•We developed a rapid method of analysis of six metabolites in A. annua extracts.•9-Epi-artemisinin was quantified in raw extracts of A. annua for the first time.•We show the method to be robust, sensitive, adequate for high throughput analysis.A rapid high-pressure liquid chromatography (HPLC) tandem mass spectrometry (TQD) method for the determination of artemisinin, 9-epi-artemisinin, artemisitene, dihydroartemisinic acid, artemisinic acid and arteannuin B in Artemisia annua extracts is described. Detection and quantification of 9-epi-artemisinin in crude extracts are reported for the first time. In this method all six metabolites are resolved and eluted within 6 min with minimal sample preparation. A recovery of between 96.25% and 103.59% was obtained for all metabolites analysed and the standard curves were linear (r2 > 0.99) over the concentration range of 0.15–10 μg mL−1 for artemisinin, 9-epi-artemisinin, artemisitene and arteannuin B, and the range of 3.75–120 μg mL−1 for dihydroartemisinic acid and artemisinic acid. All validation indices were satisfactory, showing the method to be robust, quick, sensitive and adequate for a range of applications including high throughput (HTP) analysis.A rapid method for quantification of several important co-metabolites of artemisinin in raw extracts is presented.

•Co-metabolites negatively impacting crystallization of artemisinin were identified.•Flavonoid artemetin has strongest impact on crystallization of artemisinin.•Elevated levels of artemetin were found in the problematic East African biomass.Methoxylated flavonoids casticin, artemetin and retusin were identified as putative causative factors for low crystallization yields of artemisinin from extracts. Comparative profiling of biomass grown in different countries found elevated levels (∼60% higher) of artemetin in the East African biomass, which also demonstrates poor crystallization yields. The single compound and the combined doping experiments at 0, 25 and 50 μg mL−1 doping levels showed that artemetin (at 50 μg mL−1) caused a reduction in the amount of artemisinin crystallized by ca. 60%. A combination of the three flavonoids at 50 μg mL−1 almost completely inhibited crystallization, reducing the yield by 98%. Treatment of extracts by adsorbents efficiently resolves the problem of low crystallization yield.