In general, a comprehensive approach is highly sensitive and specific for the simultaneous determination of thiopurine metabolites still missing important. Therefore we have tried to create the first LC MS / MS assay of eleven thiopurine nucleotides in a single pass. Individual levels of nucleotides in erythrocyte samples from an ongoing clinical study of patients with IBD treated with azathioprine and measured partly in terms of concentrations of nucleotides from an independent Get ngigen method.18 鈻 EXPERIMENTAL Chemicals and reagents. All chemicals and reagents were of analytical quality t. Reference compounds of the analytes MeTGMP, MeTGDP, MeTGTP, MeTIMP, MeTIDP, Me, TITP, TGMP, TGDP, TgTp, TITP and TIMP were obtained from Jena Bioscience 10 mmol / l concentration in water. Deuterium labeled internal standards TGMP, TGDP / TgTp) MeTIMP, MeTIDP / MeTITP, and MeTGMP MeTGDP / MeTGTP by chemical synthesis have been described in the Supporting Information. EDTA was used as an L Solution of 50 mmol / L prepared and adjusted to pH 10.5 with 5 N NaOH L Solutions of dithio 1.4 D / L were Fra threitol YEARS Riger prepared prior to any experience. Pure water was obtained from a Milli Q Stamml Solutions of labeled nucleotides were dissolved in water at a final concentration of 10 mmol / l was prepared and 0th Ready-L Solutions of nucleotides to final concentrations of 10 mol / L and 1 mol / L were prepared by diluting with EDTA. Blood collection. Blood samples from 18 patients with Crohn’s disease were from an ongoing study on the Bcr-Abl inhibitor in clinical trials effects of the Ma Exception thiopurine metabolites on selected clinical management Hlt. Patients had prior to azathioprine dosage at least 6 months since study entry stable.
The study protocol was approved by the Ethics Committee of the Vienna Medical University t Vienna, approved sterreich. The patients Properties are described in SI, S summarized in Table 2. Zus Tzlich blood samples were collected from four patients were treated with azathioprine, to systematically investigate the stability of t the thionucleotides. This part of the study was approved by the Ethics Committee of the University of t h Tübingen Tal, Germany approved. All patients gave written Einverst Ndniserkl Tion. Preparation of erythrocyte lysate and the sample workup. Blood samples were collected in EDTA and immediately at 4 Packed RBC were within 4 hours after collection of blood prepared and frozen immediately at described20 0 to the analysis. To a mixture of 250 l EDTA and 15 l DTT-L Solution, 10 L Arbeitsstandardl Solution and 50 L of the internal CYP inhibitor fra YEARS Riger thawed RBC were added and vortex mixed. The proteins Were min by heating at 95 for 5 denatured in a water bath, and the samples were then followed by addition of 50 L of methanol by addition of 250 l of dichloromethane with an intimate mixture, extracted after each step. After centrifugation at 16 100 g for 20, 10 l supernatant for LC MS / MS. Balance for the subsequent End analysis by HPLC determination of thioguanosine 5 phosphates was performed as described previously.20 all the shares in TGN, MTGN and MMPR were independent of one Ngigen validated HPLC determined assay.18 All samples were analyzed in duplicate. LC-MS / MS conditions. An Agilent 6460 triple spectrometer quadrip Equipped with the Jet Stream IO electrospray.