Analysis of underivatized artemisinin and related sesquiterpene lactones by high-performance liquid chromatography with ultraviolet detection.
BACKGROUND
Although high-performance liquid chromatography with ultraviolet detection (HPLC-PAD) is widely available, it has not been used for artemisinin (1) analysis because of the lack of UV absorption reported for this lactone. Increased Artemisia annua cultivation for production of 1 requires an affordable and reliable method to analyse 1 and its precursors dihydroartemisinic acid (2) and artemisinic acid (3) simultaneously from underivatized plant extracts.


OBJECTIVE
To validate HPLC-PAD for the quantification of underivatized artemisinin from A. annua and artemisinin-based drugs.


METHODS
Dried A. annua leaves were extracted with petroleum ether, dried, reconstituted in acetonitrile, and analysed by HPLC-PAD at 192 nm using an isocratic mobile phase (60:40, acetonitrile:0.1% acetic acid). HPLC-PAD was evaluated through accuracy, precision, recovery and comparison with HPLC with evaporative light scattering detection (HPLC-ELSD).


RESULTS
HPLC-PAD proved accurate, precise and reproducible for the direct quantification of 1 and related compounds, and was more sensitive than ELSD for most of the compounds tested. The limit of quantification of 1-3 from plants was 0.048, 0.024 and 0.008 g/100 g dry weight, respectively. Recoveries were over 98%, with good intra- and inter-day repeatability. HPLC-PAD correlated significantly (r(2 )= 0.99, p < 0.001) with HPLC-ELSD for artemisinin analysis. HPLC-PAD was also reliable for the analysis of dihydroartemisinin, artesunate and artelinic acid.


CONCLUSIONS
HPLC with ultraviolet detection was validated for the quantification of underivatized 1, 2, and 3 from crude plant samples, and is readily applicable for the quality control of herbals and artemisinin-related pharmaceutical compounds.
Ferreira
Jorge F S
JF
Appalachian Farming Systems Research Center, U.S. Department of Agriculture Agricultural Research Service, 1224 Airport Road, Beaver, WV 25813, USA. jorge.ferreira@ars.usda.gov
Gonzalez
Javier M
JM
D019306:Artemisia / Q000737:chemistry; D037621:Artemisinins / Q000032:analysis*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D007783:Lactones / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012717:Sesquiterpenes / Q000032:analysis*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D037621:Artemisinins; D007783:Lactones; D012717:Sesquiterpenes; C075875:artemisin