Rapid and simple method to determine morphine and its metabolites in rat plasma by liquid chromatography-mass spectrometry

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Apr 25;787(2):243-53. doi: 10.1016/s1570-0232(02)00726-2.


A rapid and simple method for the determination of morphine (M), normorphine (NM), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) in plasma by high-performance liquid chromatographic separation with mass spectrometric detection (HPLC-MS) has been developed. Samples (40 microl) were cleaned-up by protein precipitation with two volumes (80 microl) of acetonitrile and reconstituted in formic acid 0.1% in water. Naloxone was used as internal standard. Analytes were separated on a phenyl-hexyl column using a step-gradient (1 ml/min) of acetonitrile and formic acid in water. Acetonitrile was added post-column (0.3 ml/min). Quantification of morphine and its metabolites was achieved with an Agilent 1100 series HPLC-MS system equipped with electrospray interface set to selected ion-monitoring (SIM) mode. Calibration curves covered a wide range of concentrations (2.44-10,000 nM) and were best fitted with a weighed quadratic equation. The limits of quantification achieved with this method were 2.44 nM for M and 4.88 nM for NM, M3G and M6G. The method proved accurate (85-98%), precise (C.V.<10%) and was successfully applied to a wide range of in vitro and in vivo pharmacokinetic studies in rodents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Mass Spectrometry / methods*
  • Morphine / blood*
  • Rats
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Morphine