High-throughput analysis of standardized pharmacokinetic studies in the rat using sample pooling and UPLC-MS/MS |
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Authors: | Bueters Tjerk Dahlström Jessie Kvalvågnaes Kristine Betnér Ingvar Briem Sveinn |
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Affiliation: | 1. DMPK, CNSP Innovative Medicines, AstraZeneca AB, SE-151 85, Södertälje, Sweden;2. Waters Corporation, Sollentuna, Sweden |
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Abstract: | As a consequence of a continuous demand for increased throughput of pharmacokinetic (PK) studies, industries have introduced strategies to reduce the number of samples such as cassette analysis (pooling of samples after the in-life phase). Here, we have investigated whether relevant PK parameters change as a consequence of cassette analysis, and whether there are circumstances that disqualify this technique from being used. 22 compounds were intravenously and orally administered to parallel groups of 3 rats. Each compound was administered discretely. Equal volumes of three plasma samples corresponding to each time point of three discretely dosed rats with different compounds were pooled (cassette analysis). Samples were prepared by protein precipitation followed by UPLC-MS/MS analysis using pos/neg switching when required. With cassette analysis, 4 compounds, morphine, phenytoin, rofecoxib and diclofenac, showed high limit of quantification (LOQ) values after pooling, which led to less reliable PK analyses. Of all samples with contents above LOQ, about 5% could not be detected in pool samples compared to single samples. However, an excellent correlation was seen for all PK parameters when comparing the parameters obtained from discrete analysis versus those obtained from cassette analysis, although half life showed somewhat more scatter than the others. When PK parameters were grouped as low-medium-high, clearance, volume of distribution, half life and bioavailability were similar between discrete and cassette analysis for 90%, 86%, 95% and 90% of the total number of compounds tested, respectively. Some additional improvement was achieved if compounds with a low MS response were excluded. In summary, cassette analysis is an effective strategy to reduce samples without affecting the estimated PK parameters that are important for decision-making. |
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Keywords: | ACN, acetonitrile AUC, area under the curve CL, clearance DMA, dimethylamine F, bioavailability LOQ, limit of quantification PEG400, polyethylene glycol 400 PK, pharmacokinetic rt, retention time T1/2, half life UPLC–MS/MS, ultra performance liquid chromatography–mass spectrometry/mass spectrometry V, volume of distribution |
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