Liquid chromatography–mass spectrometry

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Liquid chromatography–mass spectrometry
Acronym LCMS
Chromatography
Classification
Mass spectrometry
organic molecules
Analytes
biomolecules
Agilent
Bruker
MDS
PerkinElmer
Manufacturers
Shimadzu Scientific
Thermo Fisher Scientific
Varian, Inc.
Waters Corporation
Other techniques
Gas chromatography–mass
Related
spectrometry

Liquid chromatography–mass spectrometry (LC-MS, or alternatively HPLC-MS) is an

analytical chemistry technique that combines the physical separation capabilities of liquid
chromatography (or HPLC) with the mass analysis capabilities of mass spectrometry (MS).
LC-MS is a powerful technique that has very high sensitivity and selectivity and so is useful
in many applications. Its application is oriented towards the separation, general detection and
potential identification of chemicals of particular masses in the presence of other chemicals
(i.e., in complex mixtures), e.g., natural products from natural-products extracts, and pure
substances from mixtures of chemical intermediates. Preparative LC-MS systems can be used
for rapid mass-directed purification of specific substances from such mixtures that are
important in basic research, and pharmaceutical, agrochemical, food, and other industries.

Contents
 1 Liquid chromatography
o 1.1 Flow splitting
 2 Mass spectrometry
o 2.1 Mass analyzer
o 2.2 Interface
 3 Applications
o 3.1 Pharmacokinetics
o 3.2 Proteomics/metabolomics
o 3.3 Drug development
 4 See also
 5 References
 6 Bibliography
Liquid chromatography
Present day liquid chromatography generally utilizes very small particles packed and
operating at relatively high pressure, and is referred to as high performance liquid
chromatography (HPLC); modern LC-MS methods use HPLC instrumentation, essentially
exclusively, for sample introduction. In HPLC, the sample is forced by a liquid at high
pressure (the mobile phase) through a column that is packed with a stationary phase generally
composed of irregularly or spherically shaped particles chosen or derivatized to accomplish
particular types of separations. HPLC methods are historically divided into two different sub-
classes based on stationary phases and the corresponding required polarity of the mobile
phase. Use of octadecylsilyl (C18) and related organic-modified particles as stationary phase
with pure or pH-adjusted water-organic mixtures such as water-acetonitrile and water-
methanol are used in techniques termed reversed phase liquid chromatography (RP-LC). Use
of materials such as silica gel as stationary phase with neat or mixed organic mixtures are
used in techniques termed normal phase liquid chromatography (NP-LC). RP-LC is most
often used as the means to introduce samples into the MS, in LC-MS instrumentation.

Flow splitting

This section does not cite any references or sources. Please help improve this
section by adding citations to reliable sources. Unsourced material may be challenged
and removed. (November 2013)

When standard bore (4.6 mm) columns are used the flow is often split ~10:1. This can be
beneficial by allowing the use of other techniques in tandem such as MS and UV detection.
However splitting the flow to UV will decrease the sensitivity of spectrophotometric
detectors. The mass spectrometry on the other hand will give improved sensitivity at flow
rates of 200 μL/min or less.

Mass spectrometry
Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of
charged particles.[1] It is used for determining masses of particles, for determining the
elemental composition of a sample or molecule, and for elucidating the chemical structures of
molecules, such as peptides and other chemical compounds. MS works by ionizing chemical
compounds to generate charged molecules or molecule fragments and measuring their mass-
to-charge ratios.[1] In a typical MS procedure:

A sample is loaded onto the MS instrument and undergoes vaporization.

The components of the sample are ionized by one of a variety of methods (e.g., by impacting
them with an electron beam), which results in the formation of charged particles (ions).

The ions are separated according to their mass-to-charge ratio in an analyzer by

electromagnetic fields

The ions are detected, usually by a quantitative method.

The ion signal is processed into mass spectra.

Additionally, MS instruments consist of three modules:

An ion source, which can convert gas phase sample molecules into ions (or, in the case of
electrospray ionization, move ions that exist in solution into the gas phase)

A mass analyzer, which sorts the ions by their masses by applying electromagnetic fields

A detector, which measures the value of an indicator quantity and thus provides data for
calculating the abundances of each ion present

The technique has both qualitative and quantitative uses. These include identifying unknown
compounds, determining the isotopic composition of elements in a molecule, and determining
the structure of a compound by observing its fragmentation. Other uses include quantifying
the amount of a compound in a sample or studying the fundamentals of gas phase ion
chemistry (the chemistry of ions and neutrals in a vacuum). MS is now in very common use
in analytical laboratories that study physical, chemical, or biological properties of a great
variety of compounds.

Mass analyzer

There are many different mass analyzers that can be used in LC/MS. Single quadrupole, triple
quadrupole, ion trap, time of flight (TOF) and quadrupole-time of flight (Q-TOF).

Interface

Understandably the interface between a liquid phase technique which continuously flows
liquid, and a gas phase technique carried out in a vacuum was difficult for a long time. The
advent of electrospray ionization changed this. The interface is most often an electrospray ion
source or variant such as a nanospray source; however atmospheric pressure chemical
ionization interface is also used.[1] Various deposition and drying techniques have also been
used such as using moving belts; however the most common of these is off-line MALDI
deposition.[2][3] A new approach still under development called Direct-EI LC-MS interface,
couples a nano HPLC system and an electron ionization equipped mass spectrometer.

