title: 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.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: David
authors.initials: D
authors.lastname: Sartori
authors.affiliation: Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Tamorah
authors.initials: T
authors.lastname: Lewis
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Autumn
authors.initials: A
authors.lastname: Breaud
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: wclarke@jhmi.edu.
authors.forename: William
authors.initials: W
authors.lastname: Clarke
mesh_terms: D000701:Analgesics, Opioid / Q000097:blood*; D002851:Chromatography, High Pressure Liquid / Q000379:methods* / Q000592:standards; D017408:Guidelines as Topic; D006801:Humans; D004091:Hydromorphone / Q000097:blood; D016014:Linear Models; D009020:Morphine / Q000097:blood; D009022:Morphine Derivatives / Q000097:blood; D015588:Observer Variation; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods* / Q000592:standards
chemical_list: D000701:Analgesics, Opioid; D009022:Morphine Derivatives; C035349:morphine-6-glucuronide; D009020:Morphine; C018108:morphine-3-glucuronide; D004091:Hydromorphone; C010616:normorphine
keywords: Glucuronide; HPLC–MS/MS; Method validation; Morphine; Neonatal abstinence syndrome