| Article ID | Journal | Published Year | Pages | File Type |
|---|---|---|---|---|
| 1165160 | Analytica Chimica Acta | 2012 | 6 Pages |
A capillary zone electrophoresis (CZE) method for separation of adenosine and N6-isopentenyladenosine (cytokinin) nucleotides was developed, optimized and validated. Aqueous solutions of several amino acids were evaluated as the background electrolyte constituents. Separation of six nucleotides in less than 20 min with high theoretical plate number (up to 400 000 for isopentenyladenosine triphosphate) was achieved using a 100 mM sarcosine/ammonia buffer at pH 10.0. The detection limits of the CZE-UV method are in the low micromolar range (0.69–1.27 μmol L−1). Good repeatability of migration times (within 1.3%), peak areas (within 1.8%) and linearity (R2 > 0.999) was achieved over the concentration range 5–1000 μmol L−1. The method was used to assay the activity of the recombinant Arabidopsis thaliana isopentenyltransferase 1 (AtIPT1). Baseline separation of isopentenylated nucleotides by CE–ESI-MS using a volatile buffer (30 mM ammonium formate; pH 10.0) was accomplished. The identities of the reaction products – isopentenyladenosine di- and triphosphate were confirmed by HPLC-QqTOF-MS. Dephosphorylation of ATP was observed as a parallel reaction.
Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideHighlight► We describe a capillary electrophoresis based enzymatic assay. ► Cytokinin mono-, di- and triphosphates were separated by CE for the first time. ► The products of enzymatic reactions were unambigously identified by HPLC-QqTOF-MS. ► Undesirable changes in substrates/products were observed.
