Small Molecule Analysis

SSmall 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, samples are analyzed, and compounds are identified through comparison to chemical standards or theoretical compound databases.

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

For direct injection, 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 chromatographic separation. This technique requires a relatively minimal 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, not suitable for quantitation

For chromatographic separation, samples are run through an UHPLC column (column choice and eluent conditions are dependent on the 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, suitable for quantitative applications when a pure standard is available
Cons – slower data acquisition, potentially requires optimization of separation methodology

In either case the use of chemical standards is required for further 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

Mass spectrum of inset compounds (A), extracted ion chromatograms with peak integration for monoisotopic forms of compounds (B), and representative calibration curves for compounds (C).
(Click on image for expanded view)