High-Throughput Analysis of Cyanotoxins Using Automated Sample Preparation and SPE
An automated solid-phase extraction (SPE) method has been developed to detect cyanotoxins in water.
A team of scientists from Friedrich Schiller University Jena and Analytik Jena both in Jena, Germany, developed an automated sample preparation method to facilitate the detection of microcystins (MCs) and nodularin-R (Nod), two cyanotoxins commonly associated with harmful algal blooms (1). The approach features a fully automated solid-phase extraction (SPE) workflow in a 96-well plate format that integrates liquid chromatography–mass spectrometry (LC–MS) analysis. The paper was published in the Journal of Chromatography A.
Dense green algae bloom covering lake surface, aquatic plant, surface, lake © Kathy - stock.adobe.com
Cyanobacterial blooms are becoming increasingly prevalent due to climate change and anthropogenic nutrient pollution. These blooms can produce a range of cyanotoxins, including microcystins, which have been linked to severe hepatotoxic effects in humans and wildlife (2). The World Health Organization (WHO) has set provisional guideline values for microcystin-LR (MC-LR) at 1 µg/L for drinking water and 24 µg/L for recreational water (3). However, the detection and quantification of microcystins remain challenging because of their structural diversity and the presence of complex environmental matrices.
LC–MS is the gold standard for microcystin analysis due to its high sensitivity and specificity. However, effective sample preparation is crucial to mitigate matrix interferences and concentrate low-abundance analytes. The approach presented in this study integrates SPE into a miniaturized, high-throughput workflow using a liquid handling robot.
The automated method enables the preparation of 1 to 96 water samples within an hour, improving throughput while reducing manual intervention by 90%. The process eliminates the need for drying and reconstitution steps typically required in traditional SPE protocols, allowing direct LC–MS analysis of eluted samples. This ensures faster turnaround times and minimal sample handling errors. Utilizing only 5 mL of sample for triplicate analysis, the method minimizes solvent consumption and reduces plasticware (1).
The method was validated for the detection of eight major microcystins and nodularin-R in various water matrices, including river, lake, pond, and high performance liquid chromatography (HPLC)-grade water. The results demonstrated excellent accuracy and compliance with recommended guidelines. Four internal standards were incorporated to correct for matrix effects and improve extraction recovery rates.
As a proof of concept, the method was applied to quantify cyanotoxins in two lake water samples and a laboratory culture of Microcystis aeruginosa. Even at low concentrations, the automated workflow identified and quantified microcystins.
The authors state that the automated SPE workflow holds promise for forensic and clinical chemistry applications. Similar protocols have been successfully employed in drug analysis, including fentanyl (4) and morphine (5), demonstrating its broad applicability beyond water safety.
While the current method focuses on environmental water analysis, further enhancements are possible. The integration of triple quadrupole mass spectrometry could enhance sensitivity and selectivity, making the method even more robust for trace-level detection of cyanotoxins. Additionally, future research could explore the adaptation of this workflow for applications such as forensics and medicine (1).
References
(1) Melzer, T.; Wichard, T.; Möller, R.; Ueberschaar, N.; Pohnert, G. Automation and Miniaturization of Solid-phase Extraction for High-throughput Analysis of Cyanotoxins. J. Chrom. A 2025, 1748, 465828. DOI: 10.1016/j.chroma.2025.465828
(2) Learn about Harmful Algae, Cyanobacteria and Cyanotoxins; United States Environmental Protection Agency.https://www.epa.gov/habs/learn-about-harmful-algae-cyanobacteria-and-cyanotoxins#overview4(accessed 2025-04-09).
(3) World Health Organization, Cyanobacterial Toxins: Microcystins, WHO/HEP/ECH/WSH/2020.6, 2020.
(4) Shou, W. Z.; Jiang, X.; Beato, B. D.; Naidong, W. A Highly Automated 96-well Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry Method for the Determination of Fentanyl in Human Plasma. Rapid Commun. Mass Spectrom. 2001, 15, 466–476. DOI: 10.1002/rcm.255
(5) Shou, W. Z.; Pelzer, M.; Addison, T.; Jiang, X.; Naidong, W. An Automatic 96-well Solid Phase Extraction and Liquid Chromatography–Tandem Mass Spectrometry Method for the Analysis of Morphine, Morphine-3-glucuronide and Morphine-6-glucuronide in Human Plasma. J. Pharm. Biomed. Anal. 2002, 27, 143–152. DOI: 10.1016/s0731-7085(01)00497-6
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