Two key bottlenecks in pharmaceutical bioanalysis are sample cleanup and chromatographic separations. Although multiple approaches have been developed in the past decade to either shorten or multiplex these steps, they remain the rate limiting steps as ADME (Absorption, Distribution, Metabolism, and Excretion) property screening is being routinely incorporated into the drug discovery process. In this work, a novel system incorporating an automated Direct Analysis in Real Time (DART) ionization source coupled with a triple-quadrupole mass spectrometer has been developed and evaluated for quantitative bioanalysis. This system has the capability of directly analyzing samples from their biological matrixes and therefore potentially eliminating the need for sample cleanup and chromatographic separations. A LEAP Technologies autosampler was customized to perform the automated sample introduction into the DART beam with high precision, which significantly improved the reproducibility of the method. Additional pumping was applied to the atmospheric pressure inlet on the mass spectrometer to compensate for the increased vacuum load because of the use of high-flow helium by the DART. This resulted in an improvement of detection sensitivity by a factor of 10 to 100 times. Matrix effects for a diversified class of compounds were evaluated directly from untreated raw plasma and were found to range from approximately 0.05 to 0.7. Precision and accuracy were also tested for multiple test compounds over a dynamic range of four orders of magnitude. The system has been used to analyze biological samples from both in vivo pharmacokinetic studies and in vitro microsomal/S9 stability studies, and the results generated were similar to those obtained with conventional LC/MS/MS methods. Overall, this new automated DART-triple quadrupole mass spectrometer system has demonstrated significant potential for high-throughput bioanalysis.
SummaryBeyond their essential function as the building blocks of proteins, amino acids contribute to many aspects of plant biochemistry and physiology. Despite this, there are relatively large gaps in our understanding of the biochemical pathways and regulation of amino acid synthesis in plants. A rapid (1.5 min versus 20-90 min for standard methods) HPLC-MS/MS assay for separating 19 amino acids was developed for quantifying levels of free amino acids in plant tissue. This assay was used to determine the free amino acid content in the seeds of 10,000 randomly mutagenized Arabidopsis lines, and 322 Arabidopsis lines with increased levels of one or more amino acids were identified. The heritability of the mutant phenotype was confirmed for 43 lines with increased seed levels of the aspartate-derived amino acids Ile, Lys, Thr, or Met. Genetic mapping and DNA sequencing identified a mutation in an Arabidopsis threonine aldolase (AT1G08630, EC 4.1.2.5) as the cause of increased seed Thr levels in one mutant. The assay that was developed for this project has broad applicability to Arabidopsis and other plant species.
Fasiglifam (TAK-875), a Free Fatty Acid Receptor 1 (FFAR1) agonist in development for the treatment of type 2 diabetes, was voluntarily terminated in phase 3 due to adverse liver effects. A mechanistic investigation described in this manuscript focused on the inhibition of bile acid (BA) transporters as a driver of the liver findings. TAK-875 was an in vitro inhibitor of multiple influx (NTCP and OATPs) and efflux (BSEP and MRPs) hepatobiliary BA transporters at micromolar concentrations. Repeat dose studies determined that TAK-875 caused a dose-dependent increase in serum total BA in rats and dogs. Additionally, there were dose-dependent increases in both unconjugated and conjugated individual BAs in both species. Rats had an increase in serum markers of liver injury without correlative microscopic signs of tissue damage. Two of 6 dogs that received the highest dose of TAK-875 developed liver injury with clinical pathology changes, and by microscopic analysis had portal granulomatous inflammation with neutrophils around a crystalline deposition. The BA composition of dog bile also significantly changed in a dose-dependent manner following TAK-875 administration. At the highest dose, levels of taurocholic acid were 50% greater than in controls with a corresponding 50% decrease in taurochenodeoxycholic acid. Transporter inhibition by TAK-875 may cause liver injury in dogs through altered bile BA composition characteristics, as evidenced by crystalline deposition, likely composed of test article, in the bile duct. In conclusion, a combination of in vitro and in vivo evidence suggests that BA transporter inhibition could contribute to TAK-875-mediated liver injury in dogs.
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