Sensitive determination of microcystins in drinking and environmental waters

Significance of the topic

Objectives and Study Overview

Methodology

• Online SPE uses a small Acclaim PolarAdvantage II (PA2) column (3.0×33 mm, 3 µm) to trap microcystins from 2.5 mL filtered water samples.
• Analytes are eluted by reversing SPE flush direction and applying a gradient of phosphate buffer (22.5 mM KH₂PO₄–2.5 mM K₂HPO₄) and acetonitrile.
• Separation is performed on a PA2 analytical column (3.0×150 mm, 3 µm) with a phosphoric acid (0.05% v/v) and acetonitrile gradient at 40 °C.
• Precise valve-switching times isolate the target analyte fraction before and after the SPE cut, minimizing matrix interferences.

Instrumentation

• Thermo Scientific UltiMate 3000 Dual Gradient Standard LC system including DGP-3600 pump, WPS-3000TSL autosampler, TCC-3200 column compartment, VWD-3400RS UV detector.
• Chromeleon Chromatography Data System for automated control and data processing.
• Orion 420A+ pH meter for buffer preparation.

Main Results and Discussion

• The target-cut method co-elutes all three microcystins from the SPE column in one fraction, reducing co-eluting impurities compared to the traditional SPE flow scheme.
• Optimal valve switching at 7.00 min (start) and 7.45 min (end) ensures complete analyte transfer without loss, even when using different pH mobile phases for SPE (pH 6.0) and separation (pH 2.2).
• Column temperature of 40 °C improved resolution (Rs from 0.50 to 1.94) between microcystins YR and LR.
• Method detection limits were 0.028 µg/L for each analyte, with calibration linearity from 0.1 to 10 µg/L (r>0.999).
• Precision: retention time RSD <0.04%, peak area RSD <1.6% (n=6).

Benefits and Practical Applications

• Fully automated on-line SPE–HPLC workflow reduces manual handling, operator variability, and analysis time.
• Dual-function SPE column provides both analyte concentration and partial cleanup, enhancing selectivity and sensitivity for trace-level microcystins.
• Validated in tap, lake, and bottled spring water with recoveries of 92–110%, demonstrating robust performance across matrices.

Future Trends and Potential Applications

• Coupling the target-cut SPE approach with mass spectrometry for enhanced specificity and lower detection limits.
• Application to other environmental toxins or contaminants using tailored SPE phases and gradients.
• Development of immunoaffinity on-line SPE columns for even greater selectivity in complex matrices.

Conclusion

Reference

1. World Health Organization. Cyanobacterial Toxins: Microcystin-LR in Drinking-Water, 2003.
2. Lee H.S., et al. J. Chromatogr. A 848, 179–184 (1999).
3. Aguete E.C., et al. Talanta 59, 697–705 (2003).
4. Kondo F., et al. Toxicon 38, 813–820 (2000).
5. Tsutsumi T., et al. Food Chem. Toxicol. 38, 593–600 (2000).
6. Thermo Fisher Scientific. Application Note 72491 (2018).