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Simultaneous quantification of erlotinib, gefitinib and imatinib in human
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plasma by liquid chromatography tandem mass spectrometry
A. Chahbouni, A.J. Wilhelm , J.C.G. den Burger, R.M. Vos, A. Sinjewel.
Free University medical center, Department of Clinical Pharmacology and Pharmacy, De Boelelaan 1117, 1081 HV,
Amsterdam, The Netherlands
Introduction
Results
Imatinib (Gleevec®), erlotinib (Terceva®) and gefitinib (Iressa®) are drugs
which compete with ATP for binding at the tyrosine kinase receptor. Erlotinib
and gefitinib are selective inhibitors of the epidermal growth factor receptor
(EGFR), which is often overactive in tumors cells. Erlotinib and gefitinib are
common used for treatment of non-small cell lung cancer. Imatinib is an
inhibitor of the platet-derived growth factor (PDGF)-receptor and is mainly
used by chronic myelogenous leukemia and gastrointestinal stromal tumors.
The PDGF (Fig. 1) is one of the growth factors that regulate cell growth and
division. Interindividual differences in absorption and metabolism exist and
pharmacokinetic interaction with other drugs are described. In case of
suspicion of nonadherence, drug–drug interaction, excessive side effects and
poor effectiveness, therapeutic drug monitoring (TDM) is useful.
The calibration curves in plasma were linear for erlotinib, gefitinib and
imatinib over the concentration range of 5-3000; 5-3000 and 5-5000 ng/ml,
respectively. The intra- and interday accuracy ranged from 90%-110% and the
intra- and interday precision of the method was within 5%. The reported
method provided the necessary linearity, precision and accuracy to
determine three tyrosine kinase inhibitors in clinical research and for
therapeutic drug monitoring.
Table 1. Accuracy and precision of the method.
Imatinib
Erlotinib
Gefitinib
Intraday
Conc.
(ng/ml)
Av.
CV (%)
Acc. (%)
Conc.
Av.
(ng/ml)
CV (%)
Acc. (%)
Conc.
Av.
(ng/ml)
CV (%)
Acc. (%)
77.4
78.3
1.6
101.2
79.0
80.3
2.7
101.7
84.3
90.2
2.7
107.0
348
347
1.8
99.6
356
364
2.2
102.2
379
417
2.3
110.0
3481
3347
1.8
96.2
2608
2690
1.2
103.1
2782
2994
1.3
107.6
77.4
75.5
1.6
97.6
79.0
77.8
2.2
98.5
84.3
87.1
2.0
103.3
348
338
0.9
97.0
356
361
2.3
101.4
379
402
1.7
106.0
3481
3361
0.6
96.5
2608
2719
3.8
104.2
2782
2894
3.2
104.0
Interday
Fig 1. Growth factors, such as PDGF, EGF and FGF.
Aim
Implementing and developing a method for the simultaneous quantification
of imatinib, erlotinib and gefitinib by LC-MS/MS. A simultaneous method for
the analysis of these tyrosine kinase inhibitors is more efficient, improves
clinical usefulness and makes frequent assaying possible.
Method
Pre-treatment of the samples was achieved by using liquid-liquid extraction
using D-8 imatinib as internal standard. Separation was performed on a
Waters® Alliance 2795 LC-system using an XBridge® RP18 column. The mass
spectrometer Micromass® was operating in the positive electro spray
ionization mode. The analytes were detected by multiple reaction monitoring
(MRM), monitoring the transition of the m/z precursor to the m/z of the
product ion. Figure 2. shows a representative chromatogram of the tyrosine
kinase inhibitors after pre-treatment.
Fig 2. Representative chromatogram of internal standard D8-imatinib
(A), imatinib (B), gefitinib (C) and erlotinib (D).
Av.= average, Acc. = accuracy
Applicability of the assay in clinical practise
The laboratory of clinical pharmacology and pharmacy participates in the
European treatment outcome study (EUTOS) of chronic myelogenous
leukaemia initiated by Novartis. The aim of this study is providing the
availability of imatinib monitoring on an European level. For this study 15
plasma samples were analyzed, the results of the laboratory of clinical
pharmacology and pharmacy (NL01) are described in figure 3. In a study by
Picard et al is mentioned that the effectively doses for imatinib a plasma
threshold of 1000 ng/ml is considered.
Routinely the method is mainly used for TDM of patients which treat with
erlotinib. Based on the study of Hidalgo a plasma concentration of 500 ug/L
have been estimated to provide a level of EGFR inhibition associated with a
relevant degree of antiproliferative activity.
Fig. 3. The results of the EUTOS round of standardization.
Conclusion: The developed method is sensitive and specific for the simultaneous quantification of erlotinib, gefitinib and imatinib in
human plasma by LC-MS/MS. The data confirms that the developed method is robust and has a satisfying accuracy and precision for the
quantification of imatinib, erlotinib and gefitinib. This method is suitable for therapeutic drug monitoring and for clinical research.
E-mail: [email protected]