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B Quantitative Analysis
Considering the Variables of Quantitative Analysis by LC/MS and MS/MS
In contrast to optical detectors, mass spectrometers can often successfully detect individual
analytes even when two or more compounds coelute as a single chromatographic peak.
Coeluting compounds are ionized in the mass spectrometer, and the parent or fragment ions
specific to the analyte or analytes are detected and measured for quantitative analysis. Because
the mass spectrometer detection is so specific, you can reduce the chromatographic resolution
and shorten the run times.
Using MS/MS for Analyte Detection
To optimize detection of ions by MS/MS, consider the following variables when working with
a mass spectrometer interfaced to an LC:
• Solution chemistry and polarity of the analyte of interest
• Probe selection (H-ESI, ESI, APPI, or APCI)
• Collision energy to fragment parent ions inside the mass spectrometer (optimized by
experiment)
The LC provides a stream of analyte solution (eluate) into the mass spectrometer where the
analyte is detected. You must consider the content of modifiers, salts, and contaminants in the
eluate. Specifically, you must ensure that what goes into the inlet of the mass spectrometer
does not suppress ionization of the compound of interest. Salt concentrations above 10 mM
and strong acids and bases damage the LC column. Modifier concentrations greater than
10 mM are not usually necessary for chromatographic stability and can suppress ionization of
other compounds. For best results, use volatile modifiers whenever possible.
Volatile modifiers include the following:
• Acetic acid
• Ammonium acetate
• Ammonium formate
• Ammonium hydroxide
• Formic acid
• Trifluoroacetic acid
Your analyte solution can contain neutral particles or ions. If the solution contains ions, the
ions can carry a single charge or multiple charges. The number of charges depends on the
structure of the analyte and the composition and pH of the mobile phase. Solvent systems are
generally composed of organic solvents, water, and volatile modifiers. The ionization
characteristics of your analyte influence your decisions about the optimum pH of your solvent
system and the type of probe to use at the interface of the LC and the mass spectrometer.
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