Introduction to MS Quantitation

and
Modes of LC/MS Monitoring
Introduction
This tutorial applies to the quantitation of small molecules using mass spectrometry in pharmacokinetic
analysis. In addition these very same principles can be applied to the quantitation of peptides and
proteins in biological matrices.
Traditionally before the advent of modern-day mass spectrometry quantitation was accomplished using
HPLC and UV detection. HPLC PK analysis relied on: retention time, peak area and UV spectral
character. Unfortunately the HPLC assay suffered from lack of sensitivity and specificity. We have seen
examples where a molecule was extensively metabolized and yet the retention time and UV spectral
character remained the same as the parent compound. This lack of specificity will from time to time
mislead the investigator. MS characterization is now a vital new tool in pharmacokinetic analysis.
The accepted way of performing mass spec quantitation is by using a mass spectrometer capable of
MS/MS fragmentation. MS/MS used in conjunction with quantitation is commonly accomplished with a
triple quadrupole or ion trap mass spectrometer. The reason MS/MS is required is because many
compounds have the same intact mass. While many researchers use the first dimension of MS to
perform quantitation, that technique again suffers from lack of specificity, especially in very complex
matrices like blood. The second dimension of MS fragmentation in the majority of cases provides a
unique fragment. The combination of the specific parent mass and the unique fragment ion is used to
selectively monitor for the compound to be quantified. Below we will discuss the method for acquiring
and visualizing LC/MS data.
Modes of acquiring and visualizing LC/MS data, or how MS data is represented in an LC/MS run.
Typically the mass spectrometer is set to scan a specific mass range. This mass scan can be wide as in
the full scan analysis or can be very narrow as in selected ion monitoring. A single mass scan can take
anywhere from 10 ms to 1 s depending on the type of scan. Many scans are acquired during an LC/MS
analysis. LC/MS data is represented by adding up the ion current in the individual mass scans and
plotting that "totaled" ion current as an intensity point against time. The resulting plot is a plot that
looks very much like an HPLC UV trace, see Figure 1 below.
Below we will discuss the modes of scanning and acquiring MS data and their relevance to MS
quantitation.
The most common modes of acquiring LC/MS data are :
(1) Full scan acquisition resulting in the typical total ion current plot (TIC)
(2) Selected Ion Monitoring (SIM)
(3) Selected Reaction Monitoring (SRM) or multiple reaction monitoring (MRM). MRM and SRM are
essentially the same experiment.

Full Scan Analysis

Figure 1

The total ion current full mass range plot from a PK analysis is a wild place. The MS total ion current plot
is a plot much like an HPLC UV trace except for the fact that the mass spectrometer can detect many
more components, UV transparent components. The total ion current is a plot of the total ion current in
each MS scan plotted as an intensity point. As a small molecule or peptide elutes from the HPLC column
the relative intensity goes up and a peak appears in the total ion current plot as the points (TIC of each
scan) are plotted against time. Compounds of every mass are plotted in the TIC plot above . Finding the
compound of interest can be difficult since many compounds have the same mass. The intact mass of a
compound is not a unique identifier. Using the data set above, a specific mass can be selectively plotted,
however the sensitivity will be less than what is observed in the SIM experiment described below.
(Note: The term TIC refers to graph of mass scan data collected over time and does not specify a scan
mass range or a type of MS experiment. For example one could have a TIC plot of a SIM or SRM
experiment. However, to simplify matters we will be referring to these plots as SIM and SRM plots
respectively rather than for example SIM TIC plots.)
Selected Ion Monitoring (SIM)
In selected ion monitoring the mass spectrometer is set to scan over a very small mass range, typically
one mass unit. The narrower the mass range the more specific the SIM assay. The SIM plot is a plot of
the ion current resulting from this very small mass range. Only compounds with the selected mass are
detected and plotted. The plots in Figures 1 and 2 can be from the very same sample however the plots
look very different. The reason for this is because the peaks seen in the SIM plot may only be very minor
components in the TIC plot above. The SIM plot is a more specific plot than the full scan TIC plot.
Figure 2

Still, the SIM plot shows a number of peaks leaving us without a unique identifier for our
compound. This is typical for a SIM plot of a very complicated sample, like a serum or plasma
sample. Many compounds have the same mass and in electrospray where there are multiply
charged species the likelihood is even higher for compounds having the same m/z value. The SIM
experiment is more sensitive than the full scan experiment because the the mass spectrometer
can dwell for a longer time over a smaller mass range.
Multiple Reaction Monitoring (MRM) also called Selected Reaction Monitoring (SRM)
Multiple reaction monitoring is the method used by the majority of scientists performing mass
spectrometric quantitation, see Figure 3. MRM delivers a unique fragment ion that can be
monitored and quantified in the midst of a very complicated matrix. SRM plots are very simple,
usually containing only a single peak. This characteristic makes the MRM plot ideal for sensitive
and specific quantitation.

Source
http://www.ionsource.com/tutorial/msquan/intro.htm