Capillary Electrophoresis–Mass
Spectrometry for Metabolomics:
Extracting Chemical Informationfrom Less
By Rawi Ramautar
Metabolomics has become
an important tool for addressing biological and clinical questions (1). The analytical techniques commonly used formetabolomics studies often
require relatively large amountsof biological material, notably
for sample preparation and injection. As many studies havenot been focused on limited amounts of sample material, relatively little effort has been paid to downscale theanalytical workflow for metabolomics.
However, more and more biological questions aredealing with small sample amounts. For example, microfluidic three-dimensional (3D)-cell culture models,
which can mimic physiological tissues by arranging different cell types in a 3D environment within a proper
micro-environment, are increasingly being used to address biological questions. These microfluidic cell culture systems inherently deal with relatively low numbersof cells, for example, those in the range of hundreds
to thousands of cells. Another example concerns theunravelling of the behaviour of a single cell within apopulation of cells, and, as such, obtaining a better understanding on the role of cell heterogeneity in tumour
biology. To address these questions with a metabolomics approach, the development of new microscale
analytical techniques and workflows is needed.

Capillary electrophoresis–mass spectrometry (CE–MS) is an attractive microscale analytical technique foraddressing biological questions inherently dealing withlow amounts of material. In CE, nanolitre injection volumes are often employed from just a few microlitres ofsample. As such, CE–MS is well-adapted for the profiling of especially polar and charged metabolites in tiny
sample amounts, as demonstrated for mouse cerebrospinal fluid (CSF) (2,3).
CSF can only be obtained in a few microlitres underproper experimental conditions. Using only a 1:1 dilution of CSF with water, and therefore fully retaining
sample integrity, more than 300 compounds could beobserved. As only 45 nL of the sample were consumed
from a vial containing only 2 µL of a 1:1 diluted CSF,
the proposed CE–MS approach allows multiple analyses on a single highly valuable mouse CSF sample, enabling repeatability studies and the analysis of the same
sample at different separation conditions to further enhance metabolic coverage. Performing multiple analyses on a single scarcely available biological sample isnot possible with conventional analytical techniques
used in metabolomics.
Alongside the low sample and solvent requirementof CE, the separation mechanism of CE, in which compounds are separated on the basis of their chargeto-size ratio, is fundamentally different from chromatographic-based separation techniques, thereby
providing a complementary view on the composition

of endogenous metabolites present in a given biological sample. In comparison to chromatographic-based
methods, the separation efficiency of CE is very highbecause there is no mass transfer between phases,
and under well-designed experimental conditions only
longitudinal diffusion contributes to band broadening.
An overview of the analytical features of CE–MS for
metabolomics studies, especially for those dealing withlimited sample amounts, will be given during the shortcourse “Advanced CE–MS approaches for metabolomics” on Sunday 16 June at HPLC 2019 in Milan, Italy.
Until now, various research groups have developed
CE–MS approaches for metabolic profiling of limited
sample amounts (4,5), and also for single cell analysis(6–9). Concerning the latter, the metabolomics studies were often focused on the analysis of a relatively
large single non-mammalian cell with a diameter in therange of 100–1000 µm and a cellular sample content
ranging from 100 to 500 nL. To profile metabolites in asingle mammalian cell is clearly an enormous analyticalchallenge; for example, the content of a single HepG2cell is only around 3 pL and a diameter of ~12 µm.
In my group, low-flow CE–MS approaches utilizing asheathless porous tip interface are examined for metabolic profiling of low numbers of mammalian cells using
HepG2 cells as a model system (10). The aim is to beable to profile a wide range of endogenous metabolites
in just a few cells and ultimately a single cell; the latter
will really enable the effect of cell heterogeneity to bestudied—a subject that really matters in key fundamental biological questions. In my keynote lecture at HPLC
2019, I will address the current state-of-the-art of ourCE–MS platform for metabolic profiling of low numbersof mammalian cells by presenting results obtained for
HepG2 cells.
Metabolomics studies dealing with small amountsof biological sample have to critically consider preanalytical steps because adsorption effects, notably withsample volumes far below 1 µL, may result in significant

analyte losses. Moreover, another challenge is how toeffectively get the compounds or the fraction of interest from ultra-small sample amounts into the CE–MSsystem. Various strategies that have been explored in
our laboratory and by others will also be outlined during
HPLC 2019.
References
1. R. Ramautar, R. Berger, J. van der Greef, and T. Hankemeier,Curr. Opin. Chem. Biol. 17, 841­–846 (2013).
2. R. Ramautar, R. Shyti, B. Schoenmaker, L. de Groote, R.J.E.
Derks, M.D. Ferrari, A.M.J.M. van den Maagdenberg, A.M.
Deelder, and O.A. Mayboroda, Anal. Bioanal. Chem. 404, 2895–2900 (2012).
3. R. Ramautar, LCGC Europe 30, 658–661 (2017).
4. E. Sanchez-Lopez, G.S.M. Kammeijer, A.L. Crego, M.L. Marina,
R. Ramautar, D.J.M. Peters, and O.A. Mayboroda, Sci. Rep. 9,806 (2019).
5. A.N. Macedo, S. Mathiaparanam, L. Brick, K. Keenan, T. Gonska, L. Pedder, S. Hill, and P. Britz-McKibbin, ACS Cent. Sci. 3,904–913 (2017).
6. R.M. Onjiko, E.P. Portero, S.A. Moody, and P. Nemes, J. Vis. Exp.
130 (2017).
7. R.M. Onjiko, E.P. Portero, S.A. Moody, and P. Nemes, Anal.
Chem. 89, 7069–7076 (2017).
8. J.X. Liu, J.T. Aerts, S.S. Rubakhin, X.X. Zhang, and J.V. Sweedler,Analyst 139, 5835–5842 (2014).
9. P. Nemes, S.S. Rubakhin, J.T. Aerts, and J.V. Sweedler, Nat. Protoc. 8, 783–799 (2013).
10. W. Zhang, F. Guled, T. Hankemeier, and R. Ramautar, J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 1105, 10–14 (2019).

Rawi Ramautar obtained his Ph.D. on the development of capillary electrophoresis–mass spectrometry
methods for metabolomics from Utrecht University,Netherlands, in 2010. In 2013 and 2017, he received
the prestigious Veni and Vidi research grants from theNetherlands Organization for Scientific Research forthe development of CE–MS approaches for volumerestricted metabolomics. Currently, he is a principal investigator at Leiden University, Netherlands, where hisgroup is developing microscale analytical workflows forsample-restricted biomedical problems.

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