Applying the Green and Simple Moka-Pot Extraction to detect Performance Enhancing Drugs Via HPLC-MS/MS In Dietary Supplements

In recent years, quick, green and effective extraction strategies became a need in analytical laboratories to support environmental sustainability. Along with the development of more powerful hyphenated instrumental techniques, this led to advanced technologies enabling extremely efficient procedures to be achieved with very low limits of detection. Moka-Pot Extraction (MPE) is a new strategy recently tested for Dietary Supplements (DS) [1]. Moka pots consist of three principal aluminium parts: the boiling chamber, which is filled in water; the funnel filter, in which ground coffee is added; and the collecting chamber, where the drink is collected. DS are widely used worldwide; however, they are often the cause of unintended doping for professional athletes due to the presence of prohibited substances, sometimes not declared in the product information label [2,3]. As a first proof-of-concept, MPE conditions for the extraction of 17 polar compounds from DS were optimised. A 24-1 fractional factorial design of experiments was implemented, considering the following factors: heating temperature; solvent pH; content of organic modifier (acetonitrile); amount of sample within the funnel filter. The multivariate approach allowed easy determination of the most influential variables (which resulted in the amount of sample, and the interaction between temperature and organic modifier content). From the response surface plots, potentially optimal conditions were suggested [1]. These were firstly validated, and both the recoveries (between 52 and 134 %) and matrix effects (always negligible or moderate at 100-fold dilution) were calculated from the peak areas obtained after filtration, dilution and analysis. Through a first Hydrophilic Interaction Liquid Chromatography – tandem Mass Spectrometry (MS) analysis 7 of the 17 tested compounds, including artificial sweeteners, methylxanthines and taurine, were detected and quantified [1]. The obtained concentrations were compared to those declared on the labels (when available) to check for any possible mislabelling, and to the maximum recommended doses for caffeine and taurine, considering also the daily servings. Interestingly, one DS sample contained caffeine and taurine above the permitted limits [1]. The same extracts were then analysed via a Liquid Chromatography - High Resolution MS, being screened for about two hundred of Performance Enhancing Compounds (PECs). The preliminary results highlighted several possible ‘hits’, and confirmation studies are ongoing to check for the presence and eventually quantify the suspect substances. To sum up, this work proved that a Moka-pot, a simple household product, could be used as an effective extraction tool. After the optimization of the method for a few compounds belonging to different classes, the list of targeted analytes was widened, focusing on PECs included in the World Anti-Doping Agency’s prohibited list. Thanks to the promising results, the application of such a simple household device could be encouraged, by exploring the extraction of different classes of compounds in other matrices. References: [1] M. Baglietto, B. Benedetti, M. Di Carro, E. Magi, Assessing the potentialities of an easy-to-use sample treatment strategy: Multivariate investigation on “Moka extraction” of typical ingredients from dietary supplements, Advances in Sample Preparation 10 (2024) 100110. [2] J.M. Martínez-Sanz, I. Sospedra, C.M. Ortiz, E. Baladía, A. Gil-Izquierdo, R. Ortiz-Moncada, Intended or Unintended Doping? A Review of the Presence of Doping Substances in Dietary Supplements Used in Sports, Nutrients 9 (2017) 1093. [3] S. Odoardi, E. Castrignanò, S. Martello, M. Chiarotti, S. Strano-Rossi, Determination of anabolic agents in dietary supplements by liquid chromatography-high-resolution mass spectrometry. Food Additives & Contaminants: Part A, 32(5), (2015) 635–647.