HPLC Method

HPLC Chromatogram of microcystins from Lake Mokoan

click here for chromatogram (this file is large -you might like to make a cup of coffee!)

Freeze dried Lake Mokoan scum (approx. 300 g) was extracted in Milli-Q water with sonication (3 times). The extract was filtered through Whatman No1 filter paper and ammonium sulphate was added to the filtrate to 40% saturation (0.246 g/ml). The addition of ammonium sulphate clears the extract considerably with little loss of microcystin (data not shown). Ammonium sulphate should not be used, however, for quantitative recovery of microcystins. The suspension was left to settle for 1 hour at 4 oC and filtered again through a Whatman No 1 followed by a 0.45 micron and then a 0.22 micron filter. This crude extract was divided into 50 ml aliquots and stored in the dark at 4oC until use.

A C18 gravity column (Aldrich Cat No - 37,763-5, 10 g) was wetted with 100 ml of HPLC grade methanol and then washed with 50 ml of Milli-Q water. A 50 ml aliquot of crude microcystin extract was added to the column and allowed to adsorb. The column was washed with 100 ml of Milli-Q water to remove water soluble compounds, and then with 50 ml of 30% methanol to remove less polar material. A 70% methanol solution was then added to the column and 2.0 ml fractions collected. Microcystin-LR was confined to the 6th fraction and no further concentration was required.

Fractions were analysed on a Spherisorb ODS-2 C18 analytical column (250 x 4.6 mm, 5 micron) using a 20 minute linear gradient of 15-35% acetonitrile in 8 mM ammonium acetate, with a 10 minute isocratic elution at 35% acetonitrile (8 mM ammonium acetate). Peak detection was achieved using a GBC LC 1250 fast scanning UV/Vis detector set at 238 nm. Eluting peaks were scanned between 200 nm and 300 nm with 1 nm intervals to determine absorbance maxima and minima. Microcystin-LR eluted at approx.16.7 minutes with two other major peaks at 15.8 and 17.5 minutes.