Bioanalytical methods for the quantification of hydromorphone, fentanyl, norfentanyl, morphine, morphine-3ß-glucuronide and morphine-6ß-glucuronide in human plasma

•Sensitive methods for the quantitation of hydromorphone, morphine, fentanyl and the metabolites norfentanyl, morphine-3ß-glucuronide and morphine-6ß-glucuronide in human potassium EDTA plasma by UPLC–MS/MS.•The methods are currently successfully applied for the bioanalysis of opioid concentrations in plasma of cancer patients in which opioids are administered for the treatment of moderate to severe cancer-related pain.•Method is sensitive enough when fentanyl is administered in cancer patients sublingually or change in opioid regimen with subcutaneous and transdermal fentanyl.•Less laborious method for the determination of hydromorphone, morphine, fentanyl and the metabolite norfentanyl in human potassium EDTA plasma. The aim of this study was to develop an assay for the quantification of hydromorphone, morphine, fentanyl and the metabolites norfentanyl, morphine-3ß-glucuronide and morphine-6ß-glucuronide in human plasma to support pharmacokinetic studies investigating the large interpatient variability in response to opioid treatment. For the quantitation of hydromorphone, morphine, fentanyl and its metabolite norfentanyl aliquots of 200μL human potassium EDTA plasma were deproteinized with deuterated internal standards in a mixture of acetonitrile and acetone, followed by a liquid-liquid extraction with 4% ammonium hydroxide and ethyl acetate. Morphine-3ß-glucuronide and morphine-6ß-glucuronide were extracted by a solid phase extraction using 10mM ammonium carbonate pH 8.8 and a deuterated internal standards solution. Morphine, hydromorphone, fentanyl and norfentanyl were separated on an Aquity UPLC® BEH C18 column 1.7μm, 100mm×2.1mm at 50°C. Separation, was achieved on a gradient of methanol with an overall run time of 6min. The compounds were quantified by triple-quadrupole mass spectrometry in the positive ion electrospray ionization mode. Morphine-3ß-glucuronide and morphine-6ß-glucuronide were separated on a VisionHT C18-P; 3μm 2.1×50mm, column at 40°C on a gradient of acetonitrile, with an overall run time of 10min. Both methods were precise and accurate, with within-run and between-run precisions within acceptable limits and accuracy ranging from 84.0 to 105.5%. The methods were successfully applied to support clinical pharmacological studies in patients treated with opioids for the treatment of moderate to severe cancer-related pain.