Quantification of Microcystins and Nodularins

Of all the cyanotoxins, most experience exists with the methodology for the extraction and detection of microcystins. Furthermore, due to its chemical similarity, many of the methods for microcystins will also readily detect nodularin (Lawton et al., 1994b); hence, it will be included in this section. In general, the term “microcystin” will be taken to refer to both these related classes of toxins unless the differentiation is required.

Methods range in complexity and sophistication, spanning the well- established “tried and tested” approaches through to preliminary research findings on novel detection strategies. While many of these novel methods offer exciting opportunities for the future, this chapter focuses on a few of the most relevant approaches for establishing routine methods suitable for the more widely available resources and common requirements.

Extraction Methods for Microcystins and Nodularins

Cyanobacterial Cells

All cell/bloom samples will require extraction as these toxins tend to be retained inside healthy cells. Many extraction protocols for microcystins have been described (e.g., various solvent combinations, cycles of freeze/ thawing, sonication, freeze drying, including combined methods). Among these solvent combinations, aqueous methanol (typically 50-80%; (Barco et al., 2005)) has proven to be very effective for extracting microcystins in face of their wide range of polarities. This solvent can be used for extracting cell pellets once a sample has been centrifuged (and the supernatant discarded or assayed for extracellular microcystin) as well as for extracting cells concentrated on filters. Depending on the volume of cells, around 90% recovery of microcystins (Barco et al., 2005) can be achieved with the first extraction. Often this is sufficient, as this has to be balanced against the further time required for processing a second extraction, as this will typically yield less than 10% of the total microcystin; also if the two extractions are combined, this reduces the detection limit due to the additional volume of solvent used in the second extraction. Extraction time of around 1 h is sufficient for good recovery. With the increased availability of dispersive extractor systems (automated vortex- ers that shake samples vigorously at defined speeds and timed duration), however, extraction can be achieved in just a few minutes and with high reproducibility. Where samples are extracted in centrifuge tubes (typically 1.5-mL microfuge), these can be spun and the supernatant then directly analysed using instrumental methods.

When designing an extraction protocol, it is good to keep it as simple as possible as this will limit error and also potential workplace exposure to microcystins: for example, freeze drying is sometimes reported as a step during sample preparation if a specific dry weight of cells is to be determined, but this can produce powders that are difficult to contain and prone to static charge. Other methods also reported the use of a sonicator probe which may cause cross-contamination, but also produce aerosols.

The use of organic solvents (e.g., methanol) is not compatible with biochemical assays such as ELISA and enzyme inhibition tests. Some ELISA kit manufacturers provide a cell lysis kit, while other analysts have advocated aqueous extraction or dilution to limit the concentration of solvent: for example, a 1 in 10 dilution of a 50% aqueous methanol extract may be tolerated but should be checked with controls for the specific kit used. Since microcystins demonstrate high temperature stability, a brief exposure (5 min) of a small sample (e.g., lmL) to about 80 °C in a water bath followed by centrifugation (13 OOOxg; microfuge) can result in simple solvent- free extraction (Metcalf & Codd, 2000). Extracts can then be diluted in water or buffer as required.

Similarly, high organic solvent content in extracts to be analysed by chromatographic systems needs to be tested for compatibility, in particular when gradient elution is applied that generally starts with hydrophilic conditions.

Water Samples

Some very sensitive methods (e.g., LC-MS/MS) may be able to detect microcystins at environmental concentrations. However, even then it may be desirable to carry out solid phase extraction (SPE) to limit matrix effects.

The most commonly used SPE material is end-capped C18 cartridges, which have demonstrated high recovery and reliability. Some users prefer newer resins (e.g., polymeric phases), which are good where MS is the detector of choice; however, the high recovery of polar compounds by these cartridges can interfere with the more polar microcystins (e.g., microcystin- RR) if detection is with photodiode array (PDA). Several published methods provide a good detail on establishing SPE extraction of microcystins (Lawton et al., 1994b; Triantis et al., 2017c).

Some researchers have developed online sample concentration for fully automated extraction and analysis of microcystins. This is typically an advanced option including LC/MS(MS) and a quite specialised approach; however, it may be desirable particularly for laboratories that need a high throughput, such as those of public authorities monitoring compliance to regulations or of drinking-water suppliers.

Recoveries are best if sample handling is limited, processing time is kept to a minimum and samples are analysed immediately or stored at -20 °C when this is not possible. There is some evidence that samples may change when stored for longer periods of time even at -20 °C, but further studies are required to clarify the extent of this problem. If samples are stored, they should be vortexed if a subsample is to be removed after storage.

Tissue Samples

It is becoming increasingly important to evaluate microcystins in more complex matrixes such as animals that have become intoxicated, fish and aquaculture products that may be contaminated or even plant materials. Much work is still required to fully understand the efficiency of different extraction and toxin recovery protocols. This is particularly challenging for microcystins and nodularins as they are known to bind to proteins; furthermore, microcystins, in particular, can bind covalently to certain protein phosphatases in living cells. Further, the recovery of standards spiked to the material to be tested will only represent unbound toxin recovery efficiency.

While a range of processing strategies with varying degrees of complexity have been used, all of these strategies need to be tested and tailored to the specific requirements of the material to be studied. Simple blending of fresh tissue (mussels) followed by a single aqueous methanolic (80% methanol) extraction was found to give good recoveries in the range of 61-97% for 11 microcystins and nodularin (Turner et al., 2018). Very poor recovery was observed for hydrophobic microcystins when either the samples were acidified or water alone was used. The solvent extracts can be directly analysed by instrumental systems. In contrast, for biochemical tests (ELISA or protein phosphatase inhibition), samples will need to be dried to remove the solvent or sufficiently diluted with water or buffer.

Due to the difficulties in detecting bound microcystin, a method was developed which is designed to cleave part of the microcystin at the first double bond of the ADDA moiety liberating 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB; see Figure 14.3). The assumption behind this approach is that one molecule of MMPB is liberated for each molecule of microcystin, hence predicting the total microcystin content. An oxidation step is used to liberate the MMPB fragment from the parent microcystin, which is assumed to be simpler than digesting the microcystin bound to protein. While this

ADDA moiety of microcystins and nodularins with an indication of the site of MMPB cleavage. For the full structure, see section 2.1

Figure 14.3 ADDA moiety of microcystins and nodularins with an indication of the site of MMPB cleavage. For the full structure, see section 2.1.

method has been used in a range of studies, it is very difficult to determine the degree of sample recovery as spiking will only represent free toxin. Most reported studies currently use MS detection of MMPB (m/z 208); however, this mass is not unique to this oxidation product (ChemSpider shows >6300 compound with this or very similar mass). Others have augmented the method to search for a product ion at 131, which again may not provide confident detection. However, Foss and Aubel (2015) have successfully used the MMPB method in comparison with the ADDA-ELISA, indicating good agreement.

In summary, the detection of microcystins and nodularins in tissue is important for assessing their possible role in animal poisoning or occurrence in food (fish, shellfish, vegetables, etc.), and while no current method will recover the total amount of microcystins, aqueous methanol extraction will give a good indication of whether microcystin is present.

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