Erlotinib

Cetuximab (Erbitux), solution 5 mg/ml, was purchased from Merck, stored at 4 °C; erlotinib, dry powder, was purchased from Sigma-Aldrich, stored at -20 °C as 10 mM solution in DMSO. EGF, dry powder, was purchased from PanEco, stored at -20 °C.
Peripheral blood samples from seven unrelated healthy 23–64 years old female donors were collected in two 8-ml vacuette tubes containing pro-coagulant and gel (Greiner), and serum was prepared within 3–4 h upon blood collection: tubes were centrifuged at 2500 rpm for 15 min, sera were aliquoted and stored at -75 °C. For all the human biomaterials investigated, informed written consents to participate in this study and to communicate the results in the form of a scientific report were collected from the corresponding donors. The procedure of taking human materials, the consent procedure, and the design of this study were approved by the ethical committee of the Vitamed Clinical Center, Moscow.

Cells were seeded in 96 well culture plates at a density of 3 to 9 × 10³ cells/well depending on cell line. After overnight incubation the MCF7, BT474, MDA-MB-361, ZR-75-30 and SKBR3 cells were treated with either mock treatment, trastuzumab (Roche) as previously described^{46 (link),47 (link)} or GSK2830371 (Sigma-Aldrich) alone or combined. The HCC1954 cells were treated with either mock treatment, erlotinib (Sigma-Aldrich) as previously described^{48 (link)} or GSK2830371 alone or combined. The drugs were added at a final concentration of 10 μgr/ml, 50 μgr/ml and 100 μgr/ml for trastuzumab; 0.1 μM, 0.5 μM and 1 μM for erlotinib; 1 μM and 2.5 μM for GSK2830371. Cell viability was measured on day 7 using the MTT proliferation assay kit (Promega, Madison, WI, USA) according to manufacturer instructions. The absorbance was measured at 595 nm on the Tecan sunrise microplate reader (Tecan Group Ltd, Mannedorf, Switzerland). The data shown are representative of at least three independent experiments and each treatment was performed in pentaplicate.

Chemicals (mainly acetic acid, formic acid, trichloroacetic acid, sodium hydroxide solution (0.1 mol·L⁻¹), methanol, isopropanol, water) and standards (dasatinib, erlotinib, canertinib, and bosutinib), all of analytical grade or higher (solvents of HPLC-grade) purity, were bought from Sigma-Aldrich (St. Louis, MO, USA). Deionized water with resistivity of 18.2 MΩ.cm was prepared by the MilliQ system from Millipore (Molsheim, France).
Background electrolytes (BGEs were prepared by dissolving corresponding volumes of acids in HPLC-grade water. The pH was measured using an inoLab (WTW, Weilheim, Germany) pH meter. The ionic strength was calculated using Peakmaster software [49 ]. Finally, all the BGEs were filtered using 0.45 µm PTFE syringe filters (Labicom, Czech Republic).
The blood plasma sample was obtained from Sigma-Aldrich (St. Louis, MO, USA). The sample was spiked with the model TKIs and deproteinated as follows: 100 µL of the sample was mixed with 15 µL of cold trichloroacetic acid and shaken for 15 min. Then, it was centrifuged at 12,000× g for 5 min. Finally, the supernatant (50 µL) was carefully transferred to the sample vial for CE analysis and diluted with BGE–methanol mixture (80:20, v/v, 50 µL).