Anal Bioanal Chem (2006) 385: 1545–1551 DOI 10.1007/s00216-006-0545-8 ORIGINAL PAPER Pavel Babica . Jiří Kohoutek . Luděk Bláha . Ondřej Adamovský . Blahoslav Maršálek Evaluation of extraction approaches linked to ELISA and HPLC for analyses of microcystin-LR, -RR and -YR in freshwater sediments with different organic material contents Received: 17 March 2006 / Revised: 6 May 2006 / Accepted: 11 May 2006 / Published online: 8 July 2006 # Springer-Verlag 2006 Abstract The efficiencies of conventional extraction techniques and analytical methods (HPLC–DAD and ELISA) were investigated for analyses of microcystins (MCs) in sediments. Our results showed several limita- tions. First, the extraction efficiency strongly depends on the extraction solvent, and extraction with 5% acetic acid in 0.2% trifluoroacetic acid (TFA)–methanol was confirmed as being the most appropriate for three different sediments (recovery: 33.1–44.9% of total MCs according to HPLC analyses). Second, the recovery of MCs was affected by the type of sediment but did not clearly correlate with the content of organic carbon. These results suggest that the sorption of MCs onto inorganic materials such as clay minerals is probably a more important process than interactions of the MCs with organic sediment matter. Third, the structure of the MCs is another crucial factor that affects the sorption of MCs and their recovery from sediments. Hydrophilic MC-RR gave much lower recov- eries (20.0–38.8%) than MC-YR (44.1–59.5%) or MC-LR (55.3–77.8%) from three different types of spiked sediments. Recovery results analysed with HPLC–DAD correlated well with ELISA analyses. Further, extraction with 5% acetic acid in 0.2% TFA–methanol was used for analyses of MCs in 34 natural sediment samples collected from Brno reservoir (Czech Republic) from April to October 2005. Concentrations of MCs in sediments ranged from 0.003 to 0.380 μg/g sediment d.m. (ELISA results) or 0.016–0.474 μg/g d.m. (HPLC results). These values are equivalent to 0.63–96.47 μg/L of sediment (ELISA) or 4.67–108.68 μg/L (HPLC), respectively. Concentrations of sediment MCs showed both temporal and spatial variabil- ity, with the highest MC contents observed in the spring (April and May) and the lowest concentrations in July and August. Our results demonstrate the suitability of the methods described here for studying the occurrence, fate and ecological role of MCs in the aquatic environment. Keywords Microcystin . Sediment . ELISA . HPLC–DAD Introduction Microcystins (MCs) and nodularins (NODs) belong to a diverse group of cyclic oligopeptides produced by cyano- bacteria [1, 2]. Over the last few decades, MCs and NODs have become a serious ecological and health issue due to the massive cyanobacterial water blooms that have devel- oped in eutrophied waters worldwide [1, 2]. The toxicity of and risks from some MC variants to vertebrates, including humans, have been studied in detail [3–5], and the World Health Organization recommends a provisional guideline of 1 μg/L for drinking waters for one of the MCs (MC-LR) [6]. On the other hand, the natural functions of these unique non-ribosomally synthesized peptides produced in high amounts in cyanobacterial biomass (up to 10 mg/g d.w.), as well as their fate in the aquatic environment, are poorly understood [7, 8]. MCs are biosynthesized and preferably found inside cyanobacterial cells [9, 10]. The majority of extracellular MCs are probably released after cell lysis and decay, but active transport from growing cyanobacterial cells has also been suggested recently [11]. MCs and NODs are highly stable under laboratory conditions, but they have been shown to be rapidly degrad- ed in the presence of natural microorganisms [12–19] or by indirect photolysis [20, 21]; their fate depends on their structure and is variant-specific [13, 22–26]. However, there are also reports describing continuously elevated concentrations of MCs in natural reservoirs [27], including P. Babica (*) . J. Kohoutek . L. Bláha . O. Adamovský . B. Maršálek Centre for Cyanobacteria and Their Toxins, Institute of Botany, The Academy of Sciences of the Czech Republic & RECETOX, Masaryk University, Kamenice 126/3, 62500 Brno, Czech Republic e-mail: pavel.babica@centrum.cz Tel.: +420-549495967 Fax: +420-549495967