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

Abstract

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.

Figure 1

HPLC chromatogram of murine plasma spiked with nutlin-3a (50 ng/mL) and ketoconazole (800 ng/mL) after protein precipitation.

Figure 2

Positive-ion full-scan MS1 spectra for A) nutlin-3a, and C) ketoconazole, and fragment ion MS2 spectra of B) the precursor [M+H]⁺ ion of nutlin-3a, and D) the precursor [M]⁺ ion of ketoconazole.

Figure 3

Ion suppression of nutlin-3a (A, C) and ketoconazole (B, D). A neat mixture of nutlin-3a (2000 ng/mL for PP and 20 ng/mL for LLE) and ketoconazole (800 ng/mL for PP and 8 ng/mL for LLE) was infused, post-column at a rate of 5.0 µL/min into the LC effluent while mobile phase, commercially-available pooled murine plasma, and murine plasma from 4 different mice were injected into the column after LLE (A,B) or PP (C, D). The top line in each panel is from the injection of mobile phase.

Figure 4

Nutlin-3a plasma concentration-time profile of mice administered oral nutlin-3a (100 mg/kg). Individual plasma nutlin-3a concentrations from 4 mice per time-point are plotted along with the solid line representing the best-fit curve from the pharmacokinetic analysis.