Analysis of Microcystins in Urine with 2D-LC-MS/MS – Part III

Significance of the topic

Microcystins are potent cyclic heptapeptide toxins produced by cyanobacteria during freshwater “super blooms.” These toxins resist degradation, threaten aquatic ecosystems, and pose health risks to humans and wildlife when they bioaccumulate in water and seafood. Sensitive, rapid forensic assays to detect microcystins in biological fluids such as urine are needed for exposure assessment and public health monitoring.

Study objectives and overview

The study aimed to develop and optimize a high-throughput workflow combining captive solid-phase extraction (SPE) with two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC-MS/MS) to quantify microcystins LR, RR, and YR in urine. Key goals included minimizing sample preparation time, eliminating evaporation/reconstitution, and achieving reliable recoveries in a complex biological matrix.

Methodology and instrumentation

Sample preparation employed a captive SPE scheme using Waters Oasis HLB cartridges to trap analytes directly from acidified urine. At-column dilution (ACD) enabled large-volume injections (100 µL) of organic extracts without breakthrough.

2D-LC-MS/MS instrumentation

• Waters ACQUITY UPLC with 2D Technology (trap-and-elute configuration with ACD)
• ACQUITY UPLC BEH C18 analytical column (1.7 µm, 2.1 × 50 mm)
• Oasis HLB SPE 3 cc 60 mg cartridge
• Waters Xevo TQ-S triple quadrupole mass spectrometer with electrospray ionization

Chromatographic optimization involved a 6 × 6 grid testing three trap chemistries (C8, C18, HLB), three loading pH values (3, 7, 10), and two elution solvents (acetonitrile or methanol) at low/high pH. Multiple reaction monitoring (MRM) transitions were established for each toxin and an internal standard (nodularin).

Main results and discussion

The 2D grid search identified a single method (C18 trap, pH 7 loading, pH 10 acetonitrile elution) that delivered sharp Gaussian peaks for all three microcystins in aqueous and organic standards. Solubility studies revealed significant signal loss for LR and RR in high-percentage acetonitrile; switching the elution solvent to methanol overcome these losses.

Extraction profiling across sequential elution cuts at pH 3 and pH 10 with acetonitrile, methanol, and acetone characterized the toxins’ retention behaviour. The final SPE protocol comprised:

• Wash 1: 40% methanol, pH 3
• Wash 2: 40% methanol, pH 10
• Elution: 70% methanol, pH 10

This workflow (<30 min total) achieved recoveries of 95% for YR, 94% for LR, and 92% for RR in fortified urine.

Benefits and practical applications

• High throughput: total sample preparation under 30 minutes
• Large-volume organic injection without evaporation or reconstitution
• Robust quantitation of low-level microcystins in urine for forensic and public health investigations
• Applicability to other peptide toxins and complex biological matrices

Future trends and possibilities of use

Further integration of 2D-LC-MS/MS with automated at-column dilution platforms can enhance clinical toxicology assays. Extending captive SPE and method optimization grid designs to additional environmental peptides and emerging toxins will broaden monitoring capabilities. Advances in miniaturized multidimensional LC systems may facilitate on-site testing during bloom events.

Conclusion

The combined captive SPE and 2D-LC-MS/MS approach delivers a fast, sensitive, and reproducible method for detecting microcystins LR, RR, and YR in urine. By eliminating evaporation steps and enabling large-volume organic injections, the workflow streamlines forensic toxin analysis while ensuring high recovery and peak quality.

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