Small Molecule Analysis

Small molecule analysis services involve the accurate mass determination and/or quantification of a given reactions substrate(s) and/or product(s). In this type of experiment, relatively simple sample mixtures (such as in vitro enzymatic reactions) are analyzed, and compounds are identified/quantified through comparison to chemical standards.

Two distinct approaches can be employed in this methodology, “direct injection” and “chromatographic separation”.

For direct injection, relatively pure samples (at low concentrations) are directly run through the mass spectrometer. This methodology aims to measure all compounds present in a given sample without any form of separation. This technique requires a relatively substantial amount of a given sample and is highly susceptible to ion suppression in complex mixtures.

Pros – faster data acquisition
Cons – susceptible to ion suppression, increased background

For chromatographic separation, samples are run through a HPLC column (column choice and eluent conditions dependant on analyte(s) to be observed) prior to detection by mass spectrometry. This methodology aims to separate all compounds in a given sample and measure each independently. This technique requires relatively small amounts of sample and is less susceptible to ion suppression in complex mixtures.

Pros – less susceptible to ion suppression, greater number of analytes typically can be observed
Cons – slower data acquisition, potentially requires optimization of separation methodology

In either case the use of standards is required for validation of compounds due to the potential for isomeric and isobaric species. Additionally, parent ion fragmentation (MS/MS) can be employed for further compound validation in both techniques.

reaction-monitoring

Accurate mass determination of inset compounds (A), extracted ion chromatograms with peak integration (B), and representative calibration curves (C).
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