Chemically Modified Eggshell Membrane as an Adsorbent for Solid-Phase-Extraction of Morphine Followed by High Performance Liquid Chromatography Analysis

In this project, eggshell membrane was used as a solid phase extraction sorbent for extraction of morphine followed by HPLC analysis. At first, raw eggshell membrane was used and then, because of low adsorption of morphine on untreated eggshell membrane (<70%), modification of eggshell membrane surface was carried out using different surfactants and amines. The amine modified eggshells characterized by Furrier transform infrared spectroscopy and UV-Vis diffuse reflectance spectroscopy resulted in better adsorption of morphine. The effects of different factors on extraction efficiencies were investigated. Based on experimental results 20 ml of glutaraldehyde (35% V/V) and 2 mmoles of normal hexane amine have been chosen as the best condition for chemical modification. The optimum pH values for sample solution and washing buffer were 8. Application of 300.0 mg sorbent, 6 ml methanol as desorbing solvent, 0.5 ml of 5 mg l-1 morphine sample solution and 6 min for sorption and desorption times were among the best conditions for morphine pre-concentration. At the optimum conditions, the calibration plot of morphine was obtained with good linearity (R2 = 0.9990) and using this plot, figures of merit of the proposed method were evaluated. The proposed method presents suitable working linear ranges (0.5-10, mg l-1), good limit of detection (0.095 mg l-1), acceptable repeatability (%RSD ≤ 4.50) and good accuracy (recovery = 101.6%). Finally, the amount of spiked morphine to saliva as a real sample was determined using this method.