Quantification of Glucosinolates by HPLC

A C18 column (Phenomonex, SphereClone 5μ ODS(2)) was equilibrated for 3 h with a mobile phase which consisted of 80 mL (0.02 M) TBA (tetrabutylammonium bromide) and 20 mL ACN (acetonitrile) with detection at 229 nm. The flow rate was set at 1.0 ml/min and separated according to programme for desulfoglucosinolates detailed in Table 3.

Solution A: 100% TBA (0.02 M)

Solution B: 70:30, TBA (0.02 M):acetonitrile

Glucosinolates were quantified using the chromatogram from 229 nm and standard curves were constructed using pure sinigrin (sigma aldrich), glucotropaeolin, glucoraphenin, glucoraphanin, glucerucin, glucobrassicin, gluconasturtiin, sinalbin, progoitrin and glucoiberin (phytoplan).
In the case of glucoraphasatin in R. sativus leaves and glucotropaeolin in B. juncea minor alterations were made to avoid peaks co-eluting. The mobile phase programme for R. sativus leaves was 100% A for 5 min, followed by a 35 min linear gradient to 66% B followed by a 5 min linear gradient to 100% B followed by a 5 min linear gradient to 100% A. For B. juncea leaves, an isocratic 85:15, TBA (0.02 M):acetonitrile mobile phase for 70 min was used.