Method 544 is used for the analysis six microcystins and nodularin. The six microcystins are microcystin-LA, microcystin-LA, microcystin-LR, microcystin-LY, microcystin-RR, and microcystin-YR.
In this method, the sample is initially filtered, and both the filter and the filtrate are collected. The filter is placed in a solution of methanol containing 20% reagent water and held for at least one hour at -20 ºC. This releases the intracellular toxins from cyanobacteria cells captured on the filter. The methanol solution and the filtrate are processed through a solid phase extraction cartridge. This allows for recovery and concentration of the microcystins and nodularin.
The samples are then injected into a liquid chromatograph, where the analytes are separated based on polarity, and then pass through two mass spectrometers in tandem for identification based on the molecular masses of the fragmented compounds. This analysis provides a highly precise quantitation of these compounds.