Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase system switching capillary electrospray liquid chromatography tandem mass spectrometry

Using an electrospray tandem mass spectrometer as a concentration-sensitive detector, a method has been developed to quantify femtomole amounts of plant growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeatin, isopentenyladenine and their respective riboside and glucoside analogues) and the second messenger adenosine 3':5'-cyclic monophosphate (3':5'-cAMP). Miniaturisation of the chromatographic setup using capillary high performance liquid chromatographic (HPLC) ion spray mass spectrometry increased the sensitivity to the low femtomole region. Application of automated capillary column switching allowed the introduction of large injection volumes into the HPLC system. Aliquots (25 μL) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile phase switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisation was used and resulted in 2.5-25 fmol detection limits. Cyclic nucleotides were separated under ion-pair conditions using tetrabutyl ammonium bromide as ion-pair reagent and were detected under negative electrospray ionisation conditions. Here a 25 fmol detection limit was determined. Following this approach, cytokinins and 3':5'-cAMP extracted from only mg amounts of apical shoot meristem and chloroplasts obtained from Nicotiana tabacum cv. Petit Havana SR1 were identified and quantified.