Many dietary supplements claim the ability to enhance sports performance and to improve the fitness of the consumers. Athletes can turn to this kind of help, but most of them are supposed to avoid illegal compounds included as dopants in the World Anti-Doping Agency’s prohibited list. Occasionally, producers may add illicit compounds without labelling them, leading to unintended doping [1]. Hence, the aim of this study was to develop an analytical method to determine drugs such as diuretics, stimulants and β2-agonists along with legal ingredients (like caffeine and sweeteners). The selected analytes are very polar (logKow -0.7 – 2.3) and present ionizable functions. Thus, Hydrophilic Interaction LIquid Chromatography (HILIC) was chosen as separation strategy, coupled with tandem Mass Spectrometry (MS), operating in polarity switching mode and multiple reaction monitoring (MRM) to optimise sensitivity. The instrumental method was optimized using Design of Experiments (DoE). First, a Plackett-Burman (PB) DoE was performed to identify the more influent variables affecting peak areas and resolution. Temperature, water percentage in the eluent, flow rate, as well as type and concentration of buffers as phase modifiers (both formic and acetic acid and ammonium salts) were investigated. A satisfying chromatographic separation was obtained, even resolving theophylline and paraxanthine peaks (isomers presenting the same MRM transition and thus not distinguished by the MS detector). Nevertheless, since the regression models were often not validated by the central point replicates, quadratic models were required. Thus, a D-optimal DoE was set, considering only the most significant variables from PB-DoE results to limit the number of experiments to be performed. Indeed, the buffer choice was quite important for the sensitivity: since anion-forming analytes have lower ionization efficiency and their peak area decreased more with formic acid, acetic acid was chosen (and its amount fixed at 0.1%). Regarding sample treatment, Homogeneous Liquid-Liquid MicroExtraction (HLLME) is a recent trend. It involves the preparation of a single-phase solution, which allows a theoretical infinite surface area between sample and extraction solvent. Then, an event induces a phase separation and the fraction of interest could be directly analysed. Among HLLMEs, the use of switchable hydrophilicity solvents and acetonitrile extraction via salting-out effect were preliminarily tested on blank, spiked, aqueous matrixes (mimicking those obtained by the athletes), but further optimisation is still required prior to application to real samples. [1] K. Walpurgis, A. Thomas, H. Geyer, U. Mareck, M. Thevis, Foods. 9 (2020) 1012.
Multivariate Optimisation of a HILIC-MS/MS Method to Quantify Both Polar Doping Substances and Legal Ingredients in Dietary Supplements
Matteo Baglietto;Barbara Benedetti;Marina Di Carro;Emanuele Magi
2022-01-01
Abstract
Many dietary supplements claim the ability to enhance sports performance and to improve the fitness of the consumers. Athletes can turn to this kind of help, but most of them are supposed to avoid illegal compounds included as dopants in the World Anti-Doping Agency’s prohibited list. Occasionally, producers may add illicit compounds without labelling them, leading to unintended doping [1]. Hence, the aim of this study was to develop an analytical method to determine drugs such as diuretics, stimulants and β2-agonists along with legal ingredients (like caffeine and sweeteners). The selected analytes are very polar (logKow -0.7 – 2.3) and present ionizable functions. Thus, Hydrophilic Interaction LIquid Chromatography (HILIC) was chosen as separation strategy, coupled with tandem Mass Spectrometry (MS), operating in polarity switching mode and multiple reaction monitoring (MRM) to optimise sensitivity. The instrumental method was optimized using Design of Experiments (DoE). First, a Plackett-Burman (PB) DoE was performed to identify the more influent variables affecting peak areas and resolution. Temperature, water percentage in the eluent, flow rate, as well as type and concentration of buffers as phase modifiers (both formic and acetic acid and ammonium salts) were investigated. A satisfying chromatographic separation was obtained, even resolving theophylline and paraxanthine peaks (isomers presenting the same MRM transition and thus not distinguished by the MS detector). Nevertheless, since the regression models were often not validated by the central point replicates, quadratic models were required. Thus, a D-optimal DoE was set, considering only the most significant variables from PB-DoE results to limit the number of experiments to be performed. Indeed, the buffer choice was quite important for the sensitivity: since anion-forming analytes have lower ionization efficiency and their peak area decreased more with formic acid, acetic acid was chosen (and its amount fixed at 0.1%). Regarding sample treatment, Homogeneous Liquid-Liquid MicroExtraction (HLLME) is a recent trend. It involves the preparation of a single-phase solution, which allows a theoretical infinite surface area between sample and extraction solvent. Then, an event induces a phase separation and the fraction of interest could be directly analysed. Among HLLMEs, the use of switchable hydrophilicity solvents and acetonitrile extraction via salting-out effect were preliminarily tested on blank, spiked, aqueous matrixes (mimicking those obtained by the athletes), but further optimisation is still required prior to application to real samples. [1] K. Walpurgis, A. Thomas, H. Geyer, U. Mareck, M. Thevis, Foods. 9 (2020) 1012.
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