Determination of nutlin-3a in murine plasma by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)

J Pharm Biomed Anal. 2010 Mar 11;51(4):915-20. doi: 10.1016/j.jpba.2009.10.016. Epub 2009 Oct 30.


A sensitive and precise LC-ESI-MS/MS method for determination of nutlin-3a in murine plasma using ketoconazole as an internal standard was developed and validated. Plasma nutlin-3a samples were prepared by either a simple protein precipitation (PP) for the high concentration range (10-20,000ng/mL) or by liquid-liquid extraction (LLE) for the low concentration range (0.25-300ng/mL). Nutlin-3a and ketoconazole were separated on a modified C18 analytical column (4microm, 75mmx2mm) with an isocratic mobile phase (acetonitrile/5mM HCOONH(4)=70/30, v/v). The retention times of nutlin-3a and ketoconazole were 1.14 and 1.45min. Detection was achieved by a tandem MS system, monitoring m/z 582/99 and m/z 532/82 for nutlin-3a and ketoconazole, respectively. The PP method was linear in a range of 10-20,000ng/mL (R(2)>or=0.993) and the LLE method was linear in a range of 0.25-300ng/mL (R(2)>or=0.992). The mean recoveries for PP and LLE were 24% and 78%, respectively. Within-day and between-day precisions were <or=4.5% for PP and were <or=4.9% for LLE. Within-day and between-day accuracies (% error) ranged from 4.8 to -7.9 for PP, and from -0.2 to -8.4 for LLE. The two extraction methods produced equivalent results, allowing use of both within the same study. This method has been applied to the measurement of nutlin-3a concentrations in murine plasma samples obtained from a preclinical pharmacokinetic study.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Administration, Oral
  • Animals
  • Antineoplastic Agents / administration & dosage
  • Antineoplastic Agents / blood*
  • Antineoplastic Agents / pharmacokinetics
  • Buffers
  • Chemical Precipitation
  • Chromatography, Liquid* / standards
  • Hydrogen-Ion Concentration
  • Imidazoles / administration & dosage
  • Imidazoles / blood*
  • Imidazoles / pharmacokinetics
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Piperazines / administration & dosage
  • Piperazines / blood*
  • Piperazines / pharmacokinetics
  • Reference Standards
  • Reproducibility of Results
  • Spectrometry, Mass, Electrospray Ionization* / standards
  • Tandem Mass Spectrometry* / standards


  • Antineoplastic Agents
  • Buffers
  • Imidazoles
  • Piperazines
  • nutlin 3