a b s t r a c t
A high performance liquid chromatography method with refractive index detection (HPLC–RI), for simultaneous
determination of glucose, fructose, sucrose and sorbitol in leaf and/or apple peel samples from
nine apple (Malus domestica Borkh.) cultivars and rootstocks, originating from a germplasm collection,
has been developed and validated. Box–Behnken design of response surface methodology was applied
for the method optimization. The Carbosep Coregel 87H3 column was used under the optimum conditions
predicted: mobile phase of H2SO4 0.005 mol L1 solution, flow rate of 0.3 mL min1 and column
temperature of 35 C. The method was validated for linearity (R2 > 0.99), limits of detection (2.67–
4.83 lgmL1) and quantification (8.9–16.1 lgmL1), precision (%RSD < 5.05) and recovery (93.94–
103.06%) and satisfactory results obtained. The sugars content varied across micropropagated plants
in vitro, plants regenerated after cryostorage, growing trees in vivo, and fruit peel.