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. Opioids include medicines used for pain treatment, but also highly potent synthetic drugs that are increasingly found on illicit drug markets, creating challenges for their detection in both healthcare and forensic settings. Detecting these substances accurately is particularly challenging as new compounds, such as fentanyl analogues and nitazenes, a group of highly potent synthetic opioids, continue to emerge. This review summarizes recent analytical advances for detecting and measuring opioids in alternative biological samples, such as oral fluid, hair, sweat, exhaled breath condensate, meconium and fluids collected after death. These samples can provide useful information when blood or urine is difficult to collect, unavailable or insufficient. Recent studies have improved how these samples are processed and how opioids are detected using increasingly sensitive laboratory methods. These advances can help healthcare and forensic professionals identify opioid exposure more accurately and provide useful information for clinical care, forensic investigations, and public health monitoring. Oral fluid and hair are the most widely studied types of samples, while others remain less well studied. Further research is needed to ensure that results obtained from these samples can be compared reliably across laboratories and interpreted correctly, particularly when determining the extent and timing of opioid exposure.