Development of an Efficient Extraction Method to Quantify Microcystin–LR from Natural Microcystis Bloom Samples from the Colombo Lake, Sri Lanka

Abstract

Microcystins are a diverse group of hepatotoxic secondary metabolites produced bycyanobacteria (blue-green algae). Up to date more than 90 different Microcystins wereidentified. Among them, Microcystin-LR (MC-LR) is one of the most toxic and mostcommonly encountered Microcystin variants worldwide. Although detection and extractionmethods for Microcystins have been previously reported, there are no such techniquesavailable in Sri Lanka regarding the water safety monitoring. Therefore in this study, we aimto develop an efficient extraction and quantification method of intracellular and extracellularMicrocystins from natural Microcystis bloom samples. Collection of bloom material from theColombo Lake, Sri Lanka was carried out for a period of six months from January to June2013. Samples were filtered through GF-C filters to retain cyanobacterial cells (intracellularMicrocystins) and extracellular Microcystins to pass through. Intracellular Microcystins (GFCfilters) were extracted repeatedly (x2) in 80% methanol, rotary evaporated and the residuewas reconstituted in 100% methanol. Extracellular Microcystins were extracted by C18 solidphaseextraction cartridges and eluted in 80% methanol. The cool-dry method carried out wassimilar to the GFC-filter extraction; however, the filter was subjected to heating at 550C for15 min followed by cooling (15 min) prior to the 80% methanol extraction. During thecentrifugation method, 1 litre of bloom material was centrifuged (3,000 g, 20 min). The pelletwas extracted in 100% methanol, rotary evaporated (40 0C) and the residue was reconstitutedin 80% methanol. The supernatant was solid phase extracted using C18 cartridges and elutedin 80% methanol. Identification and determination of the intracellular and extracellularMicrocystin concentration were performed by photodiode array – high performance liquidchromatography. Recovery of 17.57±0.73, 15.81±0.44 and 14.66±1.04 ppm of totalmicrocystin-LR (intracellular and extracellular) was recorded for the filter method. Incontrast, total MC-LR extracted by the cool-dry method was 0.48±0.005, 0.32±0.03 and0.22±0.07 ppm. However, compared to other two methods, centrifugation method achievedthe highest total recovery of MC-LR (16.86±2.11, 15.71±1.73 and 13.77±0.04 ppm). Thus, itreveals that the filter method and centrifugation method are the most efficient extractionmethods for MC-LR from natural bloom samples. Further optimization of these methodswould enhance the monitoring and safety measures for the drinking and recreational waterreservoirs in Sri Lanka.