Ion mobility spectrometry combined with ultra performance liquid chromatography/mass spectrometry for metabolic phenotyping of urine: Effects of column length, gradient duration and ion mobility spectrometry on metabolite detection (original) (raw)

Metabolic profiling by ion mobility mass spectrometry (IMMS)

Metabolomics, 2008

Ion Mobility Mass Spectrometry (IMMS) was evaluated as an analytical method for metabolic profiling. The specific instrument used in these studies was a direct infusion (DI)-electrospray ionization (ESI)-ambient pressure ion mobility spectrometer (APIMS) coupled to a timeof-flight mass spectrometer (TOFMS). The addition of an ion mobility spectrometer to a mass spectrometer had several advantages over direct infusion electrospray mass spectrometry alone. This tandem instrument (ESI-IMMS) added a rapid separation step with high-resolution prior to mass spectrometric analysis of metabolite mixtures without extending sample preparation time or reducing the high through put potential of direct mass spectrometry. Further, IMMS also reduced the baseline noise common with ESI-MS analyses of complex samples and enabled rapid separation of isobaric metabolites. IMMS was used to analyze the metabolome of Escherichia coli (E. coli), containing a collection of extremely diverse chemical compounds including hydrophobic lipids, inorganic ions, volatile alcohols and ketones, amino and non-amino organic acids, and hydrophilic carbohydrates. IMMS data were collected as twodimensional spectra showing both mobility and mass of each ion detected. Using direct infusion ESI-IMMS of a nonderivatized methanol extract of an E. coli culture, more than 500 features were detected, of which over 200 intracellular metabolites were tentatively assigned as E. coli metabolites.

Intra- and Interlaboratory Reproducibility of Ultra Performance Liquid Chromatography–Time-of-Flight Mass Spectrometry for Urinary Metabolic Profiling

Analytical Chemistry, 2012

Liquid chromatography coupled to mass spectrometry (LC−MS) is a major platform in metabolic profiling but has not yet been comprehensively assessed as to its repeatability and reproducibility across multiple spectrometers and laboratories. Here we report results of a large interlaboratory reproducibility study of ultra performance (UP) LC−MS of human urine. A total of 14 stable isotope labeled standard compounds were spiked into a pooled human urine sample, which was subject to a 2-to 16-fold dilution series and run by UPLC coupled to time-of-flight MS at three different laboratories all using the same platform. In each lab, identical samples were run in two phases, separated by at least 1 week, to assess betweenday reproducibility. Overall, platform reproducibility was good with median mass accuracies below 12 ppm, median retention time drifts of less than 0.73 s and coefficients of variation of intensity of less than 18% across laboratories and ionization modes. We found that the intensity response was highly linear within each run, with a median R 2 of 0.95 and 0.93 in positive and negative ionization modes. Between-day reproducibility was also high with a mean R 2 of 0.93 for a linear relationship between the intensities of ions recorded in the two phases across the laboratories and modes. Most importantly, between-lab reproducibility was excellent with median R 2 values of 0.96 and 0.98 for positive and negative ionization modes, respectively, across all pairs of laboratories. Interestingly, the three laboratories observed different amounts of adduct formation, but this did not appear to be related to reproducibility observed in each laboratory. These studies show that UPLC−MS is fit for the purpose of targeted urinary metabolite analysis but that care must be taken to optimize laboratory systems for quantitative detection due to variable adduct formation over many compound classes.

Gas-Phase Ion Mobility-Mass Spectrometry (IM-MS) and Tandem IM-MS/MS Strategies for Metabolism Studies and Metabolomics

Encyclopedia of Drug Metabolism and Interactions, 2012

This chapter describes the current and potential impact of ion mobility-mass spectrometry (IM-MS) for addressing the challenges of sample complexity, dynamic range and throughput for metabolomics experiments. The four primary ion mobility techniques are described with regard to their defining analytical figures-of-merit. Tandem MS methods are reviewed and novel tandem experiments made possible from multi-stage activation in IM-MS instrumentation are discussed in light of the additional information such experiments provide for the analysis of metabolites.

Optimization of Data Acquisition and Sample Preparation Methods for LC-MS Urine Metabolomic Analysis

Open Chemistry, 2015

Nowadays, chromatographic methods coupled with mass spectrometry are the most commonly used tools in metabolomics studies. These methods are currently being developed and various techniques and strategies are proposed for the profiling analysis of biological samples. However, the most important thing used to maximize the number of entities in the recorded profiles is the optimization of sample preparation procedure and the data acquisition method. Therefore, ultra high performance liquid chromatography coupled with accurate quadrupoletime- of-flight (Q-TOF) mass spectrometry was used for the comparison of urine metabolomic profiles obtained by the use of various spectral data acquisition methods. The most often used method of registration of metabolomics data acquisition – TOF (MS) was compared with the fast polarity switching MS and auto MS/MS methods with the use of multivariate chemometric analysis (PCA). In all the cases both ionization mode (positive and negative) were studied ...

A method for comparative metabolomics in urine using high resolution mass spectrometry

Journal of chromatography. A, 2016

Developing a workflow for metabolite profiling from biological fluids using mass spectrometry is imperative to extract accurate information. In this study, urine samples from smokers (n=10) and nonsmokers (n=10) were analyzed using an ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) system. For the analysis, two different chromatographic methods [Reversed phase chromatography (RPC) and Hydrophilic interaction liquid chromatography (HILIC)], in two ionization modes (positive and negative) were used. Spiked reserpine (positive ion mode) or taurocholate (negative ion mode) were used for data extraction and normalization. Quality controls (QCs), prepared by pooling urine samples from both smokers and non-smokers (each n=10), were used to assess the reproducibility of the method. The final data output from SIEVE 2.2 after applying a cut-off for QC coefficient of variation (CV) <20% and p-value <0.05 showed 165, 83, 177 and 100 unique compon...

Global metabolic profiling procedures for urine using UPLC���MS

2010

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ~48 h: 1 h for system setup, 8-10 h for sample preparation, 34 h for UPLC-MS analysis and 2-3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.