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Electrospun poly(vinyl alcohol)-(PVA)-poly(acrylic acid) (PAA)/carbon nanotubes(CNTs)-cellulose nanocrystal (CNC) (PVA-PAA/CNT-CNC) composite nanofibers were prepared and characterized using Fourier transform-infrared spectroscopy and field emission scanning electron microscopy. The resultant composite was used as an effective and novel sorbent for pipette-tip micro-solid phase extraction (PT-μSPE) of seven opioid analgesics (OAs) in biological samples followed by HPLC-UV analysis. Addition of CNT-CNC with the high specific surface area and plenty of OH-functional groups endows the nanofibers with considerable extraction efficiency. Under the optimum conditions, the linearity was obtained in the range 1.5 to 700.0 ng mL⁻¹ for morphine, codeine, oxycodone, and tramadol, and 0.5 to 1000.0 ng mL⁻¹ for nalbuphine, thebaine, and noscapine with coefficient of determination (r²) ≥ 0 …