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 pla nt growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeati n, 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 perfor mance liquid chromatographic (HPLC) ion spray mass spectrometry increased t he sensitivity to the low femtomole region. Application of automated capill ary column switching allowed the introduction of large injection volumes in to the HPLC system, Aliquots (25 mu L) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile pha se switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisa tion was used and resulted in 2.5-25 fmol detection limits. Cyclic nucleoti des were separated under ion-pair conditions using tetrabutyl ammonium brom ide as ion-pair reagent and were detected under negative electrospray ionis ation 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. Copyright (C) 1999 John Wi ley & Sons, Ltd.