Tropical Journal of Pharmaceutical Research

Original Research Article | OPEN ACCESS

Sensitive and Selective Reversed-Phase High Performance Liquid Chromatographic-UV Spectrophotometric Determination of Dextromethorphan and its CYP2D6 Mediated Metabolite, Dextrorphan in Human Urine

Benjamin U Ebeshi^1,2 , Obiageri O Obodozie³, Oluseye O Bolaji²

¹Department of Pharmaceutical & Medicinal Chemistry, Faculty of Pharmacy, Niger Delta University, Wilberforce Island; ²Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Obafemi Awolowo University, Ile-Ife; ³Department of Medicinal Chemistry & Quality Control, National Institute for Pharmaceutical Research & Development, Abuja, Nigeria.

For correspondence:- Benjamin Ebeshi Email: ben.beshi@gmail.com Tel:+2348059817538

Received: 4 December 2012 Accepted: 24 December 2013 Published: 20 February 2014

Citation: Ebeshi BU, Obodozie OO, Bolaji OO. Sensitive and Selective Reversed-Phase High Performance Liquid Chromatographic-UV Spectrophotometric Determination of Dextromethorphan and its CYP2D6 Mediated Metabolite, Dextrorphan in Human Urine. Trop J Pharm Res 2014; 13(2):281-286 doi: 10.4314/tjpr.v13i2.18

© 2014 The authors.
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Abstract

Purpose: To develop a simple, sensitive and selective method for the determination of dextromethorphan and its metabolite, dextrophan in human urine using reversed-phase high performance liquid chromatography with UV-spectrophotometric detection (RP-HPLC-UV).

Methods: Pre-column sample clean-up was carried out by liquid-liquid extraction of the analytes with chloroform: isopropanol (70:30) solution after alkalization of 1000 µL sample and spiking of internal standard, morphine. The samples were chromatographed in a reversed-phase (C-18) ultra sphere silica (5µm particle size and 250 x 4.6 mm I.D). The mobile phase consisted of methanol: acetonitrile: 0.5% w/v ammonium acetate (10:10:80) adjusted to pH 2.8 with orthophosphoric acid and pumped through the column at 1ml/min flow rate. The analytical method was validated for accuracy and precision as well as the recovery of the analytes, dextromethorphan and its metabolite, dextrophan over the concentration range of 0.20 to 5.0µg/ml.

Results: The standard curves were linear over the concentration range of 0.2 to 5.0µg/ml for dextromethorphan and dextrorphan. The regression coefficients (R²) of the analytes were >0.99. The method was reproducible with coefficient of variation for the analytes being < 10 %. Dextromethorphan was well resolved from its metabolite, dextrorphan and the internal standard, morphine. The limits of detection of dextromethorphan and dextrorphan were 50ng/ml and the recoveries and accuracies were greater than 85 and 90 %, respectively.

Conclusion: The analytical assay method exhibits good precision and selectivity and it was applied to the analysis of dextromethorphan and dextrorphan in urine for the assessment of CYP2D6 activity.

Keywords: Dextromethorphan, Dextrophan, Reversed-phase high performance liquid chromatography, CYP2D6 activity, Human urine