Simple and Selective Method for the Determination of Various Tyrosine Kinase Inhibitors Used in the Clinical Setting by Liquid Chromatography Tandem Mass Spectrometry

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 microl sample volume of biological matrixes can be extracted at 4 degrees 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 x 100 mm, 3 microm) column and eluted at 200 microl/min with a tertiary mobile phase consisting of 20mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 microl to 1 microl 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 degrees 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.