Opioid detection and quantification in alternative biological matrices: recent analytical advances

The continuous emergence of synthetic opioids, particularly fentanyl analogues and nitazenes, has created major analytical challenges for clinical and forensic toxicology. Alternative biological matrices have gained relevance as complementary specimens for opioid detection and quantification alongside conventional blood and urine. This review critically examines analytical advances reported between 2020 and 2026 for opioid determination in oral fluid, sweat, exhaled breath condensate, hair, meconium, vitreous humor, bile, pericardial fluid, nasal swab and teeth. Emphasis is placed on sample preparation strategies, miniaturized extraction approaches, chromatographic and mass spectrometric platforms, sensor-based methods and matrix-specific analytical performance. Oral fluid and hair remain the most established alternative matrices for assessing recent and retrospective exposure, respectively. Recent progress has involved improved sample preparation workflows, including microextraction by packed sorbent, fabric phase sorptive extraction, thin-film microextraction, dispersive micro-solid-phase extraction, supramolecular solvent extraction, cryogenic grinding and one-pot extraction strategies. Liquid Chromatography tandem mass spectrometry remains the predominant analytical platform, while high-resolution mass spectrometry-based approaches support broad screening, suspect analysis and retrospective data interrogation. Overall, alternative matrices are becoming increasingly valuable in opioid analysis, although methodological harmonization, matrix-specific interpretation and emerging synthetic opioids remain important challenges.