Used consisting of 0.1% formic acid in Gamma-Secretase Inhibitors acetonitrile for linear gradient elution were as follows: 10 to 90% B in 10 min, hold 90% B for 3 min back to 10% B in 0.1 min, 10% B and for 3.9 min and hold to the S column into balance. The flowsheets rate was 0.4 ml / min. Detection by mass spectrometry was performed on a mass spectrometer with electrospray ionization source is a 3200 QTRAP equipped. 17 was in the positive ionization mode, DMAG detected, w During AAG and GA were detected 17 in the negative ionization mode. The transition from multiple reaction monitoring ions of m / z 617 3 58-17 DMAG, m / z 5593516 for GA and m / z 5843541 for 17 AAG. The temperature of the ion source set at the 650th Curtain gas, gas 1 and gas 2 were set at 30, 50, and 50 arbitrary units. Ion spray voltage was set at 5500 V to 4500 V and 17 DMAG for 17 AG and AAG. Found neutral loss of 43 Da, probably due to loss of 17 N methylenemethanamine cha, no page. Therefore, MRM Trnsfer Length ion at m / z 561 3,518,864 3821, 866 and 3823 were used to GAH2, glutathionyl GA 19 and 19 detect glutathionyl geldanamycin hydroquinone. MRM Ionenüberg length At m / z 586 3543, 3846889, 891 and 3848 were used to demethoxygeldanamycin demethoxygeldanamycin hydroquinone 17 17 19 17 17 19 17 17 glutathionyl and detect glutathionyl demethoxygeldanamycin hydroquinone. Meanwhile, the tertiary easier Re amine of 17 DMAG its protonation and resulting in high sensitivity MS in positive ionization. Consequently, the MRM Trnsfer Length of ions at m / z 619 3 58 922 3 58 924 3 and 58 were used to 17 17 demethoxygeldanamycin hydroquinone, 19 17 17 demethoxy geldanamycin glutathionyl and glutathionyl recognize 19 17 17 demethoxygeldanamycin hydroquinone, respectively. Screening and characterization of metabolites. GA, 17 AAG, DMAG and 17 were infused into a mass spectrometer to obtain their MS, MS2, MS3 and spectra. Based on the similarities and differences between their mass spectra, the structures of the fragment ions of protonated 17 DMAG were proposed on an interim basis.
MRM ion Trnsfer were Length used 617 3 58 3 617.3 159, 617 and 3524, around 240 additionally USEFUL ion Trnsfer Length for ZD-1839 MRM detection of metabolites with Metabolite ID software, including 40 processes generate biotransformation Commons. To identify metabolites, were the scan and full scan precursor Shore also performed. Only components in the sample is detected and the lack of controlled in all samples Were the metabolites of m Held possible. To the m Resembled metabolites that characterize both the sample and controlled They were injected onto the LC-MS for EPI and MS3 scans for their MS2 and MS3 spectra. Based on the MS2, MS3 spectra of metabolites and the proposed structures of fragment ions were 17 DMAG, 17-DMAG metabolites. Inhibition of 17 DMAG oxidative metabolism by cytochrome P450 inhibitors. The incubation mixtures containing 10 liters of pooled HLM, were in 0.1 M phosphate buffer in the presence of reduced NADPH, MgCl 2 and a chemical inhibitor for 3 min at 37 in a bath agitated water preincubated. Incubations contr The parallels were carried out in the absence of chemical inhibitors. Reactions were initiated by addition of 17 DMAG. The reaction mixtures were incubated for 20 min at the 37th The P450 isoform itself.