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.