Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue

Abstract

A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.

Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging

“…Based on the results above, it appears that the sensitivity and/or analyte extraction to the matrix is not sufficient for the successful imaging of the liver tissue sections of the mice gavaged with MC-LR in 50,100, and 1000 µg/kg concentrations using a MALDI TOF mass spectrometer. In contrast to LC-ESI-MS [18], protonated MC-LR-Cys was not detected in tissue sections in any of MALDI TOF-MS experiments. Protonated MC-LR-Cys was detected by MALDI-TOF-MS when its solution with c = 100 µg/L was analyzed (data not shown), which was a much higher concentration than the minimal concentration of MC-LR solution detected by MALDI-MS (1 µg/L).…”

“…The next step was to compare the intensities of MC-LR ions in a WT vs. a NAFLD mouse. Previously, the extraction of MC-LR from tissue followed by its quantification by LC-MS indicated that a higher amount of MC-LR is accumulated in the liver of NAFLD mouse than in the liver of apparently healthy WT mouse [18]. Figure 4C indicates that protonated MC-LR was detected in liver tissue from NAFLD mouse gavaged with 100 µg MC-LR/kg.…”

“…Damaging effects of MCs in the liver have been monitored by tissue staining followed by light microscopy [10,15]. However, MCs in liver have been studied mostly after their extraction from tissue so information about their localization is lost [16][17][18]. We recently developed a methodology for extraction and LC-MS quantification of the six most common MCs spiked in liver tissue, and MC-LR and its metabolite MC-LR-cysteine (MC-LR-Cys) were detected and quantified in liver tissue derived from mice gavaged with MC-LR [18].…”

“…However, MCs in liver have been studied mostly after their extraction from tissue so information about their localization is lost [16][17][18]. We recently developed a methodology for extraction and LC-MS quantification of the six most common MCs spiked in liver tissue, and MC-LR and its metabolite MC-LR-cysteine (MC-LR-Cys) were detected and quantified in liver tissue derived from mice gavaged with MC-LR [18]. While LC-MS provided sub-ppb limits of quantification in small amounts (~40 mg) of liver, it is important to gain insight into the localization and spatial distribution of MCs in liver tissue since compartmentalization of those toxins within the organ can be visualized and related to their content within tissue.…”

Harmful Algal Bloom Toxicity in Lithobates catesbeiana Tadpoles

“…In addition, microcystins have been known to have significant bioaccumulation within various organ systems of several species, and subsequently cause severe toxicity [ 3 , 4 , 5 , 6 ]. Recent studies, including several of our investigations, have used a combination of Solid Phase Extraction and Liquid Chromatography–Mass Spectrometry (LC–MS) methods to detect various congeners of microcystins in plasma, urine, and liver tissues of mice exposed to chronic, low doses of MC-LR [ 7 , 8 , 9 ] and others have reviewed MC-LR’s accumulation within animals and plants [ 1 , 10 ].…”