Both Actin and Tubulin stabilization contributed to Ifn induced aggregation of APECs

ermining glibenclamide levels in small volumes of plasma and CSF. Glibenclamide measurement We used a LC-MS/MS mass spectrometry method to measure glibenclamide. Samples were prepared by reverse phase C18 solid phase extraction and d11-glibenclamide was used as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19757875 an internal standard. Selected reaction monitoring on an Ion Trap mass spectrometer, was used to enhance sensitivity. The mass transitions detect fragmentation of protonated precursor ions to a common fragment ion for glibenclamide and IS. Based on the limit of detection we estimate that this method provides a ~100-fold gain in sensitivity relative to detection in full scan MS mode. No interfering signals were detected at the retention time of the analyte and IS. The relationship between the spiked glibenclamide concentration and the CJ-023423 measured response was linear over the concentration range 104000ng/ml. Calibration curves obtained for three separate analytical runs on different days were linear with a correlation coefficient greater than 0.99. The accuracy and precision of the assay were within the 15% throughout the concentration 6 / 18 Glibenclamide Administration Fails to Reach Effective Levels in Brain Fig 1. Mass spectra of glibenclamide and d11-glibenclamide. Mass spectra and structural formulae of glibenclamide and d11-glibenclamide in positive ionization mode obtained by direct injection ion trap mass spectrometry. Protonated molecular ions m/z 494.1, 505.1. Fragment ion m/z, 369.0. doi:10.1371/journal.pone.0134476.g001 range 10ng/ml to 4000ng/ml. The performance PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19755095 of the assay was tested with four separately spiked QC samples at three different concentration levels: 3500, 350 and 40 ng/ml. Intra-day assay accuracy and precision were checked by running calibration standards and QC samples on the same day, and were within 20% on all three days of analysis. Inter-day accuracy and relative standard deviation and assay precision were within 15%. A lower limit of quantification in plasma of 10ng/ml, as defined by <20% accuracy limit, was achieved reproducibly. 7 / 18 Glibenclamide Administration Fails to Reach Effective Levels in Brain Fig 2. Plasma glibenclamide concentrations. Glibenclamide concentration in plasma from individual female and male mice implanted with a 21-day slow release 2.5mg glibenclamide pellet or a vehicle pellet. Data are meanSEM of triplicate measurements. MeanSEM determined glibenclamide concentration in plasma from mice treated with a vehicle or a 21-day slow release 2.5mg glibenclamide pellet. MeanSEM determined glibenclamide concentration in plasma from female and male mice implanted with a vehicle or a 21-day slow release 2.5mg glibenclamide pellet. P<0.0001. doi:10.1371/journal.pone.0134476.g002 Glibenclamide concentration in plasma Having determined we can quantify glibenclamide accurately at concentrations greater than 10ng/ml in ~30l samples, we next analysed plasma samples from mice and rats. This revealed the glibenclamide concentration in mouse plasma, 710 days after subcutaneous implantation with a 2.5mg/21-day slow-release glibenclamide pellet, was between 350 and 2000ng/ml. No signal was detected in mice implanted with a vehicle pellet. This drug concentration was chosen as it approximates the maximal oral dose in neonatal diabetes patients treated with glibenclamide. Interestingly, there was a significant difference between male and female mice, with females having approximately twice the mean plasma drug concentration than males. Sex diff