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RESULTS AND DISCUSSION
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96-Well Plate Based Method Development
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Since an LC/MS method had already been developed,18 the focus of this method development was on evaluating the consistency of the 96-well plate based sample preparation and uniformity of results from the various wells in the 96-well plate, namely the edge effect. However we choose to compare the results, it is necessary to evaluate the precision and accuracy of the 96channel pipettor rst. Precision and Accuracy of Apricot Personal Pipettor Dilution of Octanol Layer. The Apricot personal pipettor can operate in 96-channels simultaneously. Since octanol is a very viscous solvent, it is necessary to ensure that the right amount can be aspirated and delivered by the pipettor. An experiment was designed to check the linearity of the dilution by adding Rhodamine 6G, a dye with an absorbance at 525 nm, to the octanol. Several dilutions (100 , 200 , 400 , 800 , 1600 , 3200 , and 16,000 ) were performed on a 96-well plate. A SpectraMax 96-well plate reader was used to measure the dyed octanol. Figure 17.3A shows a linear dilution curve when using the Apricot personal pipettor.
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high-throughput determination of log D values by lc/ms method
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1.2 1 y = 96.275x + 0.0452 2 R = 0.9875
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absorbance (525 nm)
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0.8 0.6 0.4 0.2 0
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0.00E+00 2.00E-03 4.00E-03 6.00E-03 8.00E-03 1.00E-02 1.20E-02
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B absorbance (525 nm) 1.200 1.000 0.800 0.600 0.400 0.200 0.000 0 0.1 0.2 0.3 concentration
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Figure 17.3. Linearity of the serial dilution by 96-needle Apricot pipettor: (A) Octanol serial dilution by Apricot pipettor and (B) manual dilution compared with Apricot pipettor dilution of aqueous buffer.
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In addition the precision of the dilution was evaluated at different concentration levels with another set of samples (data not shown here). For the samples 10-fold diluted from four levels of original samples at 0.125 mM, 0.167 mM, 0.25 mM, and 0.5 mM, the %RSD (n = 4) of the dilution with the Apricot pipettor were 1.5%, 2.0%, 6.4%, and 3.5%, respectively. The %RSDs of manual dilution for the same set of samples were 7.5%, 5.1%,
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results and discussion
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4.6%, and 6.1%, respectively. There is no signi cant difference between manual dilution and the Apricot pipettor. Dilution of Buffer. Precision and accuracy of the dilution by the Apricot personal pipettor for aqueous buffer were also evaluated using the same dye. The original samples at four different concentrations, 0.125, 0.167, 0.25, and 0.5 mM, were diluted 10-fold and 100-fold by both 12-channel pipettor and the Apricot personal pipettor. These results show that the Apricot pipettor picked up 90% to 99% of the amount picked up by the manual pipettor. The %RSD (n = 4), which is the precision of the dilutions, varied between 2% and 9%. A reasonably good correlation between these two methods at the four levels of concentrations is shown in Figure 17.3B. It can be concluded from previous data that the Apricot dilution is equivalent to the manual dilution. Therefore it is expected that using the Apricot will not introduce signi cant errors to the nal log D determination. Different Types of Mixing One of our main concerns was how well the samples were mixed when a 96-well plate was used instead of micro-centrifuge tubes. Several mixing methods were tested and the log D values were compared. As a control the samples were also prepared in micro-centrifuge tubes and mixed by vortexing for 30 seconds followed by 1 hour end-over-end mixing. The mixing methods used were (1) end-over-end for 1 hour, (2) end-over-end for 4 hours, (3) horizontal shaker for 1 hour, and (4) vertical shaker for 1 hour. A Laboratory Rotator at a speed setting of 70% was used for end-over-end mixing, while a VX-2500 multi-tube vortexer (VWR Scienti c, Brisbane, CA) was used for both the horizontal and vertical shaking. Figure 17.4 shows that log D values obtained from the four mixing methods were very close. One mixing method, namely the end-over-end for 1 hour, was used for the sample determinations because it results in less emulsion formation during the mixing process. Comparison of Different Sample Preparations In order to evaluate the sample preparation procedures in the 96-well plate based assay, several commercial compounds along with two Theravance compounds were prepared using both the manual (tube) method and the 96-well plate method. Among the compounds tested, the differences are extremely small, with the biggest difference being 0.29, which is within the expected variation range of the method. Therefore the results indicate that the log D values obtained by these two sample preparations are identical.
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