Analysis of Total Microcystins using LC-MS/MS: Method Improvements and Sample Applications

Microcystin variants (MCs), including microcystin-LR (MC-LR) and related variants, are produced by cyanobacteria and are regulated due to their potential health risks. Current methods for MC analysis predominantly employ LC-MS/MS, limited by the availability of reference materials necessary for quantification. As an alternative method for total MC quantification, the analysis of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), formed by oxidation of the common ADDA structure in MCs, offers economic and practical advantages by avoiding the need for multiple MC reference materials. This method allows total MC quantification across various matrices, independent of specific MC variants. In this study, we evaluated the utility of MMPB-d3 as a surrogate to assess recovery rates throughout the processes critical for MMPB quantification. Comparative analysis with 4-phenylbutyric acid (4-PB) highlighted the stability of MMPB-d3 under oxidative conditions, which is critical for its application as a surrogate or internal standard. The oxidation process was optimized to achieve consistent recovery rates, averaging 101%, ensuring reliable conversion of MCs to MMPB for subsequent analysis. The limit of quantification (LOQ) has been improved due to the large volume injection of up to 0.1 µg/L. Comparison of MMPB concentrations from field samples with conventional LC-MS/MS results showed a significant correlation (r = 0.80, p < 0.005), but with variations in the absolute concentrations. Calculations using a formulation model with MMPB concentrations allowed us to estimate total MC levels, demonstrating the potential of the proposed method to complement existing target measurement techniques.