1. Sources and Significance
Microcystins (MCs) and nodularins (NODs), commonly found in surface waters, are cyanobacterial metabolites that can be toxic to animals and humans. The accurate and precise quantitation of MCs and NODs is critical to evaluating risk and to addressing health advisory and provisional guideline thresholds (Table 10110:1). More than 250 MC and 10 or more NOD structurally related congeners (variants) exist,1,2 making a one-method-fits-all approach difficult to achieve. The most common substitutions observed in MCs are the X2 and Y4 amino acids and Z2 in NODs, but modifications are possible on every amino acid, including the Adda (3S-amino-9S-methoxy-2S,6,8S-trimethyl-10-phenyldeca-4E,6E-dienoic acid, Figure 10110:1).3 This is important as many analysis techniques rely on specific structural components, such as the Adda, for identification and quantitation.
To overcome challenges in quantitating constituents with structural variability, broadly specific techniques such as protein phosphatase inhibition assays (PPIAs), enzyme-linked immunosorbent assays (ELISAs), and oxidative cleavage and analysis (e.g., the MMPB method) have been useful for evaluating total MCs and NODs. However, PPIAs are not specific for MCs and NODs because they react with other inhibitors. Also, ELISA congener cross reactivity is variable and depends on the manner in which the antibodies are generated. Although broadly specific, these 3 methods do not allow for congener identification.
To identify and quantitate specific congeners, a method using a technique such as liquid chromatography tandem mass spectrometry (e.g., LC-MS/MS) is required. However, targeted methods are limited by the absence of a sufficient number of reference materials (RMs) for calibration and accurate identification of analytes. In addition, bias is unknown due to a lack of RMs that are certified in concentration (Table 10110:2). This can result in underestimations of MC and NOD presence and risks. Broadly specific tests confer calibration benefits because they use an MC surrogate (e.g., MC-LR) to represent all MCs and NODs. However, the ELISA and oxidative cleavage methods do not fully account for differences in the molecular weight and toxicity of the surrogate compared to other MCs and NODs.
There are calibration benefits to broadly specific tests as they employ a surrogate MC (e.g., MC-LR) to account for all MCs/NODs, but differences in their molecular weight and toxicity are not fully accounted for in ELISA and oxidative cleavage methods. Regardless of the drawbacks of each method, these methods are nonetheless useful for obtaining semi-quantitative to quantitative data for use when assessing the safety of drinking and surface waters. Implement at least two methods to confirm, compare, and provide context to analytical data achieved in one method.