Absolute & Relative Quantitation

Absolute Quantification

Absolute quantification uses isotopically-labeled (e.g. 13C, 2H, 15N) homologues of the metabolites to be measured as internal standards. Usually these isotopically-labeled versions are spiked into the samples prior to extraction or added in the final extract. Limits of detection and quantification (LOD and LOQ) are determined, as well as the linearity of response to determine accurate quantification of absolute values.

This type of quantification provides quantitative values, such as nmol/mg, μM, or other units.

aTRAQ labeling
An example of absolute quantification of amino acids using SCIEX's aTRAQ kit. Tagged amino acids are compared to isotopically-labeled amino acids of known concentrations.

Relative Quantification

Relative quantification uses surrogate internal standards that are chemically similar to a class of compounds, but may not be exactly the same. One or a few surrogate internal standards are included to account for many metabolites. These types of standards may or may not be isotopically-labeled depending on the need or availability.

This type of quantification is useful for determining “up/down” changes between sample types, such as wildtype vs. mutant or treated vs. control. This quantification may also be desired if absolute quantification is unnecessary for the research goals. Values are often represented as “Relative amount” or “peak area ratio” or other arbitrary units.

An example of relative quantification in the Targeted Lipidomics Panel. A single internal lipid standard is used to normalize and relatively quantify a class of lipids containing tens to hundreds of lipid molecular species.