Michal P. Dybowski

Purpose: This study aimed to develop and optimize a novel analytical approach utilizing the transient matrix effect (TME) to simultaneously enhance the determination of varying drug classes (tramadol, doxepin, diazepam) and anabolic-androgenic steroids (testosterone, nandrolone) in human blood plasma. Methods: A GC-MS method combined with QuEChERS extraction was applied. To controllably induce TME, varying classes of analyte protectants (PEG-400, C14-NH2, IPM-98, and Triton X-100) were evaluated both individually and as multicomponent mixtures. The optimized procedure was subsequently evaluated for inter-source matrix variability across authentic human plasma samples. Results: Combining protectants with different affinities significantly modified their protective action. While a four-component mixture provided the highest enhancement in pure solvent, it generated competitive adsorption in plasma extracts. A binary combination of PEG-400 and C14-NH2 was identified as the most effective mixture for plasma - yielding increases of 314% and 221% for nandrolone and diazepam, respectively, compared to standard samples. The induced matrix effect was strictly transient - protectants were completely flushed from the column between analyses. Conclusion: Intentional modulation of TME using a binary mixture of high-boiling protectants (PEG-400 and C14-NH2) is proposed to initiate the formation of a transient condensation film at the beginning of the chromatographic column. Analytes condense onto this film, leading to the strong focusing of their concentration zone. This phenomenon significantly enhances the detection sensitivity of structurally diverse drugs and steroids in plasma. By normalizing the sorption environment, the method neutralizes endogenous matrix variability, representing a robust and reproducible tool for forensic toxicology and routine casework.

The determination of benzodiazepines and opioids in whole blood by gas chromatography (GC) remains challenging due to low analyte concentrations and matrix‐related effects that may compromise analytical response. In this study, the use of analyte protectants (APs) was investigated as a means of enhancing detector response in GC–tandem mass spectrometry (GC–MS/MS) analysis of selected benzodiazepines and opioids following Quick, Easy, Cheap, Effective, Rugged, and Safe‐based sample preparation. A series of high‐boiling compounds, including linear alcohols, amines, carboxylic acids, and polyethylene glycols (PEGs), were evaluated as APs capable of modulating the chromatographic response. Among the tested compounds, PEG‐400 and PEG‐600 provided the most consistent signal enhancement, increasing responses of benzodiazepines by 413%–475% and opioids by up to 628% under optimized conditions. Long‐chain amines showed the highest enhancement for selected opioids, reaching up to 1080%. The influence of key experimental parameters on APs efficiency was systematically assessed, demonstrating that acetonitrile as the extraction solvent, elevated injector temperatures (up to 325°C), and low initial oven temperatures favored signal enhancement. The developed GC–MS/MS procedure exhibited satisfactory analytical performance, including high linearity ( R ² > 0.998), good precision (relative standard deviation <5%), and low limits of quantification. Application of the approach to authentic whole‐blood samples confirmed that signal enhancement can also be achieved in complex biological matrices, although its magnitude was reduced by the presence of residual matrix constituents. In particular, bromazepam exhibited a 593% increase in signal following the addition of PEG‐400. The results demonstrate that the implementation of APs can serve as a simple means of improving analytical response in GC–MS/MS analysis of benzodiazepines and opioids without modification of routine sample preparation procedures. The findings further contribute to the understanding of protectant‐mediated signal enhancement mechanisms in the analysis of biological samples.

Purpose: Anabolic–androgenic steroids (AAS) enhance athletic performance, giving athletes an unfair advantage and disrupting fair competition. Banned in sports and listed by World Anti-Doping Agency, they require precise detection. This study aimed to develop a method using the transient matrix effect to improve AAS identification in biological samples. Methods: Gas chromatography–tandem mass spectrometry (GC–MS/MS) method for determination of AAS samples was developed and validated. Biological samples were prepared using the QuEChERS technique. Results: The optimised and validated method enhances AAS signals using high-boiling protectants. It ensures good linearity, low detection limits, and reliable precision. Optimal QuEChERS extraction and multiple reaction monitoring transitions in GC–MS/MS were evaluated, confirming applicability with blood plasma samples. The addition of a protectant to the analysed sample results in several notable effects. High-boiling protectants, such as polyethylene glycol (PEG-400), tetradecanoic acid (C14-COOH), n -tetradecylalcohol (C14-OH), and n -tetradecylamine (C14-NH₂), significantly enhance AAS’s signal in blood plasma. This enhancement, however, is accompanied by a transient matrix effect induced by the protectants. PEG-400 produced the most substantial signal increase, with the response for nandrolone rising by as much as 912%. Conclusions: The results demonstrate the potential offered by the utilisation of PEG-400 as a protectant to generate a transient matrix effect. The outcome of this process is an increased analytical signal from AAS in blood plasma, enabling their identification even at trace concentrations. The methodology developed and applied during the study can be used to reduce the detection limit of steroids and thus improve antidoping measures in sport. Graphical

Purpose: The purpose of this paper was to determine 3- and 4-chloromethcathinone (3- and 4-CMC) binding degree and possible binding interaction modes with human serum albumin (HSA) using analytical and theoretical methods. Methods: Experimental determination of 3- and 4-CMC binding degree with HSA was performed using gas chromatography–tandem mass spectrometry preceded by the equilibrium dialysis (ED) and ultrafiltration (UF). Nuclear magnetic resonance (NMR) spectroscopy was used to determine 3- and 4-CMC epitope-binding maps and possible binding sites in HSA. The molecular docking and molecular dynamics were employed to obtain detailed information about binding modes of 3- and 4-CMC enantiomers in HSA. Results: As follows from the presented data, the degree of binding of 3- and 4-CMC is at a similar level of approx. 80%. This indicates a relatively strong binding of CMC to plasma proteins. The model studies employing the NMR spectroscopy and molecular simulations indicate that both CMCs bind to HSA. The whole 3- and 4-CMC molecules are embedded in the binding sites, with aromatic moieties being in the closest contact with the HSA residues. Moreover, conducted experiments show that Sudlow site II is the main binding center for 3- and 4-CMC and Sudlow site I acts as the secondary binding site. Conclusions: Although many studies describe pharmacological and toxicological properties of synthetic cathinones (SC), the data taking SCs binding in plasma into consideration are scarce. To our knowledge, this is the first report presenting comprehensive experimental and theoretical characterization of 3- and 4-CMC binding with plasma proteins.