Simultaneous measurement of heroin and its metabolites in brain extracellular fluid by microdialysis and ultra performance liquid chromatography tandem mass spectrometry

J Pharmacol Toxicol Methods. 2012 Jul;66(1):14-21. doi: 10.1016/j.vascn.2012.04.009. Epub 2012 Apr 26.


Introduction: The pharmacokinetic profile and systemic bioavailability of a substance is often described by blood or total tissue concentrations. For centrally acting drugs, like opioids, the free fraction of active compound in brain extracellular fluid (ECF) is more likely to be correlated to the pharmacodynamic effects than the blood concentrations. Drugs of abuse, like heroin, are often administered intravenously as bolus injections, and the blood concentrations might change rapidly due to metabolism and distribution. The aim of our study was to establish a method to measure the free fraction of heroin and its metabolites in brain ECF, and follow their fast changes in concentration.

Methods: Sprague-Dawley rats were injected intravenously with a bolus of heroin. Heroin and its main metabolites 6-monoacetylmorphine, morphine and morphine-3-glucuronide were measured simultaneously. Brain microdialysis was used for sampling and a method for quantification using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed. Deuterated analogues for each analyte were included in the microdialysis perfusion solution as calibrators for recovery estimation.

Results: A highly sensitive UPLC-MS/MS method allowed short sampling intervals, down to one minute, and the simultaneous detection of each analyte and its specific deuterated analogues, making possible the individual recovery calculation for each compound of interest. This method allowed us to determine the pharmacokinetic profiles of heroin and its metabolites in brain-ECF in the same animal after an intravenous injection of heroin.

Discussion: Our method makes detecting concurrently the rapid changes in concentrations of heroin and its metabolites in brain ECF possible, despite the rapid metabolism of heroin. Recovery was measured specifically for each analyte in the same sample by carefully combining different deuterated analogues. This technique can be applied to pharmacokinetic studies where more than one compound of interest has to be monitored, and to study distribution of prodrugs or drugs with active metabolites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain / metabolism*
  • Brain Chemistry
  • Chromatography, High Pressure Liquid
  • Extracellular Fluid / chemistry
  • Extracellular Fluid / metabolism*
  • Heroin / analysis
  • Heroin / pharmacokinetics*
  • Injections, Intravenous
  • Male
  • Microdialysis
  • Narcotics / analysis
  • Narcotics / pharmacokinetics*
  • Rats
  • Rats, Sprague-Dawley
  • Spectrometry, Mass, Electrospray Ionization
  • Tandem Mass Spectrometry / methods*


  • Narcotics
  • Heroin