Pitfalls in microcystin extraction and recovery from human blood serum

Abstract

a b s t r a c tBackground: Microcystins (MCs) contaminate water bodies due to cyanobacterial blooms all over the world, leading to frequent exposure of humans to MCs through consumption of meat, fish, seafood, blue green algal products and water, accidental ingestion of contaminated water and scum during recre ational activities and inhalation of cyanobacterial aerosols. For monitoring of human exposure, sensitive screening methods are needed. However, during the analytical process of various matrices, such as human serum, some problems appear to regularly occur during sample preparation and storage, leading to MC loss and thus to underestimation of the true MC concentration. The aim of the current study was therefore to assess the pitfalls of the MC extraction method from human serum with more detail. Methods: Six MC congeners (MC LR, YR, RR, LA, LW, LF) and defined equimolar MC mixtures thereof were spiked into human serum, and quantified using the commercially available Adda ELISA subsequent to standard extraction (methanol extraction with subsequent SPE). To detect the potential influence of sample storage and preparation/storage materials, different types of material such as glass, standard polypropylene and surface treated polypropylene were compared. Results: Loss of MC during preparation and storage is largely dependent on (1) the handling of the stored material, (2) the 'surface' of the storage material and (3) the hydrophobicity of the MCs. Conclusions: The pitfalls described for MC analysis with the ELISA are primarily associated with sample preparation and clean up and thus also apply to other analytical techniques for MC detection beyond the ELISA used. It can be concluded that ELISA based methods are suitable tools for the detection of MCs in human sera and other samples.

Adsorption of Ten Microcystin Congeners to Common Laboratory-Ware Is Solvent and Surface Dependent

“…Corroborating observations from previous studies [19,20,21], the lipophilicity/hypdrophilicity of the MC congeners (Table 2) in our study also appeared to be governed by their adsorptivity to non-charged pipetting material, especially as MC congeners dissolved in water, and adsorbed to polypropylene laboratory-ware increasingly with the number of pipetting steps. The microscopic structure of the pipette tips used in this study (Axygen, Maxymum Recovery) is a relatively smooth surface, as protruding polypropylene chains are removed using acid treatment by the manufacturer.…”

“…However, the latter approach not only wrongly assumes that all microcystins have the same toxicokinetics and dynamics [16,17,18], but also presumes that all congeners behave similarly during sample processing and handling. Indeed, previous analyses have demonstrated that losses of different congeners can occur during sample handling [19,20,21,22]. Thus, it is crucial to analyze all congeners present in a certain sample, without loss due to pre-analysis handling.…”

“…Studies have shown that MC adsorption to plastic-ware was dependent on the solvent used (i.e., methanol or acetonitrile concentration) and differed according to the structural characteristics of the MC congeners [19,20,21]. The pH of the solvent can affect adsorption, with acidified methanol leading to less adsorption of MC to GF/C filters, which are commonly used during sample preparation, compared to non-acidified solvents [22].…”

Comparison of two ELISA-based methods for the detection of microcystins in blood serum

“…The observed d ifferential recoveries of the different MCs are therefore not due to a differential recogni tion by the antibodies but rather due to a different behavior during the extract ion procedure. This assumption is supported by the cur rent preliminary data on the effects of storage vessels and is con firmed by very recent data from our laboratory (41 ).…”

Double-sided magnetic molecularly imprinted polymer modified graphene oxide for highly efficient enrichment and fast detection of trace-level microcystins from large-volume water samples combined with liquid chromatography–tandem mass spectrometry

“…Therefore, a rapid, sensitive, and accurate analytical method is of particular importance for the determination of MCs in environmental water samples. Over the past decades, enzyme linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and high-performance liquid chromatography (HPLC) with UV or mass spectrometric (MS) detection are usually applied for analysis of MCs [17][18][19][20][21] . But these methods often provided poor qualitative and quantitative results, particularly in complex matrices.…”