Targeted metabolomics aims to measure multiple chemically characterised or biochemically annotated metabolites (mass tags) in biological samples. By using a priori knowledge to target metabolic pathways or key individual molecules, experiments can be better designed, more sensitive and selective, and hypotheses can be more readily tested than with untargeted metabolomics. Additionally, through the use of isotopically labelled standards to correct for recovery and sample matrix interferences, robust portable quantitative analytical measurements can be obtained.
Targeted MS-based metabolomics requires obtaining isotopically labelled standards for all targeted metabolites which is both impractical and prohibitively expensive. A common strategy, relying on spiking with a few representative isotopically labelled standards, is a compromise that can result in a considerable degree of imprecision. To enable the accurate measurement of target analytes at low parts per billion (ppb) amounts in complex matrices, we have developed and implemented a number of liquid chromatography mass spectrometry (LC-MS) methods based upon MS-probe modification chemistry and stable isotope coding.
In this approach, target compounds (X) are derivatised with a probe molecule (P) that increases their analysis sensitivity (X-P). An internal standard for each compound is similarly created using stable isotope coding by derivatising an analytical standard with a stable isotope labelled (e.g. deuterated) analogue of the probe (IS X-dP). Using this technique only the derivatisation reagent (labelled probe, dP) needs to be isotopically labelled and a mixture of all targeted metabolites can be derivatised with the labelled probe to prepare an internal standard for all targeted metabolite in a single reaction. Measurement of the ratio of the endogenous compound (X-P) relative to its internal standard (IS X-dP) is used for accurate quantification.
We have used this approach to measure metabolites with a range of functional chemistries including short chain fatty acids, neurotransmitters and amino acids in plasma for nutritional studies focused on gut health or cognitive function; and acidic plant hormones in kiwifruit, avocado and model plant systems to help to understand the role of plant hormones in the flowering and development of plants.