N source is coupled to an Agilent 1200 HPLC system. The ionization mode of ESI-mass spectrometer was positive. Nitrogen was used as drying gas, the destruction Via gas, sheath gas, and a collision gas. The capillary voltage was set at 3500 V, and the nozzle voltage was set at 1000 V. The Trocknungsgasstr Determination is 10 l / min at a temperature of gas 325th The destruction Via pressure was adjusted to 20 psi and the temperature of gas was 350 sheath with a sheath gas flow of 11 L / min. The mass spectrometer was operated in multiple reaction monitoring mode. Fragmentor voltage voltages and collision energies were optimized for each transition of a series of injections of pure flow standards. MRM Trnsfer length With the optimized conditions are in SI, S summarized Table 3. The chromatographic separation was carried out at 30 on a S Molecules AX Biobasic. The mobile phases A and B consisted of water: acetonitrile 7:3, 10 mM ammonium acetate at pH 6 for the mobile phase A with 1 mM ammonium acetate at pH 10.5 for mobile phase B. The gradient started at 0% B, was min at 35% B from 1 to 2.5, further to 65% B 5-7 min increased ht, then to 100% B 10 to 10.5 min increased ht ht erh what happened min to 15. The S was Column obsolete with the first mobile for 5 min. The flow rate was 0.15 ml / min for the first minute and 0.25 ml / min min to 15. Standardization and validation. The calibration curves were prepared by addition of increasing amounts of analyte to erythrocyte lysate and extraction of samples, as described above drug-free. Calibration curves based on the internal calibration standard were by weighted linear Bay 43-9006 Nexavar regression to the ratio Ratio of the Peakfl Surface of the analyte to internal standard versus the amount of the analyte obtained. The concentration of the analyte in the unknown sample was obtained from the regression line. Normalization was performed with the software MassHunter. The reproducibility and precision Pr Of the method was determined by analysis of samples with premium quality t controls, as determined prepare calibration samples.
Eight stages of the calibration samples and three levels checked The quality was t duplicates analyzed with each batch of samples. Assay Pr Precision and accuracy was evaluated by measuring the intra concentration of analytes in six aliquots of three different samples of contr The quality of t extracted and analyzed in a single day. Interassay Pr Precision and accuracy was determined from the results of three different samples of contr The quality of t extracted and analyzed six times on three different days. The detection limit was determined as the lowest concentration with a coefficient of variation and a polarization of 20%. The matrix effect was determined for all analytes enriched by comparing the Peakfl Surfaces of the extracted Imatinib CGP-57148B samples postextraction with cutting edge areas of unextracted standards at three different concentrations. The efficiency of extraction was enriched determined by comparing the Peakfl Surfaces of the extracted samples prior to extraction in areas of the tip postextraction spiked samples. Stability of t. Thionucleotides stability t has been studied in blood samples stored in EDTA-blood of four patients with up to 4 to 5 days at room temperature. The stability of t the RBC nucleotides may need during the check was examined by nucleotide doping separately.