Testing tuberculosis drug efficacy in a zebrafish high-throughput translational medicine screen.

The translational value of zebrafish high-throughput screens can be improved when more knowledge is available on uptake characteristics of potential drugs. We investigated reference antibiotics and 15 preclinical compounds in a translational zebrafish-rodent screening system for tuberculosis. As a major advance, we have developed a new tool for testing drug uptake in the zebrafish model. This is important, because despite the many applications of assessing drug efficacy in zebrafish research, the current methods for measuring uptake using mass spectrometry do not take into account the possible adherence of drugs to the larval surface. Our approach combines nanoliter sampling from the yolk using a microneedle, followed by mass spectrometric analysis. To date, no single physicochemical property has been identified to accurately predict compound uptake; our method offers a great possibility to monitor how any novel compound behaves within the system. We have correlated the uptake data with high-throughput drug-screening data from Mycobacterium marinum-infected zebrafish larvae. As a result, we present an improved zebrafish larva drug-screening platform which offers new insights into drug efficacy and identifies potential false negatives and drugs that are effective in zebrafish and rodents. We demonstrate that this improved zebrafish drug-screening platform can complement conventional models of in vivo Mycobacterium tuberculosis-infected rodent assays. The detailed comparison of two vertebrate systems, fish and rodent, may give more predictive value for efficacy of drugs in humans.

Gas pressure assisted microliquid-liquid extraction coupled online to direct infusion mass spectrometry: a new automated screening platform for bioanalysis.

In the field of bioanalysis, there is an increasing demand for miniaturized, automated, robust sample pretreatment procedures that can be easily connected to direct-infusion mass spectrometry (DI-MS) in order to allow the high-throughput screening of drugs and/or their metabolites in complex body fluids like plasma. Liquid-Liquid extraction (LLE) is a common sample pretreatment technique often used for complex aqueous samples in bioanalysis. Despite significant developments that have been made in automated and miniaturized LLE procedures, fully automated LLE techniques allowing high-throughput bioanalytical studies on small-volume samples using direct infusion mass spectrometry, have not been matured yet. Here, we introduce a new fully automated micro-LLE technique based on gas-pressure assisted mixing followed by passive phase separation, coupled online to nanoelectrospray-DI-MS. Our method was characterized by varying the gas flow and its duration through the solvent mixture. For evaluation of the analytical performance, four drugs were spiked to human plasma, resulting in highly acceptable precision (RSD down to 9%) and linearity (R(2) ranging from 0.990 to 0.998). We demonstrate that our new method does not only allow the reliable extraction of analytes from small sample volumes of a few microliters in an automated and high-throughput manner, but also performs comparable or better than conventional offline LLE, in which the handling of small volumes remains challenging. Finally, we demonstrate the applicability of our method for drug screening on dried blood spots showing excellent linearity (R(2) of 0.998) and precision (RSD of 9%). In conclusion, we present the proof of principe of a new high-throughput screening platform for bioanalysis based on a new automated microLLE method, coupled online to a commercially available nano-ESI-DI-MS.

Three-phase electroextraction: a new (online) sample purification and enrichment method for bioanalysis.

The migration and at the same time enrichment of analytes from a liquid aqueous sample donor phase through an immiscible organic solvent layer acting as a filter phase into a liquid aqueous acceptor phase is enabled by the application of an electric field between the donor and acceptor phase. The organic filter phase acts as a purification filter, which prevents, for example, proteins from migrating into the acceptor phase. Moreover, the composition of the organic filter phase influences the selectivity of the extraction. We show that analytes can be rapidly enriched from a 50 µL donor phase at the bottom of a sample vial, via an immiscible organic filter phase, into a 2 µL acceptor phase which consists of a droplet that is hanging from a (conductive) pipet tip in the organic filter phase. Acylcarnitines spiked to human plasma as a donor phase were extracted reproducibly with good linearity and a 10-fold improved limit of detection and, importantly, resulted in a stable, protein-free nanoelectrospray signal. Finally, a proof of principle toward the online integration in an automated nanoelectrospray-direct infusion-mass spectrometry platform has been realized. This makes 3-phase electroextraction (3-phase EE) a novel sample purification and enrichment method, with straightforward online integration possibility. We envision that 3-phase EE will enable new possibilities using electrokinetic sample pretreatment for fully automated, high-throughput bioanalysis purposes.

Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae.

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.