first extracted for one min with chloroform. ^cThe percentage of the chloroform which sticks to the plant material after chloroform extraction (accounting for a part of the not-extracted percentage) was gravimetrically determined. Quantities are presented as the concentration after sample preparation (multiply by 6 to obtain quantities in μg analyte/g fresh plant material).

Accuracy, precision, LOD and LLOQ

LOD and LLOQ are presented with peak-to-peak signal-to-noise ratio. Within- and between-day accuracy and precision are presented at LLOQ and for 7 spiked concentrations (0.1; 0.2; 0.4; 0.8; 1.2; 2.0 and 3.0 μg/ml) within the range of the standard curve. Within- and between-day variation was also calculated for 20 unspiked A. annua samples. Quantities are presented as the concentration after sample preparation.