Extraction and Determination of Trehalose
Trehalose was extracted according to Ferreira et al.
(1997). 0.1 g of frozen tissue (shoots or roots) was
boiled in 2 mL ethanol, evaporated at 60 ºC, the residue
was dissolved in 5 mL of 5 mM H2SO4, centrifuged at
10,000 g for 10 min and filtered. Filtrate was heated in
boiling water bath for 60 min to hydrolyze sucrose in the
extract, since the sucrose retention time is the same as
that of trehalose. The pH was adjusted to 7.0, the solution
was evaporated and the residue was dissolved in distilled
water. The content of trehalose was determined by the
method described by Cizmarik et al. (2004) using HPLC
(Hewlett Packard, HP 1090 liquid chromatograph),
Hypersil, 100 × 3 mm, 3 μm column. The separation was
carried out at 32 °C using a flow rate of 0.8 ml/ min with
acetonitrile: H2O (85: 15) as mobile phase. The elution
was detected with a Diode Array Detector (DAD).
Sucrose was extracted by grinding 0.1 g of tissue, mixing with 10 mg PVP, 80% ethanol. The homogenate has been incubated at 70 °C for 20 min. After centrifugation at 4,000 g for 10 min, the supernatant was separated and the pellet was re-extracted twice with 80 % ethanol at 70 °C for 30 min. The supernatant was pooled
and evaporated. The residue was dissolved in 1 mLof deionized H2O. The content of sucrose was determined using previously mentioned HPLC method (Cizmarik et
al., 2004).
Hi Heshmat
Detection of simple sugars with DAD is not the most sensitive means. Other detectors more routinely used for sugar analysis couple to HPLC would be refractive index (used by Ferreira et al. (1997)) and evaporative light scattering.
The extraction part using 80% to 100% EtOH at 60-70C is pretty much the usual way, and the most likely reason your methods actually then evaporate off all the extracting solvent before redissoluton of the residual samples is to pre-concentrate samples prior to HPLC-DAD. slight changes to the 85:15, MeCN:H2O mobile phase will help with the resolution of sucrose from trehalose.
hope that helps some......
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