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 Ebeshi1,2*, Obiageri O Obodozie3
and 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:
Email:
ben.beshi@gmail.com; Tel:
+234-8059817538
Received: 4 December 2012
Revised accepted: 24
December 2013
Tropical Journal of
Pharmaceutical Research, January 2014;
13(2):
281-286
http://dx.doi.org/10.4314/tjpr.v13i2.18
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
