Advanced sample preparation methods for LC-MS/MS bioanalysis of naturally derived narcotics in clinical and forensic contexts.

Naturally occurring narcotics such as morphine and codeine require careful monitoring due to the risks of tolerance, addiction, and overdose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is considered a gold standard method for the quantitative and qualitative analysis of these narcotics in biological samples. However, because of the complex structure of biological matrices, sample preparation directly affects analytical performance. Inadequate preparation can lead to matrix effects, poor recovery, and analytical uncertainties, making optimized strategies necessary for reliable results. This review presents a systematic classification of sample preparation techniques used in the LC-MS/MS-based bioanalysis of morphine and codeine. Protein precipitation, liquid-liquid extraction, solid-phase extraction, and next-generation microextraction approaches are comprehensively evaluated regarding matrix-effect reduction, ion suppression control, and analyte recovery. Current methodological limitations and literature gaps are discussed, alongside proposed innovative strategies for future research. Rather than viewing sample preparation as a simple preliminary step, this study emphasizes its role as a key component of the analytical workflow. Integrating optimized extraction with LC-MS/MS is essential for achieving reliable, sensitive, and reproducible narcotic analysis.