Principio Lcms

Basics of LC/MS

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Contents
Why Liquid Chromatography/Mass Spectrometry? Instrumentation Ion Sources Electrospray ionization Atmospheric pressure chemical ionization Atmospheric pressure photoionization Mass Analyzers Quadrupole Time-of-flight Ion trap Fourier transform-ion cyclotron resonance (FT-ICR) Collision-Induced Dissociation and Multiple-Stage MS CID in single-stage MS CIDand multiple-stage MS Adapting LC Methods Sample preparation Ionization chemistry Capillary LC/MS and CE/MS Applications Molecular Weight Determination Differentiation of similar octapeptides Determining the molecular weight of green fluorescent protein Structural Determination Structural determination of ginsenosides using MSn analysis Pharmaceutical Applications Rapid chromatography ofbenzodiazepines Detection of degradation products for salbutamol Identification of bile acid metabolites Biochemical Applications Rapid protein identification using capillary LC/MS/MS and database searching Clinical Applications High-sensitivity detection of trimipramine and thioridazine Food Applications Identification of aflatoxins in food Determination of vitamin D3 in poultry feed supplements using MS3Environmental Applications Detection of phenylurea herbicides Detection of low levels of carbaryl in food CE/MS Applications Analysis of peptides using CE/MS/MS 4 6 6 7 8 9 10 10 11 11 12 13 14 14 16 17 17 19 20 20 20 21 22 22 24 24 24 25 26 26 28 28 28 28 30 32 32 32 34 34

Why Liquid Chromatography/ Mass Spectrometry?
Liquid chromatography is a fundamental separation technique in the lifesciences and related fields of chemistry. Unlike gas chromatography, which is unsuitable for nonvolatile and thermally fragile molecules, liquid chromatography can safely separate a very wide range of organic compounds, from small-molecule drug metabolites to peptides and proteins. Traditional detectors for liquid chromatography include refractive index, electrochemical, fluorescence, andultraviolet-visible (UV-Vis) detectors. Some of these generate twodimensional data; that is, data representing signal strength as a function of time. Others, including fluorescence and diodearray UV-Vis detectors, generate three-dimensional data. Three-dimensional data include not only signal strength but spectral data for each point in time.

Mass spectrometers also generate threedimensional data. Inaddition to signal strength, they generate mass spectral data that can provide valuable information about the molecular weight, structure, identity, quantity, and purity of a sample. Mass spectral data add specificity that increases confidence in the results of both qualitative and quantitative analyses.

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CH2CH2CH2NHCOCH3 CH2CH2CH2OH CH2CH2CH(NH2)COOH

% Rel. Abundance

% Rel. Abundancem/z

Figure 1. Two-dimensional abundance data and three-dimensional mass spectral data from a mass spectrometer

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For most compounds, a mass spectrometer is more sensitive and far more specific than all other LC detectors. It can analyze compounds that lack a suitable chromophore. It can also identify components in unresolved chromatographic peaks, reducing the need for perfectchromatography. Mass spectral data complements data from other LC detectors. While two compounds may have similar UV spectra or similar mass spectra, it is uncommon for them to have both. The two orthogonal sets of data can be used to confidently identify, confirm, and quantify compounds.

Some mass spectrometers have the ability to perform multiple steps of mass spectrometry on a single sample. They cangenerate a mass spectrum, select a specific ion from that spectrum, fragment the ion, and generate another mass spectrum; repeating the entire cycle many times. Such mass spectrometers can literally deconstruct a complex molecule piece by piece until its structure is determined.

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MS TIC

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m/z 648

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m/z 376

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