Quantititative determination of glycosylated and aglycon isoprenoid cytokinins at sub-picomolar levels by microcolumn liquid chromatography combined with electrospray tandem mass spectrometry.

Microcolumn liquid chromatography (microLC) combined with electrospray tandem mass spectrometry is used for the determination of intact glycosylated cytokinins and the corresponding aglycons at picomole and sub-picomole levels in plant tissue. Routine analysis was done on C8-bonded silica using a methanol-water gradient. Data acquisition was performed by multiple reaction monitoring. Quantification was carried out by using isotopically labelled analogues and applying linear regression to the response factor versus concentration data. For routine analysis a calibration range from 0.5 to 10 pmole injected on-column was used. The limits of detection ranged from 50 to 100 fmole injected on-column. The microLC procedure was used to analyse plant tissue extracts from transgenic homozygote and hemizygote as well as wild-type Nicotiana tabacum species, and cauliflower samples. The data were compared with results obtained by conventional immunoassay and a satisfactory correlation was found. Validation data are presented.