Introduction: Artemisia afra Jacq. ex. Willd. (Asteraceae) is a popular traditional medicine in South Africa, mainly used in the form of an infusion, for the treatment of respiratory ailments. Quality control methods are limited and phytochemical variation for the infusion is not well known.Objective: To develop a sensitive quality control method for A. afra infusions by validating a liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method and quantitatively comparing six marker compounds in A. afra samples collected from different locations and over a 12-month period.Material and methods: There was a multiple reaction monitoring method optimised and validated, according to ICH and FDA guidelines, to quantify the chemical markers present in infusions. Results:The chemistry differed significantly and interestingly, with an interchangeable trend between chlorogenic acid (CGA) and 4,5-dicaffeoylquinic acid (DCQA) observed in the samples collected monthly, elevated levels of CGA during winter and elevated levels of DCQA during summer. The remaining four markers showed a steady decrease as winter approached and a steady increase as summer approached.The ranges of the six markers were the following: CGA (0.68-14.68 μg/mg), DCQA (0.005-8.110 μg/mg), quercetin (0.01-0.65 μg/mg), luteolin (0.05-1.30 ng/mg), scopoletin (0.10-1.14 μg/mg), scopolin (0.03-1.21 μg/mg).Conclusions: A sensitive LC-ESI-MS/MS method was developed, validated, and used to quantify six marker compounds. The results indicated a large degree of phytochemical variation occurred across all samples tested, which highlights the importance of producing herbal medicine under controlled conditions and the necessity of analytical quality control methods.

Artemisia afra (A. afra) is an herbal medicine, traditionally prepared as a tea infusion, used for centuries in African countries to treat a vast number of ailments. This herb contains an ample amount of known and unknown compounds and has antibacterial, anti-viral and anti-fungal properties; hence, topical treatment was considered. A liquid chromatography mass spectrometry (LC-MS/MS) method was developed and validated to detect the six selected marker compounds used during this study: two organic acids (4,5-dicaffeoylquinic acid (DCQA) and chlorogenic acid (CGA), two flavonoids (luteolin and quercetin) and two coumarins (scopoletin and scopolin). The formulations selected had to accommodate the hydrophilic nature of the A. afra infusion; therefore, there was an infusion, hydrogel and emulgel selected. The hydrogel and emulgel contained a gelling agent, xanthan gum, whereas the emulgel additionally contained a chemical penetration enhancer, evening primrose oil (EPO), to improve penetration through the lipophilic stratum corneum. The characterisation of the semi-solid formulations was to ensure skin application suitability. Membrane release studies confirmed sufficient release of the different markers from the formulations. During the in vitro skin diffusion studies, the discovery was that the infusion had the highest median flux and amount per area diffused compared to the other formulations. Thereafter, there was tape stripping performed, and established that there were markers present in the stratum corneum-epidermis (SCE) and epidermis-dermis (ED). The testing of cytotoxicity was to determine the safety of topical delivery, and the finding was that A. afra showed no cytotoxicity at the levels tested.