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Eco-friendly solvent bar microextraction based on a natural deep eutectic solvent and multivariate optimization for simultaneous determination of spironolactone and canrenone in urine and plasma samples

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Simultaneous measurement of spironolactone and canrenone in urine and plasma provides valuable insight into renal function, and therapeutic efficacy and can be utilized to identify potential health risks and ensure patient safety throughout treatment. By adopting greener methods to analyze these compounds, significant reductions in the environmental impact of such studies can be achieved. For this purpose, a sensitive and eco-friendly solvent bar microextraction method using natural deep eutectic solvent (NDE) followed by high-performance liquid chromatography-diode array detection (HPLC–DAD) was developed to determine spironolactone and canrenone in urine and plasma samples. The extraction solvents were synthesized using NDE-based terpenoids containing menthol and camphor in various ratios. The extraction efficiency percentage (EE%) of both drugs was measured using response surface methodology (RSM) based on central composite design (CCD), and 29 extraction tests were conducted to determine the optimum conditions. Although all parameters were found to be significant, the extraction and elution times were critical for isolating the target analytes. Under optimized conditions, the linear dynamic ranges for spironolactone (SPI)/canrenone (CAN) were 11.7–104/13.1–104 μg L−1 and 21.7–104/24.6–104 μg L−1 in urine and plasma samples, respectively with R2 ≥ 0.993. The ranges of intra-/interprecision (relative standard deviation (RSD) %, n = 5) were 1.31–9.17%/ 2.4–11% with extraction recovery ≥ 88.6% for both drugs. The comparison findings with previously published methods confirmed that the developed NDE-solvent bar microextraction (SBME)-HPLC–DAD method for spironolactone and canrenone analysis displayed confident sensitivity, feasible operation, and simple analysis. Furthermore, the method’s applicability and effectiveness were proven by successfully analyzing spironolactone and its metabolite canrenone in patients’ urine and plasma samples.

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It is acknowledged that The Hashemite University’s Deanship of Scientific Research has offered financial assistance for this study.


The financial support for the work was from the Deanship of Scientific Research at The Hashemite University, without limited details.

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Authors and Affiliations



NNAL-H: conception of the work, data collection, data analysis, interpretation, critical revision of the article, and final approval of the version to be published. HAA: data collection, data analysis, and data interpretation. AHE-S: interpretations, critical revision, and final approval of the version to be published. SHH: data collection, data analysis data, and critical revision. KAAS: data analysis, data interpretation, and critical revision. All authors read and approved the final manuscript.

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Correspondence to Nabil N. AL-Hashimi.

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No animals were used in the study; all reported humans were experimented in accordance with the ethical standards of the committee responsible for human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008 ( en/20activi-ties/10ethics/10helsinki/).

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AL-Hashimi, N.N., Alfattah, H.A., El-Sheikh, A.H. et al. Eco-friendly solvent bar microextraction based on a natural deep eutectic solvent and multivariate optimization for simultaneous determination of spironolactone and canrenone in urine and plasma samples. Environ Sci Pollut Res 31, 3031–3043 (2024).

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