Converting a Moka-Pot into a Green Extraction Tool for the Analysis of 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 based on the use of a Moka pot, that consists 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 [1]. MPE works at slightly higher pressure than the atmospheric and at high temperatures, and the extraction encompasses two different mechanisms: (i) during the normal phase a solid-liquid extraction occurs, then (ii) the volcanic phase involves a solid-liquid-vapour extraction, thus allowing a higher extraction yield [1]. This consideration led to the conception of the use of a Moka pot as a device for sample preparation for extracting targeted compounds in Dietary Supplements (DS) [2]. 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 [3,4]. This study aimed to optimise the extraction efficiency of MPE (including performance enhancing compounds - PECs) and applying the methodology to several DS samples. As a first proof-of-concept, MPE conditions for the extraction of 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 [2]. These were firstly validated for 17 target analytes by processing a spiked matrix which did not present any of them, 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. These conditions were applied to real samples: (i) through Hydrophilic Interaction Liquid Chromatography – tandem Mass Spectrometry (MS) 7 of the 17 tested compounds, including artificial sweeteners, methylxanthines and taurine, were detected and quantified [2]; (ii) via a Liquid Chromatography - High Resolution MS analysis the extracts were further screened for more than a hundred PECs. The obtained concentrations were then 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 [2]. To evaluate the performances of this method, the results were compared with those of a Salt-Assisted Liquid-Liquid Extraction (SALLE) previously developed [5], both in terms of extraction efficiencies and greenness. The two methodologies were quite comparable, even if the MPE was much more efficient in the extraction of taurine, which was not included in the subset of analytes for which SALLE has been optimized [5]. Regarding the greenness, MPE produced higher amounts of waste, but if a smaller Moka was available (e.g. 10 times smaller), it would be even greener than the SALLE [2]. 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] L. Navarini, E. Nobile, F. Pinto, A. Scheri, F. Suggi-Liverani, Experimental investigation of steam pressure coffee extraction in a stove-top coffee maker, Applied Thermal Engineering 29 (2009) 998–1004. [2] 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. [3] 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. [4] 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. [5] M. Baglietto, B. Benedetti, M. Di Carro, E. Magi, Polar licit and illicit ingredients in dietary supplements: chemometric optimization of extraction and HILIC-MS/MS analysis, Anal Bioanal Chem 416 (2024) 1679–1695.