# Analysis of Total-Forms of Cyanotoxins Microcystins in Biological Matrices: A Methodological Review
## 1 Introduction
Other organs may be targeted by MCs, including the reproductive organs, kidneys, brain, heart, gastrointestinal tract, or lungs (reviewed in [22]). This specific organo-toxicity can be explained by the tropism of MC towards organs containing organic-anion-transporting polypeptides (OATPs) [23]. The health impact of these toxins is therefore not marginal, and cases of human and animal poisoning are regularly reported (reviewed in [24]). These acute or chronic intoxications can occur after swimming in contaminated environments or after the ingestion of contaminated drinking water or aquatic organisms and cause symptoms of varying intensity ranging from gastroenteritis to kidney and liver failure, sometimes even death (reviewed in [25]). For human and environmental health risk assessments, either cyanobacteria or intracellular and extracellular cyanotoxins can be monitored from water samples. The monitoring of cyanobacterial density or cyanotoxin concentration limits the exposure of the population to cyanotoxins, particularly during swimming activities, but also in the case of the consumption of species that may bioaccumulate these toxins. For this purpose, a panel of techniques is available for the analysis of cyanotoxins in various matrices (water, sediments, and animal tissues). More specifically, the analysis of MCs can concern the free or the total (free plus covalently bound) fraction. The analyses of free forms of MCs are the most widespread and the simplest to perform (reviewed in [26]). These analyses generally consist of an extraction step with different mixtures of organic solvents (methanol, acetonitrile, butanol, acetone, water, chloroform, hexane, and ethanol) that are either pure or diluted according to the protocols. These extraction steps are sometimes followed by a purification step. Three main methods are described in the literature: liquid-liquid extraction (LLE) [27], solid-phase-extraction (SPE) [28; 29; 30], or the use of immunoaffinity columns (IAC) [31; 32; 33; 34]. The extracts obtained from the extraction and purification steps can be analyzed by various methods, including immunological, chemical, or biological methods. ELISA (enzyme-linked immunosorbent assay) tests are the most common immunological methods [35; 36], although other approaches exist, such as aptamers [37; 38; 39; 40] or biosensors (reviewed in [8]). Chemical approaches are also widely used by using equipment including high-pressure liquid chromatography (HPLC) coupled with a diode array detector (HPLC-UV-DAD) [41; 42; 43] or capillary electrophoresis [44; 45; 46; 47; 48]. However, the most used and sensitive chemical method remains liquid (LC-MS) or gas (GC-MS) chromatography coupled with mass spectrometry [29; 49]. The biological approach, including bioassays on rodents [50], plants and algae [51; 52; 53], invertebrates [54; 55], fish and amphibians [56], or in vitro bioassays [57] seems to be less and less used. The protein phosphatase inhibition assay (PPIA), which measures the inhibitory effect of MCs on the activity of PP, is still quite widespread and can be very useful for screening MCs, particularly when coupled with a chemical method such as LC-MS [58]. Complementary to these methods of analysis of free toxins, methods are available to analyze both free and bound (i.e., total) MCs in biological matrices (cyanobacteria or animal tissues). These methods of analysis of the total forms of MCs are still rarely applied as they are more complex to use than the methods of analysis of the free forms of MCs. However, the bound forms can account for the majority of the MCs in a given sample [59; 60]. The aim of this literature review is, therefore, to give an overview of existing techniques for the analysis of total forms of MCs. Sample preparation methods (extraction and purification), analysis methods, and potential matrix effects will be reported.