In the present study, a new graphene based nanofibers material (Polyacrylonitrile/Graphene Oxide (PAN/GO)) was used for microextraction by packed sorbent (MEPS). The PAN/GO nanofiber was synthesized using the electrospinning technique. MEPS online with liquid chromatography-tandem mass spectrometry (LCMS/ MS) was utilized for the extraction and determination of two local anesthetic drugs (lidocaine, prilocaine) and their major metabolites (2,6-xylidine, o-toluidine) in human plasma samples. Parameters affecting the extraction efficiency were investigated and optimized (including sample pH, washing solution and elution solution). The validation of the method was based on FDA (Food and Drug Administration) guidelines for bioanalytical methods. The calibration curve ranged from 2.00 to 2000 nmol/L for lidocaine and prilocaine, and from 10.0 to 2000 nmol/L for 2,6-xylidine and o-toluidine. The coefficient of determination (R2) values were 0.996, 0.995, 0.995, 0.996 (n=3) for lidocaine, prilocaine, 2,6-xylidine and o-toluidine, respectively. The extraction recovery was 93.0% for lidocaine, 96.0% for prilocaine, 68.0% for 2,6-xylidine and 69.0% for o-toluidine. The limits of detection (LODs) were 0.25, 0.50, 2.50, 1.25 nmol/L for lidocaine, prilocaine, 2,6-xylidine and o-toluidine, respectively. The lower limits of quantification (LLOQs) were 2.0 nmol/L for lidocaine and prilocaine, and 10 nmol/L for 2,6-xylidine and o-toluidine, respectively. The accuracy values for the quality control (QC) samples were in the range of 91.0 - 111% for lidocaine, 92.0 - 118% for prilocaine, 84.0 - 98.0% for 2,6-xylidine and 82.0 - 90.0% for o-toluidine. The inter-day precisions for QC samples ranged from 7.0% to 11.8% for lidocaine, from 8.6% to 11.7% for prilocaine, from 8.0% to 10.0% for 2,6-xylidine and from 8.0% to 9.0% for o-toluidine. The matrix effect values were in the range of -2.3% to -8.6% for lidocaine, -2.7% to -10.2% for prilocaine, 4.8% to 5.2% for 2, 6-xylidine and -8.2% t
