The antioxidant capacity measured with ABTS was carried out
according to the method described by Re et al. (1999) with some
modifications. The radicals ABTS·+ were generated by the addition
of 2.45 mM potassium persulfate to an 7 mM ABTS solution prepared
in phosphate-buffered saline (PBS, pH 7.4) and allowing the mixture
to stand in darkness at room temperature for at least 12 h before use.
The ABTS+ stock solution was adjusted with PBS to an absorbance of
0.7 (±0.02) at 734 nm in a 1 cm cuvette at 25 °C (Lambda 25 UV, VIS
spectrophotometer, Perkin Elmer Instruments, Madrid, Spain). An aliquot
of 50 μL of coffee sample diluted with demineralized water
(5:1000 to 15:1000) was added to 2 mL of ABTS+ reagent and the absorbance
was monitored for 18 min at 25 °C. Calibration was performed
with Trolox solution (a water-soluble vitamin E analogue)
and total antioxidant capacity was expressed as micromoles (μmol)
of Trolox per milliliter of coffee brew or fraction.