A sensitive assay for the quantification of morphine and its active metabolites in human plasma and dried blood spots using high-performance liquid chromatography-tandem mass spectrometry

Opioids such as morphine are the cornerstone of pain treatment. The challenge of measuring the concentrations of morphine and its active metabolites in order to assess human pharmacokinetics and monitor therapeutic drugs in children requires assays with high sensitivity in small blood volumes. We developed and validated a semi-automated LC-MS/MS assay for the simultaneous quantification of morphine and its active metabolites morphine 3 beta-glucuronide (M3G) and morphine 6 beta-glucuronide (M6G) in human plasma and in dried blood spots (DBS). Reconstitution in water (DBS only) and addition of a protein precipitation solution containing the internal standards were the only manual steps. Morphine and its metabolites were separated on a Kinetex 2.6-mu m PFP analytical column using an acetonitrile/0.1% formic acid gradient. The analytes were detected in the positive multiple reaction mode. In plasma, the assay had the following performance characteristics: range of reliable response of 0.25-1000 ng/mL (r (2) > 0.99) for morphine, 1-1,000 ng/mL (r (2) > 0.99) for M3G, and 2.5-1,000 ng/mL for M6G. In DBS, the assay had a range of reliable response of 1-1,000 ng/mL (r (2) > 0.99) for morphine and M3G, and of 2.5-1,000 ng/mL for M6G. For inter-day accuracy and precision for morphine, M3G and M6G were within 15% of the nominal values in both plasma and DBS. There was no carryover, ion suppression, or matrix interferences. The assay fulfilled all predefined acceptance criteria, and its sensitivity using DBS samples was adequate for the measurement of pediatric pharmacokinetic samples using a small blood of only 20-50 mu L.