Increasing throughput and information content for in vitro drug metabolism experiments using ultra‐performance liquid chromatography coupled to a quadrupole time‐of‐flight mass spectrometer

Abstract

The field of drug metabolism has been revolutionized by liquid chromatography/mass spectrometry (LC/MS) applications with new technologies such as triple quadrupoles, ion traps and time-of-flight (ToF) instrumentation. Over the years, these developments have often relied on the improvements to the mass spectrometer hardware and software, which has allowed users to benefit from lower levels of detection and ease-of-use. One area in which the development pace has been slower is in high-performance liquid chromatography (HPLC). In the case of metabolite identification, where there are many challenges due to the complex nature of the biological matrices and the diversity of the metabolites produced, there is a need to obtain the most accurate data possible. Reactive or toxic metabolites need to be detected and identified as early as possible in the drug discovery process, in order to reduce the very costly attrition of compounds in late-phase development. High-resolution, exact mass measurement plays a very important role in metabolite identification because it allows the elimination of false positives and the determination of non-trivial metabolites in a much faster throughput environment than any other standard current methodology available to this field. By improving the chromatographic resolution, increased peak capacity can be achieved with a reduction in the number of co-eluting species leading to superior separations. The overall enhancement in the chromatographic resolution and peak capacity is transferred into a net reduction in ion suppression leading to an improvement in the MS sensitivity. To investigate this, a number of in vitro samples were analyzed using an ultra-performance liquid chromatography (UPLC) system, with columns packed with porous 1.7 mum particles, coupled to a hybrid quadrupole time-of-flight (ToF) mass spectrometer. This technique showed very clear examples for fundamental gains in sensitivity, chromatographic resolution and speed of analysis, which are all important factors for the demands of today's HTS in discovery.

Fast analysis in liquid chromatography using small particle size and high pressure

“…The method offered three-fold decrease in retention time, up to a ten-fold increase in detected peak height, and two-fold decrease in peak width compared to HPLC-MS/MS performed on a 2.1650 mm, 3.5 lm column. Castro-Perez et al [112] exploited the potential of sub-2 lm particles in the analysis of in vitro drug metabolism of dextromethorphan in rat liver microsomes. The UPLC approach (1.7 lm) appeared to be significantly better than the conventional HPLC approach (3.5 lm).…”

High-Resolution Mass Spectrometry Elucidates Metabonate (False Metabolite) Formation from Alkylamine Drugs during In Vitro Metabolite Profiling

“…Increasingly, highresolution mass spectrometry (MS) (i.e., resolving power Ͼ10,000 full width at half maximum) is being used for metabolite profiling and characterization because accurate mass data establish the elemental composition and fragmentation characteristics of detected drug-related components, which improves structure elucidation and the identification of metabolic soft spots and routes of biotransformation (Castro-Perez et al, 2005;Sanders et al, 2006). Time-of-flight (ToF) mass spectrometers are high-resolution instruments with fast cycle times, making them compatible with the higher-resolution ultra-highpressure liquid chromatography (UHPLC) systems currently available (Castro-Perez et al, 2005).…”

“…Increasingly, highresolution mass spectrometry (MS) (i.e., resolving power Ͼ10,000 full width at half maximum) is being used for metabolite profiling and characterization because accurate mass data establish the elemental composition and fragmentation characteristics of detected drug-related components, which improves structure elucidation and the identification of metabolic soft spots and routes of biotransformation (Castro-Perez et al, 2005;Sanders et al, 2006). Time-of-flight (ToF) mass spectrometers are high-resolution instruments with fast cycle times, making them compatible with the higher-resolution ultra-highpressure liquid chromatography (UHPLC) systems currently available (Castro-Perez et al, 2005). The combination of UHPLC with ToF MS provides a robust platform for rapid profiling and characterization of metabolites formed from new drugs and enables implementation of generic analytical workflows, such as elevated energy MS (MS E ), comprising simultaneous acquisition of precursor (m/z values corresponding to intact metabolites for biotransformation assignment) and product ion data (tandem mass spectral fragmentation data for structural elucidation of metabolites) by generating full-scan mass spectral data that are subsequently deconvoluted and mined by software strategies capitalizing on the high information content associated with accurate mass (Wrona et al, 2005;Bateman et al, 2007).…”

Future of Liquid Chromatography–Mass Spectrometry in Metabolic Profiling and Metabolomic Studies for Biomarker Discovery

“…GC as a front-end separation technique is unsurpassed in terms of speed, separation efficiency, and reproducibility, although the debut of [57][58][59][60] and recent developments in [61] highpressure liquid chromatography show promise for this technique to perhaps be equal to current one-dimensional GC in terms of separation efficiency in the near future. However, continued advancements in GC should continue to place the technology above LC in terms of peak capacity, sensitivity, and number of compound identifications.…”