A platform using Dionex ion chromatography (IC) coupled to triple quadrupole mass spectrometry (MS) was developed to measure various metabolites in Brassica napus seeds. The IC tandem mass spectrometry (IC-MS/MS) technique provides a good tool to identify and quantify metabolites in a complex matrix.

Here, the author describes an ion-exchange system that provides good separation of the polar metabolities with on-line desalting to allow MS detection. This technique will allow studies of key metabolites that do not separate well on traditional reversed-phase columns. Some ionic metabolites may be positional isomers adjacent to each other in a synthetic pathway and as such, are isobaric. In such cases, MS is not sufficient to differentiate the two compounds. There is clearly a need for adequate separation techniques for isomeric metabolites.

The purpose of this application note is to provide a simple and direct assay of nucleotides and deoxynucleotides in amplification cocktails used for PCR, rolling circle amplifications, DNA and RNA polymerase reactions, and reverse-transcriptase reactions. Nucleotides are separated by anion exchange on the DNAPac PA100 column using gradient elution and detected by absorbance at 260 nm. Resolution of these components provides a means to assay their quality and help identify causes of amplification failure.

The Thermo Scientific HPLC system is applied for the determination of cyclic monophosphates. The separation is performed on a Thermo Scientific Hypercarb HPLC column, with UV detection at 254 nm.

The Thermo Scientific HPLC system is applied for the analysis of 5-monophosphates. The separation is performed on a Thermo Scientific Hypersil GOLD aQ HPLC column, with UV detection at 254 nm.