ABSTRACT
Freshwater cyanobacteria Microcystis, Oscillatoria, Anabaena, and Nostoc produce several types of toxins, among which the most commonly detected are the hepatotoxic peptides microcystins. The general structure of the microcystins is cyclo-(D-Ala1-X2-DMeAsp3-Z2-Adda5-D-Glu6-Mdha7), in which X and Z represent variable L-amino acids, D-MeAsp3 is D-erythro-β-methylaspartic acid, Mdha is N-methyldehydroalanine, and Adda is the unusual C20 amino acid, (2S, 3S, 8S, 9S)-3-amino-9-methoxy-2,6,8-trimethyl10-phenyldeca-4(E),6(E)-dienoic acid (Fig. 1).[1] The structural differences in the microcystins mainly depend on the variability of the two L-amino acids (denoted X and Z), and secondarily on the methylation or demethylation of D-MeAsp and/or Mdha.[1] More than 60 microcystins have been isolated from bloom samples and isolated strains of cyanobacteria.[1]
Microcystins have caused the poisoning of wild and domestic animals worldwide, and in 1996, they caused the death of 76 people in Caruaru, Brazil, which was attributed to the use of microcystin-contaminated hemodialysis water.[2] Microcystins, like the welldocumented tumor promoter, okadaic acid, strongly and specifically inhibit the protein phosphatases 1 and 2A and have a tumor-promoting activity in the rat liver.[3] In addition to acute hepatotoxicity, microcystins pose problems to human health-which could result from low-level, chronic exposure to microcystins in drinking water, as suggested by the high incidence of primary liver cancer in the Qidong and Haimen regions of China.[4] In 1998, the World Health Organization (WHO) proposed a provisional guideline level of 1.0 µg/L for microcystin-LR in drinking water.[4]
OVERVIEW
In order to achieve the rapid and precise determination of microcystins in complicated matrices, a systematic procedure is seriously required. This may include screening, sample purification, identification, and quantification processes.[5] Screening is intended to rapidly check for the presence of microcystins in a small amount of sample, through sensitive and simple methods. If a sample proves positive in the screening test, it will be necessary to follow through with sample purification, identification, and quantification analyses. The purpose of sample purification is to eliminate coexisting substances by a simple operation without the loss of any analyte and, considering that the microcystin
concentration may be low, it also enables the enrichment of the analyte. Octadecyl silanized (ODS) silica gel has been extensively employed for this process because it retains microcystins and allows coexisting substances to pass through.[5] A method using ODS silica gel extraction followed by a purification on silica gel has been established to effectively eliminate the coexisting substances in lake water.[6] Although this method has been successfully applied to the analysis of microcystins in lake water samples, it had several problems. The method required a large water sample (5 L) to accumulate the low level of microcystins, resulting in a laborious and time-consuming extraction process. It was also shown that less hydrophobic coexisting substances still remained even after purification on silica gel,[6] indicating that a more effective purification method is required.
