Liquid chromatography with mass spectrometry coupling

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Liquid chromatography with mass spectrometry coupling (LC / MS, HPLC-MS) is an analytical method for the separation and determination of molecules by a combination of liquid chromatography (LC or HPLC ) with mass spectrometry (MS). Chromatography is used to separate molecules in a mixture and the subsequent mass spectrometry is used to identify and / or quantify the substances. As a rule, additional detectors such as B. UV / VIS or conductivity detectors .

principle

One of the great difficulties of LC / MS has long been the interface between the chromatography part and the mass spectrometer. At this point, excess sample volume and, above all, the solvent must be removed. As a rule, about 90% of the solution is removed from the chromatography and the remaining material is evaporated with various gas streams. While early devices showed frequent defects and soiling of the interface, there are now devices that are suitable for routine use in the analytical laboratory.

Electrospray ionization (ESI) or chemical ionization at atmospheric pressure (APCI) are used as ionization processes today . Another way to circumvent the problems associated with these methods is to reduce the sample volume in a nano-LC / MS . The chromatographic separation is carried out at a significantly lower flow rate, usually between 200 and 1000 nL / min. This means that there is no need for a carrier gas, which means that contamination occurs less frequently. However, a nano-LC unit is overall more susceptible to other types of interference such as B. Blockages in the separation column .

By coupling the methods, a mass spectrum is available as a result for each point of the chromatogram. This makes it possible to clarify the chemical structure of impurities in mixtures and to present them as impurity profiles. The information or amount of data available due to the large number of mass spectra still presents workstation computers with challenges today. In the LC / MS analysis of proteins in particular, great effort is therefore made to filter out false positive results. Since different LC / MS devices (and coupling methods) often result in incomparable mass spectra, interpretation of the spectra using spectra databases (as is usual in gas chromatography with mass spectrometry coupling (GC / MS)) is only possible to a very limited extent. The elucidation of the chemical structure with LC / MS therefore requires a high degree of experience and extensive knowledge of the possible fragment masses. The use of MSxMS coupling by triple-quad devices or ion traps therefore often pursues the goal of obtaining further information about the substances to be examined. The interpretation of the LC / MS measurements can also be supported by liquid chromatography / nuclear magnetic resonance spectroscopy measurements (LC / NMR).

Individual evidence

  1. ↑ JO Becker, AN Hoofnagle: Replacing immunoassays with tryptic digestion-peptide immunoaffinity enrichment and LC-MS / MS. In: Bioanalysis . Volume 4, Number 3, February 2012, pp. 281-290, ISSN  1757-6199 . doi : 10.4155 / bio.11.319 . PMID 22303832 . PMC 3699856 (free full text).
  2. ↑ I. Finoulst, M. Pinkse, W. Van Dongen, P. Verhaert: Sample preparation techniques for the untargeted LC-MS-based discovery of peptides in complex biological matrices. In: Journal of biomedicine & biotechnology. Volume 2011, 2011, pp. 245291, ISSN  1110-7251 . doi : 10.1155 / 2011/245291 . PMID 22203783 . PMC 3238806 (free full text).
  3. ↑ B. Zhou, JF Xiao, L. Tuli, HW Ressom: LC-MS-based metabolomics. In: Molecular bioSystems. Volume 8, Number 2, February 2012, pp. 470-481, ISSN  1742-2051 . doi : 10.1039 / c1mb05350g . PMID 22041788 . PMC 3699692 (free full text).
  4. ^ E. Ciccimaro, IA Blair: Stable-isotope dilution LC? MS for quantitative biomarker analysis. In: Bioanalysis. Volume 2, Number 2, February 2010, pp. 311-341, ISSN  1757-6199 . doi : 10.4155 / bio.09.185 . PMID 20352077 . PMC 2843934 (free full text).
  5. ↑ Gey, MH, Instrumentelle Analytics and Bioanalytik , pp. 6, 261–288, 298, 329–335; 2nd edition, Springer Verlag, Berlin, 2008