Thereafter, it was properly mixed with 50 ml of 2 % ammonia in 70 % alcohol that was taken in beaker and it was stand for an hour. After that, the sample was poured into falcon tubes and centrifuge at 2000 rpm for 20 minutes. The liquid portion of centrifuged sample was poured into a rotating flask and evaporated on rotary evaporator at 50°C till concentrated. Thereafter, the concentrated sample was mixed thoroughly with 5 ml dilute acetic acid (3:1). The 30 ml acetic acid is defined value in the method.