ABSTRACT
Pharmacological screening heavily relies on the reliability of compound libraries. To ensure the accuracy of screening results, fast and reliable quality control (QC) of these libraries is essential. While liquid chromatography (LC) with ultraviolet (UV) or mass spectrometry (MS) detection has been employed for molecule QC on small sample sets, the analytical throughput becomes a bottleneck when dealing with large libraries. Acoustic ejection mass spectrometry (AEMS) is a high-throughput analytical platform that covers a broad range of chemical structural space. In this study, we present the utilization of an AEMS system equipped with a high-resolution MS analyzer for high-throughput compound QC. To facilitate efficient data processing, which is a key challenge for such a high-throughput application, we introduce an automatic data processing toolkit that allows for the high-throughput assessment of the sample standards' quantitative and qualitative characteristics, including purity calculation with the background processing option. Moreover, the toolkit includes a module for quantitatively comparing spectral similarity with the reference library. Integrating the described high-resolution AEMS system with the data processing toolkit effectively eliminates the analytical bottleneck, enabling a rapid and reliable compound quality assessment of large-scale compound libraries.
ABSTRACT
The cycle time of a standard liquid chromatography (LC) system is the sum of the time for the chromatographic run and the autosampler injection sequence. Although LC separation times in the 1-10 s range have been demonstrated, injection sequences are commonly >15 s, limiting throughput possible with LC separations. Further, such separations are performed on relatively large bore columns requiring flow rates of ≥5 mL/min, thus generating large volumes of mobile phase waste when used for large scale screening and increasing the difficulty in interfacing to mass spectrometry. Here, a droplet injector system was established that replaces the autosampler with a four-port, two-position valve equipped with a 20 nL internal loop interfaced to a syringe pump and a three-axis positioner to withdraw sample droplets from a well plate. In the system, sample and immiscible fluid are pulled alternately from a well plate into a capillary and then through the injection valve. The valve is actuated when sample fills the loop to allow sequential injection of samples at high throughput. Capillary LC columns with 300 µm inner diameter were used to reduce the consumption of mobile phase and sample. The system achieved 96 separations of 20 nL droplet samples containing 3 components in as little as 8.1 min with 5-s cycle time. This system was coupled to a mass spectrometer through an electrospray ionization source for high-throughput chemical reaction screening.
ABSTRACT
Since the advent of modern-day screening collections in the early 2000s, various aspects of our knowledge of good handling practices have continued to evolve. Some early practices, however, continue to prevail due to the absence of defining data that would bust the myths of tradition. The lack of defining data leads to a gap between plate-based screeners, on the one hand, and compound sample handling groups, on the other, with the latter being the default party to blame when an assay goes awry.In this paper, we highlight recommended practices that ensure sample integrity and present myth busting data that can help determine the root cause of an assay gone bad. We show how a strong and collaborative relationship between screening and sample handling groups is the better state that leads to the accomplishment of the common goal of finding breakthrough medicines.
Subject(s)
Biological AssayABSTRACT
Unbound tissue-to-plasma partition coefficients (Kpuu) were determined for 56 structurally diverse compounds in rats following intravenous infusion. Five tissues were included in the study: white adipose, brain, heart, liver, and skeletal muscle. The rank ordering of the median tissue Kpuu values was: liver (4.5)â¯>â¯heart (1.8)â¯>â¯adipose (1.2)â¯>â¯skeletal muscle (0.6)â¯>â¯brain (0.05), with liver being most enriched and brain most impaired. The median Kpuu values of acids and zwitterions were lower than those of bases and neutrals in all tissues but liver. Selective tissue distribution was observed, dependent upon chemotype, which demonstrated the feasibility of targeting or restricting drug exposure in certain tissues through rational design. Physicochemical attributes for Kpuu were identified using recursive partitioning, which further classified compounds with enriched or impaired tissue distribution. The attributes identified provided valuable insight on design principles for asymmetric tissue distribution to improve efficacy or reduce toxicity.