Applications
Pharmacokinetics

LC-MS is very commonly used in pharmacokinetic studies of pharmaceuticals and is thus the
most frequently used technique in the field of bioanalysis. These studies give information
about how quickly a drug will be cleared from the hepatic blood flow, and organs of the
body. MS is used for this due to high sensitivity and exceptional specificity compared to UV
(as long as the analyte can be suitably ionised), and short analysis time.

The major advantage MS has is the use of tandem MS-MS. The detector may be programmed
to select certain ions to fragment. The process is essentially a selection technique, but is in
fact more complex. The measured quantity is the sum of molecule fragments chosen by the
operator. As long as there are no interferences or ion suppression, the LC separation can be
quite quick. It is common now to have analysis times of 1 minute or less by MS-MS
detection, compared to over 10 mins with UV detection speed and throughput when using
HPLC-MS/MS systems for drug metabolism and pharmacokinet Henion JD.[4]

Proteomics/metabolomics

LC-MS is also used in proteomics where again components of a complex mixture must be
detected and identified in some manner. The bottom-up proteomics LC-MS approach to
proteomics generally involves protease digestion and denaturation (usually trypsin as a
protease, urea to denature tertiary structure and iodoacetamide to cap cysteine residues)
followed by LC-MS with peptide mass fingerprinting or LC-MS/MS (tandem MS) to derive
sequence of individual peptides.[5] LC-MS/MS is most commonly used for proteomic analysis
of complex samples where peptide masses may overlap even with a high-resolution mass
spectrometer. Samples of complex biological fluids like human serum may be run in a
modern LC-MS/MS system and result in over 1000 proteins being identified, provided that
the sample was first separated on an SDS-PAGE gel or HPLC-SCX.[citation needed]

Profiling of secondary metabolites in plants or food like phenolics can be achieved with
liquid chromatography–mass spectrometry.[6]

Drug development

LC-MS is frequently used in drug development at many different stages including peptide
mapping, glycoprotein mapping, natural products dereplication, bioaffinity screening, in vivo
drug screening, metabolic stability screening, metabolite identification, impurity
identification, quantitative bioanalysis, and quality control.[7]

See also
 Gas chromatography–mass spectrometry
 Capillary electrophoresis–mass spectrometry
 Ion-mobility spectrometry–mass spectrometry

References
1. Arpino, Patrick (1992). "Combined liquid chromatography mass spectrometry. Part
III. Applications of thermospray". Mass Spectrometry Reviews 11: 3.
doi:10.1002/mas.1280110103.
2. Arpino, Patrick (1989). "Combined liquid chromatography mass spectrometry. Part I.
Coupling by means of a moving belt interface". Mass Spectrometry Reviews 8: 35.
doi:10.1002/mas.1280080103.
3. Murray, Kermit K. (1997). "Coupling matrix-assisted laser desorption/ionization to
liquid separations". Mass Spectrometry Reviews 16 (5): 283. doi:10.1002/(SICI)1098-
2787(1997)16:5<283::AID-MAS3>3.0.CO;2-D.
4. "Thermospray liquid chromatography/mass spectrometry determination of drugs and
their metabolites in biological fluids". Analytical chemistry 57 (2): 474–81.
doi:10.1021/ac50001a036.
5. Wysocki VH, Resing KA, Zhang Q, Cheng G (2005). "Mass spectrometry of peptides
and proteins". Methods 35 (3): 211–22. doi:10.1016/j.ymeth.2004.08.013.
PMID 15722218.
 6. Stobiecki, M.; Skirycz, A.; Kerhoas, L.; Kachlicki, P.; Muth, D.; Einhorn, J.; Mueller-
Roeber, B. (2006). "Profiling of phenolic glycosidic conjugates in leaves of
Arabidopsis thaliana using LC/MS". Metabolomics 2 (4): 197. doi:10.1007/s11306-
006-0031-5.
7. Lee, Mike S.; Kerns, Edward H. (1999). "LC/MS applications in drug development".
Mass Spectrometry Reviews 18 (3–4): 187–279. doi:10.1002/(SICI)1098-
2787(1999)18:3/4<187::AID-MAS2>3.0.CO;2-K. PMID 10568041.

Bibliography
 Thurman, E. M.; Ferrer, Imma (2003). Liquid chromatography/mass spectrometry,
MS/MS and time of flight MS: analysis of emerging contaminants. Columbus, OH:
American Chemical Society. ISBN 0-8412-3825-1.
 Ferrer, Imma; Thurman, E. M. (2009). Liquid chromatography-Time of Flight Mass
Spectrometry: Principles, Tools and Applications for Accurate Mass Analysis. New
York, NJ: Wiley. ISBN 978-0-470-13797-0.
 Ardrey, R. E.; Ardrey, Robert (2003). Liquid chromatography-mass spectrometry: an
introduction. London: J. Wiley. ISBN 0-471-49801-7.
 McMaster, Marvin C. (2005). LC/MS: a practical user's guide. New York: John
Wiley. ISBN 0-471-65531-7.
 Wilfried M.A. Niessen, Wilfried M. Niessen (2006). Liquid Chromatography-Mass
Spectrometry, Third Edition (Chromatographic Science). Boca Raton: CRC. ISBN 0-
8247-4082-3.
 Yergey, Alfred L. (1990). Liquid chromatography/mass spectrometry: techniques and
applications. New York: Plenum Press. ISBN 0-306-43186-6.

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