larity mode. The analytical column used was a Phenomenex, Synergi, Polar RP 80A, 75 × 2mm, 4 :m with a Phenomenex, SecurityGuard Polar RP, 4×2mm2 guard column. The column was held at ambient temperature. An isocratic mobile phase consisting Andarine GTX-007 of 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile was used at a flow rate of 300 :L/min. Injection volume was 10 :L, total run time was 4 min. The autosampler was held at a temperature of 5C. Under these conditions, retention time of TFV was 1.6 min and retention time of the internal standard TFV d6 was 1.58 min. The LLOQ of this method was 1.0 ng/mL. TFV in Tissues The samples prepared as described above were added to a 96 well extraction plate. Next, the working internal standard TFV d6 was added. The plate was capped and vortexed for about 30 s.
The plate was then centrifuged for approximately 5min at 4C. The samples were subsequently analyzed by LC MS MS using the same equipment for analysis of TFV in plasma except that the MS MS was operated in the negative polarity mode. The analytical column used was Sunitinib PDGFR inhibitor a BioBasic AX 50× 3.0mm, 5 :m without a guard column. A gradient solvent system consisting of mobile phase A: acetonitrile/10 nM ammonium acetate in water, pH 6.0 and mobile phase B: acetonitrile/1mM ammonium acetate in water, pH 10.5 was used. The gradient system consisting steps starting at 95% A 5% B to 100% B over 2.1min after which 100% B was used until 8min was reached. The flow rate was 400 :L/min, injection volume was 5.0 :L and the column was held at ambient temperature. The autosampler temperature was held at 5C.
Under these conditions, retention times of TFV and the internal standard were 4.9 min. The LLOQ of this method was 2.0 ng/mL. Total TFV in Tissues Total TFV was measured by using the method described above for TFV in plasma. After sonication of tissue MGCD0103 samples, freshly prepared phosphatase working solution was added to 100 :L of sample solution and mixed by vortexing for approximately 20 s in a 96 well plate. The plate was then covered with sheets of sealing film and incubated in a water batch at 40C for 40 5min. Next, a 96 well filtration plate was placed onto a 96 well sample processing manifold with a 96 well collection plate in position. Acetonitrile was added to all wells of the filtration plate. The samples were then transferred to the PPT filtration plate and allowed to stand for 5 min.
Then, vacuum was applied to the filter plate and the whole filtrate collected. This filtrate was evaporated to dryness under a stream of N2 at approximately 50C. The samples were then reconstituted with 200 :L water. The plate was capped and vortexed for 30 s. The sample was then injected onto the LC MS MS for analysis. The TFV analysis method used is that described above for analysis of TFV in rabbit plasma. Statistical Methods Group comparisons were made using a single factor analysis of variance followed by two tailed heteroscedastic t tests. MDOE Modeling Detailed methods for the MDOE modeling were described previously.24 RESULTS The overall approach to designing an SG, ISG, and BG is outlined in Figure 1. This work involved preparation of 19 gels usingMDOEsas described previously.24 The gels prepared for analysis following the MDOE are shown in Table 1. Using these gel
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