ABSTRACT
To establish the usefulness of fluorescence detection to quantify urinary concentrations of dextromethorphan and dextrorphan for oxidation phenotyping, we determined the molar concentration ratio of dextromethorphan to dextrorphan in 38 subjects by UV and fluorescence detection. Dextromethorphan and dextrorphan concentrations were quantified after overnight hydrolysis of urine samples and organic solvent extraction with heptane and butanol. The compounds were separated by high-performance liquid chromatography using a phenyl column and a mobile phase consisting of acetonitrile and an aqueous mixture of 0.01 M heptane sulfonic acid and 0.01 M phosphate buffer. The eluents were detected in series by a UV detector (280 nm) and fluorescence detector (excitation 280 nm and emission 310 nm). The dextromethorphan to dextrorphan molar concentration ratio by UV and fluorescence detection was highly correlated (r = 0.997) and not statistically different (p = 0.1036). However, increased sensitivity with fluorescence detection enabled detection of lower dextromethorphan and dextrorphan concentrations when compared with UV detection. Fluorescence detection was able to detect dextromethorphan as low as 0.02 microgram/ml, which may be helpful in phenotyping individuals with extremely rapid metabolism of dextromethorphan. Fluorescence detection also produced chromatograms with significantly less interference and allows a more accurate quantitation of dextromethorphan and dextrorphan concentrations.
Subject(s)
Dextromethorphan/urine , Dextrorphan/urine , Chromatography, High Pressure Liquid , Cytochrome P-450 CYP2D6 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Dextromethorphan/metabolism , Dextrorphan/metabolism , Humans , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/metabolism , Oxidation-Reduction , Phenotype , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
A high-performance liquid chromatographic assay was developed to determine salivary chlorhexidine concentration. Saliva sample (200 microliters) was extracted into methylene chloride. Chromatographic separation was achieved with a C18 column using a mobile phase of acetonitrile-0.05 M sodium acetate and 0.005 M heptanesulfonic acid (40:60, v/v). The standard curve was linear from 0.051 to 20.48 micrograms/ml (r2 > or = 0.997). Intra-day and inter-day coefficients of variation were < 5.5 and < or = 9%, respectively. The assay is rapid, sensitive, simple, and successfully used for quantitating salivary chlorhexidine content released from a chlorhexidine-impregnated resin worn by patients.
