Assessment, Validation and Application to Real Samples of a RP-HPLC Method for the Determination of Guayulins A, B, C and D in Guayule Shrub

Guayule (Parthenium argentatum Gray) is a shrub native of the arid regions of Mexico. In the last decades, significant attention for its cultivation has risen because it is the raw material for the production of hypoallergenic natural rubber. Guayule biomass contains also high amounts of resin, which is not normally exploited in any way. Among other sesquiterpenic esters, guayulins (i.e. the parteniol esters of cinnamic acid, guayulin A, or of anisic acid, guayulin B) are contained in resin. In addition, minor amounts of guayulin C and guayulin D are formed by degradation/oxidation of guayulins A and B, respectively. Guayulins likely act as cinnamate and p-anisate reservoirs for Guayule shrub, in addition, it has been postulated that they might have a key role in the chemical defense system of Guayule. Furthermore, it seems reasonable that guayulins can possess significant biological properties (e.g, antibacterial and anticancer activities), in close analogy with those shown by sesquiterpene lactones in many other species of Parthenum genus. As a matter of fact, guayulins A and B play an important role in the synthesis of antineoplastics used in breast cancer treatment. In this contribution we propose an original and validated RP-HPLC approach to the simultaneously quantification of guayulins A, B, C and D. The procedure of resin extraction from Guayule biomass has been optimized in terms of both nature of extraction method and solvent of extraction. RP-HPLC separation has been accomplished on an Ascentis^® C18 column under isocratic elution with a 80:20 (v:v) acetonitrile:water mixture. Validation was carried out in terms of both the limit of detection, limit of quantification, linearity, precision, and trueness. Finally, the method was tested with a number of fresh and seasoned samples of spontaneous Guayule shrub from Mexico.