Development and applications of solid-phase extraction and liquid chromatography-mass spectrometry methods for quantification of microcystins in urine, plasma, and serum

The protocols for solid-phase extraction (SPE) of six microcystins (MCs; MC-LR, MC-RR, MC-LA, MC-LF, MC-LW, and MC-YR) from mouse urine, mouse plasma, and human serum are reported. The quantification of those MCs in biofluids was achieved using HPLC-orbitrap-MS in selected-ion monitoring (SIM) mode, and MCs in urine samples were also quantified by ultra-HPLC-triple quadrupole-tandem mass spectrometry (UHPLC-QqQ-MS/MS) in multiple reaction monitoring (MRM) mode. Under optimal conditions, the extraction recoveries of MCs from samples spiked at two different concentrations (1 μg/L and 10 μg/L) ranged from 90.4% to 104.3% with relative standard deviations (RSDs) ≤ 4.7% for mouse urine, 90.4-106.9% with RSDs ≤ 6.3% for mouse plasma, and 90.0-104.8% with RSDs ≤ 5.0% for human serum. Matrix-matched internal standard calibration curves were linear with R2 ≥ 0.9950 for MC-LR, MC-RR and MC-YR, and R2 ≥ 0.9883 for MC-LA, MC-LF, and MC-LW. The limits of quantification (LOQs) in spiked urine samples were ∼0.13 μg/L for MC-LR, MC-RR, and MC-YR, and ∼0.50 μg/L for MC-LA, MC-LF, and MC-LW, while the LOQs in spiked plasma and serum were ∼0.25 μg/L for MC-LR, MC-RR, and MC-YR, and ∼1.00 μg/L for MC-LA, MC-LF, and MC-LW. The developed methods were applied in a proof-of-concept study to quantify urinary and blood concentrations of MC-LR after oral administration to mice. The urine of mice administered 50 μg of MC-LR per kg bodyweight contained on average 1.30 μg/L of MC-LR (n = 8), while mice administered 100 μg of MC-LR per kg bodyweight had average MC-LR concentration of 2.82 μg/L (n = 8). MC-LR was also quantified in the plasma of the same mice. The results showed that increased MC-LR dosage led to larger urinary and plasma MC-LR concentrations and the developed methods were effective for the quantification of MCs in mouse biofluids.