Complementing reversed-phase selectivity with porous graphitized carbon to increase the metabolome coverage in an on-line two-dimensional LC-MS setup for metabolomicsby Karin Ortmayr, Stephan Hann, Gunda Koellensperger

The Analyst





Cite this: DOI: 10.1039/c5an00206k

Received 30th January 2015,

Accepted 22nd March 2015

DOI: 10.1039/c5an00206k

Complementing reversed-phase selectivity with porous graphitized carbon to increase the metabolome coverage in an on-line twodimensional LC-MS setup for metabolomics

Karin Ortmayr,a,b Stephan Hanna and Gunda Koellensperger*b

Efficient and robust separation methods are indispensable in modern LC-MS based metabolomics, where high-resolution mass spectrometers are challenged by isomeric and isobaric metabolites. The optimization of chromatographic separation hence remains an invaluable tool in the comprehensive analysis of the chemically diverse intracellular metabolome. While it is widely accepted that a single method with comprehensive metabolome coverage does not exist, the potential of combining different chromatographic selectivities in two-dimensional liquid chromatography is underestimated in the field. Here, we introduce a novel separation system combining reversed-phase and porous graphitized carbon liquid chromatography in a heart-cut on-line two-dimensional setup for mass spectrometry. The proposed experimental setup can be readily implemented using standard HPLC equipment with only one additional

HPLC pump and a two-position six-port valve. The method proved to be robust with excellent retention time stability (average 0.4%) even in the presence of biological matrix. Testing the presented approach on a test mixture of 82 relevant intracellular metabolites, the number of metabolites that are retained could be doubled as compared to reversed-phase liquid chromatography alone. The presented work further demonstrates how the distinct selectivity of porous graphitized carbon complements reversed-phase liquid chromatography and extends the metabolome coverage of conventional LC-MS based methods in metabolomics to biologically important, but analytically challenging compound groups such as sugar phosphates. Both metabolic profiling and metabolic fingerprinting benefit from this method’s increased separation capabilities that enhance sample throughput and the biological information content of LC-MS data. An inter-platform comparison with GC- and LC-tandem MS analyses confirmed the validity of the presented two-dimensional approach in the analysis of yeast cell extracts from P. pastoris.


Liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) are still the most widely employed analytical platforms in metabolomics.1–6

Despite the rapid evolution of high-resolution mass spectrometry instrumentation and the development of very high throughput methodologies7,8 in the past decade, chromatographic separation is still a prerequisite for reliable metabolite analysis.3 In this context, intracellular metabolites are generally considered a challenging set of analytes, as they are subject to rapid turnover and have widely different physicochemical properties and abundances within the cell.6,9–11

Moreover, typical cell extract samples are of high complexity and give rise to extensive ion suppression and matrix effects.

As a consequence, the analytical method itself must tolerate samples with a dominant matrix whilst providing robustness and delivering the analytes to the mass spectrometer in a suitable solvent.

To date, a single analytical technique with comprehensive coverage of the intracellular metabolome does not exist.11–13

However, several attempts have been made on the establishment of such analytical platforms. For instance, van der Werf reported on the combination of six different complementary

GC-MS and LC-MS methods that enabled the analysis of 380 compounds relevant in microbial metabolomics.14 While such a multi-method approach is presumably the only feasible way to achieve comprehensive coverage across the multiple compound classes in the intracellular metabolome, the collective evaluation of the resulting different data sets remains aDepartment of Chemistry, University of Natural Resources and Life Sciences (BOKU)

Vienna, Muthgasse 18, 1190 Vienna, Austria bInstitute of Analytical Chemistry, University of Vienna, Faculty of Chemistry,

Waehringer Str. 38, 1090 Vienna, Austria.

E-mail:; Fax: +43 1 42779523

This journal is © The Royal Society of Chemistry 2015 Analyst

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View Journal difficult.13,15 This is an aspect of high importance in nontargeted metabolomics, where the aim is the creation of metabolic fingerprints, i.e. representative snapshots of the metabolome in a certain condition. Moreover, each sample has to be analyzed with each method, which has a multiplicative effect both on the total analysis time and the data volume.

All of the aforementioned aspects have propelled efforts towards two-dimensional (2D) chromatography, where orthogonal separation methods are combined to give the maximum peak capacity and resolution. Although the concept of twodimensional chromatography was already introduced decades ago,16–19 its establishment as standard tool in analytical laboratories was hampered by it being more demanding in terms of instrumentation, method development and data analysis.

The potential benefits of such a methodology for the field of metabolomics span from reduced total analysis time to a significant simplification of data processing, all owing to the fact that the information is provided by a single analytical platform. Moreover, higher resolving power and coverage across different classes of metabolites can be achieved. Reversedphase (RP),20–22 ion pair (IP)14,23–26 and hydrophilic interaction liquid chromatography (HILIC)14,25,27–30 are the most commonly applied chromatographic modes in the field of MSbased metabolomics. Moreover, methods employing silica hydride-based,31–33 mixed-mode34,35 and porous graphitized carbon (PGC)36–38 stationary phases have been described in literature. Ion pair LC provides sufficient selectivity to solve a variety of separation problems relevant in metabolomics, yet its practical application suffers from shortcomings in terms of robustness and contamination of the LC-MS instrumentation that limits its use for other purposes.11 While RP and HILIC represent the two most orthogonal chromatographic modes among those relevant for LC-MS based metabolomics, their coupling faces several challenges associated with mobile phase incompatibility. In most cases, suitable interfaces make use of trapping devices or make-up flows to adjust the solvent composition, resulting in an effective dilution at the expense of sensitivity. Nevertheless, the combination of RP and HILIC is invaluable in metabolomics and efficient methodologies have been described.39,40 Ultimately, even such separation systems face limitations with respect to resolving power, as important compound groups (e.g. sugar phosphates) of high relevance in metabolomics remain unresolved. Despite the potential benefits of utilizing other combinations of available chromatographic modes to obtain a higher degree of metabolome coverage, 2DLC and selectivities beyond RP and HILIC are rarely employed in LC-MS based metabolomics.