A high‐speed liquid chromatography/tandem mass spectrometry platform using multiplexed multiple‐injection chromatography controlled by single software and its application in discovery ADME screening

Abstract

RATIONALE:Multiplexed liquid chromatography (LC) coupled with multiple-injection-chromatogram acquisition has emerged as the method of choice for high-speed discovery bioanalysis, because it significantly reduces injection-to-injection cycle time while maintaining the chromatography quality. Historically, systems utilizing this approach had been custom built, and therefore relied on custom software tools to communicate with multiple vendor software for system control, which lacked transferability, flexibility and robustness. METHODS: In this study, we refined a multiplexed bioanalytical system previously reported, by implementing open-deck auto-sampler manifold and multiple-injection-chromatogram acquisition, all on a commercially available system with single software control. RESULTS: As a result of these improvements, the developed LC/tandem mass spectrometry (MS/MS) method on the system was nearly three times faster than the previous method, while demonstrating comparable analytical accuracy, precision and robustness. This system has been evaluated for in vitro ADME screening assays including metabolic stability, CYP inhibition and Caco-2. The biological data generated on the developed system displayed good correlation with those from the previous LC/MS/MS approaches. CONCLUSIONS:The developed platform demonstrated applicability to the in vitro screening assays evaluated and has been successfully implemented to support the high-throughput metabolic stability assay, with a significantly improved bioanalytical throughput, capacity and data turnaround.

Addition of Optimized Bovine Serum Albumin Level in a High-Throughput Caco-2 Assay Enabled Accurate Permeability Assessment for Lipophilic Compounds

“…11 To obtain the optimum selected reaction monitoring (SRM) conditions for sample analysis, MS/MS optimization for each compound was performed using DiscoveryQuant as reported previously. 12 A 5 µL aliquot of sample was injected and the separation was carried out on a Kinetex XB-C18 column (2.6 µm, 2.1 × 30 mm; Phenomenex, Torrance, CA) under a gradient elution using mobile phases A (0.2% formic acid in water) and B (0.2% formic acid in acetonitrile). The percentage of B was first held at 2% for 5 s and then ramped up to 98% in 25 s, where it was held for 20 s before being brought back to the starting composition.…”

Acoustic ejection mass spectrometry: Development, applications, and future perspective

“…The multiplexed LC-MS approach involves coupling two or four independent LCs into the same mass spectrometer with a switching valve, making staggered injections onto the different LCs, and collecting MS data from the retention window of each LC channel. A speed of 15-30 s can be achieved with a 2Â or 4Â multiplexed LC-MS system (Kapinos et al, 2017;Zhang et al, 2010;Zhang et al, 2013). Direct online solid-phase extraction (SPE) is another LC-based approach.…”

Acoustic Ejection/Full-Scan Mass Spectrometry Analysis for High-Throughput Compound Quality Control

“…8 In an effort to minimize MS idle time, the multiplexed LC approach combined with multiple injection chromatography for data acquisition yields a typical analysis speed of 0.5 min per sample. 9,10 An even faster injection-to-injection cycle time of 5–10 s has been achieved on the RapidFire system (Agilent Technologies, Santa Clara, CA), where the chromatographic separation is simply reduced to a minimum of online solid-phase extraction. 11,12 Sample pooling for cassette LC-MS analysis has also been seen for high-throughput nanoscale synthesis 3 and various in vitro profiling assays 13,14 to alleviate the analytical workload.…”