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. 2009 Jul 1;877(20-21):1894-900.
doi: 10.1016/j.jchromb.2009.05.034. Epub 2009 May 21.

A high-performance liquid chromatography-mass spectrometry assay for quantitation of the tyrosine kinase inhibitor nilotinib in human plasma and serum

Robert A Parise  1 , Merrill J Egorin, Susan M Christner, Dhvani D Shah, Wei Zhou, Jan H Beumer
Affiliations

A high-performance liquid chromatography-mass spectrometry assay for quantitation of the tyrosine kinase inhibitor nilotinib in human plasma and serum

Robert A Parise et al. J Chromatogr B Analyt Technol Biomed Life Sci. .

Abstract

Nilotinib (AMN-107, Tasigna) is a small-molecule inhibitor of BCR/ABL, approved for chronic myelogenous leukemia. We developed and validated, according to FDA-guidelines, an LC-MS assay for sensitive, accurate and precise quantitation of nilotinib in 0.2 mL human plasma or serum. After acetonitrile protein precipitation, separation is achieved with a hydro-Synergi column and a 0.1% formic acid in methanol/water-gradient. Detection uses electrospray, positive-mode ionization mass spectrometry. Between 5 (LLOQ) and 5000 ng/mL, accuracy (92.1-109.5%), intra-assay precision (2.5-7.8%), and inter-assay precision (0-5.6%)) were within FDA limits. We demonstrated the suitability of this assay by quantitating plasma concentrations of nilotinib in a healthy volunteer after oral administration of 400 mg nilotinib.

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Figures

Fig. 1
Fig. 1
Chemical structures of imatinib, nilotinib and stable-labeled [C132,N152]-nilotinib internal standard in their base form.
Fig. 2
Fig. 2
Representative chromatograms of (A) nilotinib (m/z 530.4; 6.5 min) added to control plasma at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace); (B) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control plasma at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace). (C) nilotinib (m/z 530.4; 6.5 min) added to control serum at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); (D) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control serum at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); and (E) nilotinib (top trace with offset of 2000 counts) and internal standard (bottom trace) in the Cmax sample of a patient 4 h after having been orally administered 400 mg nilotinib.
Fig. 2
Fig. 2
Representative chromatograms of (A) nilotinib (m/z 530.4; 6.5 min) added to control plasma at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace); (B) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control plasma at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace). (C) nilotinib (m/z 530.4; 6.5 min) added to control serum at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); (D) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control serum at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); and (E) nilotinib (top trace with offset of 2000 counts) and internal standard (bottom trace) in the Cmax sample of a patient 4 h after having been orally administered 400 mg nilotinib.
Fig. 2
Fig. 2
Representative chromatograms of (A) nilotinib (m/z 530.4; 6.5 min) added to control plasma at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace); (B) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control plasma at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human plasma (bottom trace). (C) nilotinib (m/z 530.4; 6.5 min) added to control serum at the LLOQ concentration of 5 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); (D) [C132,N152]-nilotinib internal standard (m/z 534.4; 6.5 min) added to control serum at a concentration of 25 ng/mL (top trace with offset of 500 counts) and control human serum (bottom trace); and (E) nilotinib (top trace with offset of 2000 counts) and internal standard (bottom trace) in the Cmax sample of a patient 4 h after having been orally administered 400 mg nilotinib.
Fig. 3
Fig. 3
Time course of nilotinib in plasma of a study subject given 400 mg of nilotinib orally.
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