vitro and in vivo assays are needed in order to bring a compound forward into development. In this section, we discuss some of the strategies that have been used to increase the throughput of in vivo assays. As discussed in a review of this topic by Cox et al. , there are two basic strategies for increasing the throughput of assays used for various PK studies performed in a discovery environment: assay enhancement and sample reduction.
Assay-enhancement strategies are typically procedures that speed up the rate that samples can be processed and analyzed. Examples of this include various on-line sample preparation techniques that minimize the effort needed for manual sample preparation. One common technique is based on column switching (CS); in this procedure a short column is used to extract the analyte from the plasma matrix, then a second column is used to provide the sample chromatography [39-43]. As discussed by Hopfgartner et al. , CS can also be used along with protein precipitation in a combined sample cleanup approach. Another technique for direct plasma injection is based on the use of a single HPLC column, which serves not only as the means for analyte extraction, but provides chromatographic separation as well. Hsieh et al.  have provided several examples of how this technique can be used for drug-discovery applications. For most of the applications described by these authors, a mixed-functional column was used; in a recent article, Hsieh et al.  described the use of a monolithic silica column for direct plasma injection of discovery plasma samples into an HPLC-MS/MS system.
The various methods for sample reduction include cassette dosing [38,51,52], sample pooling strategies [38,53,54], and cassette-accelerated rapid rat screen (CARRS) . As discussed recently in a review article by Papac and Shahrokh , the cassette-dosing approach has the disadvantage that drug-drug interactions can be an issue. As discussed by White and Manit-pisitkul , these drug-drug interactions can lead to both false-positive and false-negative results. Multiple-compound sample pooling without cassette dosing has also been used to reduce the number of samples to be assayed, but as discussed by Papac and Shahrokh , this process has disadvantages in that careful attention has to be taken to ensure that isomeric compounds are not mixed and that possible metabolites of compounds do not interfere with dosed compounds.
The CARRS system described by Korfmacher et al.  avoids all of these problems while still providing the capability of a higher throughput in vivo rat PK screen. In the CARRS assay, compounds are grouped into cassettes of six selected from one drug-discovery team's new compounds. Each compound is dosed orally into two rats. Six plasma samples are taken from each rat at six time points (0.5, 1, 2, 3, 4, 6 hours postdose), and samples from the two rats dosed with one compound are pooled across the two rats to give a total of six samples (one at each time point) to assay per each compound that was dosed. Multiple cassettes are dosed each week (week 1). In the second week, the plasma samples are assayed in cassettes of six compounds each. Due to the standardization of the dosing and assay regimes, various steps have been streamlined to improve their efficiency. For example, as shown in Figure 12.11, all the samples and standards needed to assay one set of six compounds can fit into one 96-well plate . Sample preparation consists
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