Development of a high-performance liquid chromatographic-mass spectrometric method for the determination of cellular levels of the tyrosine kinase inhibitors lapatinib and dasatinib

A highly sensitive and selective liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed to quantify cellular levels of the tyrosine kinase inhibitors (TKIs) dasatinib (Sprycel (TM)) and lapatinib (Tykerb (TM), Tyverb (TM)). Cellular samples were extracted with a tert-butyl methyl ether: acetonitrile (3: 1, v/v): 1 M ammonium formate pH 3.5 (8: 1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm x 2.0 mm 3 mu m) column with isocratic elution using a mobile phase of acetonitirile-10 mM ammonium formate. pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The TKIs were quantified using a triple quadrupole mass spectrometer which was operated in multi-reaction-monitoring mode employing positive electrospray ionisation. The limit of detection and limit of quantification for lapatinib was determined to be 15 and 31 pg on column, respectively. The limit of detection and quantification for dasatinib was 3 and 15 pg on column, respectively. The method allowed for sensitive and accurate determination of cellular levels of dasatinib and lapatinib. in addition, we examined the potential for this method to be utilised to quantitate other TKIs, using gefitinib, erlotinib, imatinib and sorafenib as examples. In principle, these agents were also quantifiable by this method, however, no drug specific validation studies were undertaken with these TKIs. The data indicates that in the cancer cell-line model, DLKP, significantly more lapatinib accumulates in cells in comparison to dasatinib. Additionally, over-expression of the membrane protein drug transporter, P-glycoprotein (P-gp) a common cancer drug resistance mechanism, greatly reduces the cellular accumulation of dasatinib but not of lapatinib. (C) 2009 Elsevier B.V. All rights reserved.