Development and validation of UPLC-UV-DAD method for the determination of oleandrin in Nerium oleander dried leaves collected in Eastern Algeria

Abstract

To develop and validate an ultra-high-performance liquid chromatography coupled to diode array detection (UPLC-DAD) method for the determination of oleandrin, the active and toxic principle of Nerium oleander dried leaves and to assess oleandrin concentrations in samples collected in Eastern Algeria. Plant poisoning accounts for 3 to 4% of all calls to the Anti Poison Center (CAP) in Algiers each year, of which 7% are due to Nerium oleander. Dried leaves of Nerium oleander were macerated in methanol for 18 hours at room temperature, followed by 1/5th dilution in a mixture of phosphate buffer (pH = 3.8)/ACN (90/10). Chromatographic separation was performed on a C18 2.1 × 100 mm (Kinetex 2,6 μm Polar) and a gradient of acetonitrile:phosphate buffer (initial conditions of 20:80 ACN:buffer for 0.5 min. Linear gradient to 90:10 ACN:buffer for 4.5 min. Isocratic conditions of 90:10 ACN:buffer for 1 min. Return to initial conditions within 1 min. Equilibrate for 3 min) using an UPLC-DAD system (ThermoFisher®). The method was validated for linearity, limit of detection (LOD) and of quantification (LOQ), accuracy, precision, and recovery. The method was applied to 100 samples of Nerium oleander leaves collected from wild (wadi) or cultivated (roadside) shrubs, with pink, red, or white flowers. The method is linear in the concentration range 0.1–25 mg/L, with LOD and LOQ of 0.03 mg/L and 0.1 mg/L, respectively. Accuracy (bias %) intra-day and inter-day ranged from 94.3 to 101.1% and 96.5 to 99%, respectively. Precision (CV%) intra-day and inter-day ranged from 1.7 to 3.3% and 2.1 to 9.1%, respectively. Recovery was 89%. Oleandrin levels were measured in leaves during the flowering period, as this is where the highest concentrations were described. It is also the leaves that are the most consumed, as they appear in the traditional pharmacopoeia and are therefore a potential hazard. Oleandrin concentrations varied from 0.01 to 0.48 mg/g of dried leaves. The mean oleandrin concentration for the Eastern region of Algeria, including Constantine (main city) and 4 adjacent cities (Guelma, Mila, Skikda and Oum El Bouaghi) was 0.12 ± 0.12 mg/g. There was no significant difference in the mean oleandrin levels in the leaves neither of the wild or cultivated plants ( P = 0.573), nor between the different flowering plants (pink, red and white) ( P = 0.893). Secondary metabolites of toxic plants, such as oleandrin are present, in small amounts and serve as a defence against biotic and abiotic stresses. Our study has revealed an average level of 0.012% oleandrin (leaves) in Eastern Algeria. These concentrations would be helpful to estimate the amount of oleandrin to which an individual could be exposed, in case of ingestion of this plant, which allow to assess the severity of the poisoning, and to adapt the treatments.