Simple and selective method for the determination of various tyrosine kinase inhibitors used in the clinical setting by liquid chromatography tandem mass spectrometry

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2010-05, Vol.878 (15), p.1059-1068
• Main Authors: Honeywell, R., Yarzadah, K., Giovannetti, E., Losekoot, N., Smit, E.F., Walraven, M., Lind, J.S.W., Tibaldi, C., Verheul, H.M., Peters, G.J.
• Format: Article
• Language: English
• Subjects: Acetonitriles - chemistry; Analysis; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biological materials; Cellular; Chromatography; Chromatography, Liquid - methods; Drug Stability; Erlotinib; Fundamental and applied biological sciences. Psychology; Gefitinib; General pharmacology; Humans; Inhibitors; Ionization; Kinases; LC–MS/MS; Linear Models; Liquid chromatography; Medical sciences; Neoplasms - blood; Neoplasms - drug therapy; Pharmacology. Drug treatments; Protein Kinase Inhibitors - analysis; Protein Kinase Inhibitors - blood; Protein Kinase Inhibitors - chemistry; Reproducibility of Results; Sensitivity and Specificity; Sorafenib; Sunitinib; Tandem Mass Spectrometry - methods; Tyrosine
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2010.03.010

A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC–MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 μl sample volume of biological matrixes can be extracted at 4 °C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC–MS/MS system without further clean-up and assayed across a linear range of 1–4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm × 100 mm, 3 μm) column and eluted at 200 μl/min with a tertiary mobile phase consisting of 20 mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 μl to 1 μl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 °C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1 m/ z; 127.9 m/ z), Erlotinib (393.9 m/ z; 278.2 m/ z), Sunitinib (399.1 m/ z; 283.1 m/ z) and Sorafenib (465.0 m/ z; 251.9 m/ z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2 ± 3.8%, 11.2 nM; Erlotinib: 101.6 ± 3.7%, 12.7 nM; Sunitinib: 100.8 ± 4.3%, 12.6 nM; Sorafenib: 93.9 ± 3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.