On-line clean-up and determination of tramadol in human plasma and urine samples using molecularly imprinted monolithic column coupling with HPLC

► We prepared molecularly imprinted monolithic column for the assay of tramadol. ► The column showed highly specific recognition for the template. ► The work was applied for automated analysis of tramadol in urine and plasma. The applicability of an on-line solid phase extraction method using molecularly imprinted monolithic column was developed for the assay of tramadol (TRD) in urine and plasma samples. The monolithic column was prepared by using TRD as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker and chloroform as the porogen with in situ molecular imprinting polymerization technique. Various parameters affecting the extraction efficiency of the monolithic column were evaluated. Chromatographic analysis of TRD after on-line clean-up of samples was performed by reversed-phase HPLC on an ACE column with ultraviolet detection at 218nm. The present work was successfully applied for automated simple analysis of TRD in urine and plasma samples with high recoveries between 90.5–93.1% and 93.3–96.0%, respectively. The results revealed that in concentration up to 500ng/mL of dextromethorphan (DEX), timolol (TMO) and O-desmethyltramadol (M1), the recoveries were not reduced more than 4.3% and 4.0% for plasma and urine samples, respectively. The limit of detection (S/N=3) and limit of quantification (S/N=10) for TRD in urine samples were 0.03ng/mL and 0.10ng/mL, and in plasma samples were 0.3 and 1.0ng/mL, respectively. Inter-column precision of the assays (n=3) for urine and plasma samples at the 100ng/mL TRD level were 4.0% and 4.2%, respectively.