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How is mass spectrometry used for quantitative analysis?

Researchers are not always interested in obtaining full mass spectra in cases where the compounds are already known. Rather, they may want to confirm the presence of specific substances or measure how much is present. This is commonly done in environmental pollutant work and in pharmacokinetic studies where the goal is quantitation at very low concentrations in complex mixtures. The mass spectrometer is set to monitor only m/z values of ions representative of the molecules of interest so that valuable detection time is not wasted. In many cases, the form of ionization is chosen to favor production of a single type of ion, thus maximizing sensitivity by keeping the ion signal in a single m/z value. In Figure 16, only the signals at m/z 158 and m/z 160 were monitored during a gas chromatographic run. This procedure is known as selected ion monitoring (SIM) and is different from the situation shown schematically in Figure 15 where compete mass spectra are obtained and used to construct selected ion profiles.

A quantitative mass spectral analysis of the dimethyl derivative of 5-fluorouracil in blood plasma is illustrated in Figure 16. In this analysis, the response from the substance of interest is measured relative to that from an internal standard added to the sample. The top trace shows the SIM profile of the molecular ion at m/z 158. The compound of interest produces the center peak. Two minor peaks arise from other sample components that also produce ions at m/z 158. The lower trace is the SIM profile of the same molecule having 15N substituted for the normal 14N at both nitrogen positions. Since both nitrogens are substituted, the signal from its molecular ion is observed at m/z 160, 2 Da higher. Although both the sample and standard have the same retention time, they are detected separately by virtue of their different masses. Standards may be a closely related substance or may be chemically identical but synthesized by substituting a isotope of one of the elements as in this example.