Automated Determination of Dextromethorphan and Its Main Metabolites in Human Plasma by High-Performance Liquid Chromatography and Column Switching

Abstract

An automated column-switching technique coupled to isocratic high-performance liquid chromatography (HPLC) with fluorescence detection was developed for simultaneous determination of dextromethorphan and its three major metabolites, dextrorphan, hydroxymorphinan, and methoxymorphinan. After cleavage of conjugates by incubation with glucuronidasearylsulfatase at 37 degrees C for 15 h, plasma samples were injected directly into the HPLC system. Dextromethorphan and metabolites were retained on a cleanup column (10 x 4.6 mm internal diameter [ID]) filled with cyanopropyl (CN) material (Hypersil CPS, 10-microns article size) while interfering proteins and lipids were washed to waste. After column switching, the drugs were eluted from the cleanup column and separated on Spherisorb CN material (5-microns particle size, column size 250 x 4.6 mm ID). Fluorescence detection was carried out with an excitation wavelength of 220 nm and an emission wavelength of 305 nm. Sample cleanup and HPLC separation were completed within 20 min. Regression analyses found linearity (r > 0.99) between drug concentration and detector response over a wide range-5-220 ng/ml for dextromethorphan, 5-550 ng/ml for dextrorphan, 5-500 ng/ml for hydroxymorphinan, and 5-200 ng/ml for methoxymorphinan. The limit of quantification was approximately 5 ng/ml, and the recovery was > 90% for all compounds. At concentrations of 20-500 ng/ml, the intra- and interassay coefficients of variation ranged from 3.5 to 14.6% and from 7.0 to 14.0%, respectively. The method is suitable for in vivo phenotyping of CYP2D6 activity, which catalyzes the O-demethylation of dextromethorphan to dextrorphan, and is also applicable to pharmacokinetic studies in man.

Inhibition of dextromethorphan metabolism by moclobemide

“…Determination of dextromethorphan and its metabolites dextrorphan, hydroxymorphinan, and methoxymorphinan, was performed as reported previously (Härtter et al 1996). Brießy, plasma samples were mixed with the b-glucuronidase / arylsulphatase solution (1 : 1; v : v) and incubated for 18 h at 37°C to hydrolyze the glucuronide conjugates.…”

A validated SIM GC/MS method for the simultaneous determination of dextromethorphan and its metabolites dextrorphan, 3‐methoxymorphinan and 3‐hydroxymorphinan in biological matrices and its application to in vitro CYP2D6 and CYP3A4 inhibition study

“…Various analytical methods for the determination of dextromethorphan and/or its metabolites in biological matrices have been reported, such as high-performance liquid chromatography combined with fluorescence detection (East and Dye, 1985;Johansson and Svensson, 1988;Chen et al, 1990;Lam and Rodriquez, 1993;Hartter et al, 1996;Bartoletti et al, 1996;Kimiskidis et al, 1996;Bendriss et al, 2001;Hendrickson et al, 2003;Afhsar et al, 2004;Lin et al, 2007) or ultraviolet detection (Park et al, 1984;Vielnascher et al, 1996), capillary electrophoresis (Kristensen, 1998), gas chromatography coupled with different detectors (Pfaff et al, 1983;Bauman and Jonzier-Perey 1988;Wu et al, 2003;Bagheri et al, 2005;Rodriques et al, 2008) and liquid chromatography-mass spectrometry (Vengurlekar et al, 2002;Arellano et al, 2005;Constanzer et al, 2005). GC/MS in the SIM mode and LC-MS/MS are the preferred methods in terms of sensitivity and selectivity.…”

Polymeric Membrane Sensors for the Selective Determination of Dextromethorphan in Pharmaceutical Preparations

“…Most of those analytical methods imply chromatographic techniques, such as high-performance liquid chromatography, [5][6][7][8] high-performance liquid chromatography-mass spectrometrymass spectrometry, 9,10 gas chromatography, 11,12 gas chromatography-mass spectrometry 13,14 and high-performance thin-layer chromatography. 15 Capillary electrophoresis 16,17 and multiwavelength and derivative spectrophotometry 18,19 have also been reported for the determinations of DXM.…”