Comprehensive optimization of LC–MS metabolomics methods using design of experiments (COLMeD)

Abstract

Introduction Both reverse-phase and HILIC chemistries are deployed for liquid-chromatography mass spectrometry (LC-MS) metabolomics analyses, however HILIC methods lag behind reverse-phase methods in reproducibility and versatility. Comprehensive metabolomics analysis is additionally complicated by the physiochemical diversity of metabolites and array of tunable analytical parameters. Objective Our aim was to rationally and efficiently design complementary HILIC-based polar metabolomics methods on multiple instruments using Design of Experiments (DoE). Methods We iteratively tuned LC and MS conditions on ion-switching triple quadrupole (QqQ) and quadrupole-time-of-flight (qTOF) mass spectrometers through multiple rounds of a workflow we term COLMeD (Comprehensive optimization of LC-MS metabolomics methods using design of experiments). Multivariate statistical analysis guided our decision process in the method optimizations. Results LC-MS/MS tuning for the QqQ method on serum metabolites yielded a median response increase of 161.5% (p<0.0001) over initial conditions with a 13.3% increase in metabolite coverage. The COLMeD output was benchmarked against two widely used polar metabolomics methods, demonstrating total ion current increases of 105.8% and 57.3%, with median metabolite response increases of 106.1% and 10.3% (p<0.0001 and p<0.05 respectively). For our optimized qTOF method, 22 solvent systems were compared on a standard mix of physiochemically diverse metabolites, followed by COLMeD optimization, yielding a median 29.8% response increase (p<0.0001) over initial conditions. Conclusions The COLMeD process elucidated response tradeoffs, facilitating improved chromatography and MS response without compromising separation of isobars. COLMeD is efficient, requiring no more than 20 injections in a given DoE round, and flexible, capable of class-specific optimization as demonstrated through acylcarnitine optimization within the QqQ method.

Extraction and Analysis of Pan-metabolome Polar Metabolites by Ultra Performance Liquid Chromatography–Tandem Mass Spectrometry (UPLC-MS/MS)

“…Polar metabolites were then analyzed through UPLC-MS utilizing a method optimized through the COLMeD approach as described in (Rhoades and Weljie, 2016). The following procedure should be used as guidelines in acquiring data by individuals trained in using a mass spectrometer.…”

“…Although both NMR and MS are essential tools in metabolomics, mass spectrometry can analyze samples with greater sensitivity (Liu and Locasale, 2017). Within mass spectrometry, various approaches are available, however a rapid approach to profile the global metabolome is needed (Lv et al ., 2011; Rhoades and Weljie, 2016). Advancements in triple quadrupole mass spectrometers have made them well suited for ion-switching methods (scanning in both positive and negative ion modes within a single analysis) in addition to enabling reproducible and sensitive targeted profiling of numerous metabolites (Lv et al ., 2011; Gika et al ., 2012; Yuan et al ., 2012; Rhoades and Weljie, 2016).…”

“…Within mass spectrometry, various approaches are available, however a rapid approach to profile the global metabolome is needed (Lv et al ., 2011; Rhoades and Weljie, 2016). Advancements in triple quadrupole mass spectrometers have made them well suited for ion-switching methods (scanning in both positive and negative ion modes within a single analysis) in addition to enabling reproducible and sensitive targeted profiling of numerous metabolites (Lv et al ., 2011; Gika et al ., 2012; Yuan et al ., 2012; Rhoades and Weljie, 2016). Additionally, LC typically does not require extensive sample preparation, nor derivatization, which allows for the detection of a broader range of metabolites (Gika et al ., 2012; Liu and Locasale, 2017).…”

“…Nonetheless, development of LC-MS methods to comprehensively analyze small polar metabolites is nontrivial, and requires advanced modeling to optimize both LC and MS factors simultaneously. Thus, the COLMeD approach aimed to address this challenge and enabled a more comprehensive metabolite analysis across platforms including on a triple quadrupole mass spectrometer (Rhoades and Weljie, 2016). The LC-MS workflow on a triple quadrupole is described here and has been used to successfully study metabolomics in a circadian context in which 179 metabolites were successfully profiled and analyzed (Krishnaiah et al ., 2017).…”

“…Liquid chromatography (LC) is advantageous, as it does not require derivatization procedures and a large diversity in physiochemical characteristics of analytes can be accommodated through a variety of column chemistries. Recently, the comprehensive optimization of LC-MS metabolomics using design of experiments (COLMeD) approach has been described and used by our group to develop robust LC-MS workflows (Rhoades and Weljie, 2016). The optimized LC-MS/MS method described here has been utilized extensively for metabolomics analysis of polar metabolites.…”