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In this work, the nickel-aluminum layered double hydroxide (Ni-Al LDH) with nitrate interlayer anion was synthesized and used as a solid phase extraction sorbent for the selective separation and pre-concentration of mefenamic acid prior to quantification by UV detection at λmax ? 286 nm. Extraction procedure is based on the adsorption of mefenamate anions on the Ni-Al(NO3? ) LDH and/or their exchange with LDH interlayer NO3? anions. The effects of several parameters such as cations and interlayer anions type in LDH structure, pH, sample flow rate, elution conditions, amount of nano-sorbent and co-existing ions on the extraction were investigated and optimized. Under the optimum conditions, the calibration graph was linear within the range of 2-1000 mg/L with a correlation coefficient of 0.9995. The limit of detection and relative standard deviation were 0.6 mg/L and 0.84% (30 mg/L, n ? 6), respectively. The presented method was successfully applied to determine of mefenamic acid in human serum and pharmaceutical wastewater samples.