Green and Emerging Microextraction Strategies for Bioanalytical Determination of Hormones: Trends, Challenges, and...

Accurate and sensitive determination of hormones in biological matrices is essential for clinical diagnostics, therapeutic monitoring, and endocrine research. However, hormone determination presents significant challenges due to their typically low concentrations, complex sample matrices, and structural diversity. In recent years, microextraction techniques have emerged as strategic tools in bioanalytical chemistry, offering advantages in terms of miniaturization, enhanced selectivity, and compatibility with the principles of green analytical chemistry (GAC). This review provides a comprehensive overview of green and emerging microextraction approaches for the determination of steroidal, thyroid, peptide, and other hormones in biological samples. Key techniques such as solid-phase microextraction (SPME) and dispersive liquid-liquid microextraction (DLLME), followed by high-performance liquid chromatography (HPLC) coupled to diode array detectors (DADs) or mass spectrometry (MS), are critically discussed. Special emphasis is placed on the use of environmentally friendly solvents, such as deep eutectic solvents (DESs), supramolecular solvents (SUPRASs), and advanced sorbents including molecularly imprinted polymers (MIPs) and nanostructured magnetic phases. Applications across various bioanalytical matrices (urine, plasma, serum, saliva, tissues…) are examined in terms of sensitivity, selectivity, and validation parameters. Finally, current challenges, method development gaps, and future directions are highlighted to support the continued advancement of sustainable hormone determination in complex biological systems.