Since its inception as an instrumental analysis method in the 1990s, capillary
electrophoresis (CE) has evolved as a prime separation tool for charged
compounds. CE is used for the analysis of low molecular weight (LMW)
pharmaceuticals (especially for the separation of enantiomers) and many
other LMW molecules of high biological relevance, and for ion analysis. CE has
become an indispensable tool for the determination of structural homogeneity,
size, charge heterogeneity, and especially glycosylation patterns of
endogenous and recombinant proteins. This is because CE pairs high resolving
power with high sensitivity, and requires minimal sample volumes.
Electrospray ionization mass spectrometry (MS) coupled with liquid
chromatography (LC) became available by the end of the 1980s and has
been a crucial tool in development of new pharmaceuticals. In life science
research, LC with MS detection gathers invaluable structural information,
resulting in unambiguous confirmation of peptide and protein identity,
structure, and heterogeneity.
Not surprisingly, soon after LC/MS became established, successful efforts
to couple CE with MS were reported in the late 1980s. The method became
commercially available with the introduction of the Agilent Technologies (then
Hewlett-Packard) CE/MS interface in 1999. It has since demonstrated wide
versatility and practical applicability in bio-analytical measurements.
Recently, capillary electrophoresis coupled with mass spectrometry (CE/MS)
evolved into an essential tool in development, characterization, and
manufacturing of biopharmaceuticals and in biomarker discovery. Currently
35 to 50 % of new drugs that are under development in the pharmaceutical
industry are proteins or protein-like molecules. These biopharmaceuticals
require more extensive bio-analytical verifications than small molecule drugs
for regulatory approval. Besides that, intellectual property rights (IPR) on firstgeneration biopharmaceuticals are running out, triggering the introduction
of biosimilars. These biosimilars require proof of identity and of structural
equivalence when introduced to the market.
More recently in metabolomics, CE/MS has gained the interest of many
groups for the screening of cationic/anionic and polar metabolites. CE/MS
has been developed into a standard method and has become available as a
These trends have given strong drive growth of the CE/MS market.
Unlike HPLC though, CE has been regarded a method for experts and specialists,
which has inhibited its broad application in bio-analysis and in life science
research. However, the factors that render a CE separation a reproducible,
repeatable, robust, and sensitive method are now well understood. Both CE and
CE/MS have therefore become a valuable separation method providing structural
and compositional information of biomolecules complementary to HPLC/MS.
In coupling CE with MS, another degree of “difficulty” is added through
the interface that is required for their connection when compared to an
It is the intention of this guidebook to help novices to step into the practical
application of CE/MS, and to provide current users with relevant information on
method development and on diagnosis of practical problems. Towards this goal,
the booklet is divided into two parts:
Part 1: Concepts of CE/MS
Description of the concept of CE/MS coupling with detailed emphasis to help
understand the technique and to choose proper starting conditions.
Part 2: Practice of CE/MS
Detailed description of the practice according to recommendations of
Agilent that help to set up the CE instrument, the interface, the MS, and to
provide current users with relevant information on method development and
on diagnosis of practical problems. Also detailed guidelines to do system
performance verification will be given.
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