Simultaneous determination of tramadol and O-desmethyltramadol in human plasma using HPLC-DAD.

A sensitive and accurate method for the extraction and quantification of tramadol (T) and its active metabolite, O-desmethyltramadol (ODT) in human plasma with high-performance liquid chromatography-diode array detection was developed and validated. The analytes were extracted from plasma samples by tert-butylmethyl ether in the presence of ammonium hydroxide as alkaline medium and back extraction with 1.0 M hydrochloric acid. Propranolol was used as internal standard. The extraction efficiencies of T and ODT were 83.51 and 78.72%, respectively. The calibration curves were linear (r(2) > 0.99) in the concentration range of 250-2000 ng/mL for T and ODT. Limits of detection and quantification were 125 and 250 ng/mL for both analytes. Intra- and interassay precision for T and ODT were ranged from 1.89 to 10.91% and 2.16 to 5.85%, respectively. Intra- and interassay accuracy for T and ODT were ranged from -13.07 to 4.99% and -2.03 to -6.98%, respectively. The method was successfully applied to quantify T and ODT from authentic plasma samples received from Hospital Sohag University. The method was completely validated and can be of interest to clinical and forensic laboratories.