Development, validation and clinical application of a HPLC-FL method for CYP2D6 phenotyping in South Brazilian breast cancer patients

Abstract

ObjectiveTo develop and validate a method for determination of dextromethorphan (DMT) and dextrorphan (DTP) in plasma samples using HPLC-FL and to apply it to CYP2D6 phenotyping of a population from the South of Brazil. MethodsSamples were prepared by hydrolysis and liquid–liquid extraction. Analysis was conducted in a reversed phase column, with isocratic elution and fluorescence detection. One hundred and forty patients being treated with tamoxifen were given 30mg of dextromethorphan and their CYP2D6 phenotypes were determined on the basis of [DMT]/[DTP] metabolic ratios in plasma samples collected after 3h. ResultsTotal chromatography running time was 12min. Precision (CV%) was below 9.7% and accuracy was between 92.1 and 106.9%. The lower limits of quantification were 1ngmL−1 for DMT and 10ngmL−1 for DTP. Mean extraction yield of analytes was 86.6%. Mean age of patients was 55.7years. Phenotype frequencies were as follows: 7.1% poor metabolizers, 13.6% intermediate metabolizers, 77.1% extensive metabolizers and 2.1 ultra-rapid metabolizers. Metabolic ratios for patients on strong (n=11) and weak (n=16) CYP2D6 activity inhibitors were different from each other and also different from ratios for patients not taking enzyme inhibitors (n=113). ConclusionsA sensitive method for determination of dextromethorphan and its metabolite in plasma samples was developed and successfully applied, providing evidence of the impact that CYP2D6 inhibitors have on the enzyme's metabolic capacity.