Simple and rapid preparation method of standard samples of pheophytins

A rapid and simple method for preparation of pheophytins from spinach was established. Spinach leaves (500g) were dipped into boiling water for 3min. The pigments were extracted from the leaves with acetone (1000cm3) by ultrasonic vibration for 10 min. Chlorophylls were precipitated with addition of dioxane (150cm3) and water(100cm3), followed by their pheophytinization with 0.1 M hydrochloric acid. Pheophytin a and b were separated by preparative reversed-phase HPLC with an ODS column (24 mm i.d.×360 mm length) and a mobile phase of acetone-acetonitrile (50/50, v/v). The purities of the fractionated pheophytins a and b were checked by analytical reversed-phase HPLC with an ODS column (4.6 mm i.d.×250 mm length) and a mobile phase of acetone-acetonitrile (50/50, v/v). The purity was 98.1% for pheophytin a, with its epimer, i.e. pheophytin a', being the sole impurity. No impurity was detected for pheophytin b. From 500 g of leaves, more than 400 mg and 120 mg of pheophytins a and b were obtained respectively. These yields were 1.53 times as high as those of the conventional methods. Effect of the storage of pheophytins at -5°C was also examined by the analytical reversed-phase HPLC. After 60 days, no purities were changed. However after 180 days, the purities of both pheophytin a and b were down to 91.6%. Hence, it is confirmed that these pheophytins are stable for at least 60 days.