The development of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of morphine, morphine-3-β-glucuronide, morphine-6-β-glucuronide, hydromorphone, and normorphine in serum.

OBJECTIVES: Development and validation of a selective, robust high-performance liquid chromatography-tandem mass spectrometric (HPLC/MS-MS) method for the quantification of morphine, morphine-3-β-glucuronide, morphine-6-β-glucuronide, hydromorphone, and normorphine in human serum. DESIGN AND METHODS: Drug-free human serum samples spiked with morphine, morphine-3-β-glucuronide, morphine-6-β-glucuronide, hydromorphone, and normorphine were prepared by protein precipitation using methanol containing the internal standards. Samples were injected onto a Thermo Scientific AccuCore PFP column for chromatographic separation. Detection was achieved using a Thermo Scientific TSQ Vantage mass spectrometer. Assay validation followed the new Clinical and Laboratory Standards Institute (CLSI) C62-A guidelines. RESULTS: The analytical measuring range for all analytes was determined to be 5 to 1000 ng/mL. Intra- and inter-assay precision for three quality control levels were ≤ 7.0% and ≤ 13.5%, respectively. Carryover, stability, linearity, matrix effects, extraction and processing efficiency and method comparison characteristics were acceptable relative to the CLSI C62 guidelines. CONCLUSION: The validation of this HPLC-MS/MS method demonstrated a robust and rapid assay for the quantification of morphine, morphine-3-β-glucuronide, morphine-6-β-glucuronide, hydromorphone, and normorphine.