Two millilitres of clear supernatan

Two millilitres of clear supernatant were filtered with 0.2 lm nylon filters (Millipore, Bedford, MA, USA) and then submitted to HPLC analysis. Twenty microlitres of the above mentioned solution were analysed by RP-HPLC, which consisted on a binary LC pump model Prostar 250 (Varian Inc., Palo Alto, CA, USA), an UV/Vis detector and an autosampler. The chromatographic column was a Novapak C18 (250 mm  4.6 mm i.d; 4 lm) from Waters (Milford, MA, USA) thermostated at 25 C. Ascorbic acid was released under isocratic conditions using bi-distilled de-ionised water acidified with sulphuric acid to pH 2.2 as solvent at a flow rate of 0.6 ml/min.