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

Palagama, Dilrukshika S.W.; Baliu-Rodriguez, David; Lad, Apurva; Levison, Bruce S.; Kennedy, David J.; Haller, Steven T.; Westrick, Judy A.; Hensley, Kenneth; Isailović, Dragan

doi:10.1016/j.chroma.2018.08.023

Cited by 28 publications

(17 citation statements)

References 39 publications

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“…The dosages used in the current study approximated the 40 μg/kg NOAEL [1] albeit with a reduced dosing regimen (every other day vs. daily) and total study duration (4 weeks vs. 13 weeks) [1,2]. This chronic low dose regimen resulted in significant increases in both circulating plasma levels of MC-LR as well as increased 24 h urinary excretion of MC-LR in the Lepr db /J mice, as measured by UHPLC-QqQ-MS/MS and HPLC-orbitrap MS as we have described [43]. Interestingly, while there was no mortality or clinically observable MC-LR induced liver injury in the non-NALFD wild type C57Bl/6J group in our study, we did note a decreasing, albeit non-significant trend in survival in the MC-LR exposed Lepr db /J mice, despite similar body and liver weights, as well as the gross morphology of the livers measured in the surviving mice.…”

Section: Discussionmentioning

confidence: 99%

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease

Lad

Breidenbach

et al. 2019

Toxins

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Microcystins are potent hepatotoxins that have become a global health concern in recent years. Their actions in at-risk populations with pre-existing liver disease is unknown. We tested the hypothesis that the No Observed Adverse Effect Level (NOAEL) of Microcystin-LR (MC-LR) established in healthy mice would cause exacerbation of hepatic injury in a murine model (Leprdb/J) of Non-alcoholic Fatty Liver Disease (NAFLD). Ten-week-old male Leprdb/J mice were gavaged with 50 μg/kg, 100 μg/kg MC-LR or vehicle every 48 h for 4 weeks (n = 15–17 mice/group). Early mortality was observed in both the 50 μg/kg (1/17, 6%), and 100 μg/kg (3/17, 18%) MC-LR exposed mice. MC-LR exposure resulted in significant increases in circulating alkaline phosphatase levels, and histopathological markers of hepatic injury as well as significant upregulation of genes associated with hepatotoxicity, necrosis, nongenotoxic hepatocarcinogenicity and oxidative stress response. In addition, we observed exposure dependent changes in protein phosphorylation sites in pathways involved in inflammation, immune function, and response to oxidative stress. These results demonstrate that exposure to MC-LR at levels that are below the NOAEL established in healthy animals results in significant exacerbation of hepatic injury that is accompanied by genetic and phosphoproteomic dysregulation in key signaling pathways in the livers of NAFLD mice.

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Section: Discussionmentioning

confidence: 99%

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease

Lad

Breidenbach

et al. 2019

Toxins

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“…Three solvents with ZnSO 4 (ES-2, ES-3, and ES-4) were chosen for extraction of MCs from liver because zinc binds to an estimated 3000 human proteins [39], including PP1 [40] and PP2A [41], and potentially displaces noncovalently bound MCs or inhibits MCs from binding. ZnSO 4 has also been used to improve extraction of MCs from plasma and serum under acidic conditions [42]. Figure 1 shows that solvent ES-3 (85:15 (v:v) CH 3 CN:H 2 O containing 100 mM ZnSO 4 and 1% FA) was the most effective, with relative abundance ≥ 97% for all seven congeners.…”

Section: Extraction Optimizationmentioning

confidence: 99%

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

Baliu-Rodriguez

Kucheriavaia

Palagama

et al. 2020

Toxins

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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.

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“…Mass spectrometry (MS) is a robust tool to detect and quantify molecules with a high degree of accuracy, resolution and information available for appropriate data processing [1,2]. MS technologies have been widely applied to diverse fields [3], including chemical analysis, forensics [4][5][6], plant sciences [7][8][9], materials [10][11][12][13] and basic life sciences [14][15][16][17][18][19][20]. In these areas of application, there is often a need for chemicals or biomolecules to be separated from a mixture using liquid chromatography (LC) methods prior to MS analysis.…”

Section: Introductionmentioning

confidence: 99%

Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI) for Voltage-Free Liquid Chromatography-Mass Spectrometry

Ranganathan

Suder

et al. 2019

J. Am. Soc. Mass Spectrom.

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Here we report a continuous flow-based ionization method, capillary Vibrating Sharp-edge Spray Ionization (cVSSI), that nebulizes liquid sample directly at the outlet of a capillary without using high-speed nebulization gas or a high electrical field. cVSSI is built upon the recently reported VSSI principle which nebulizes bulk liquid using vibrating sharp-edges. By attaching a short piece of fused silica capillary on top of the vibrating glass slide in VSSI, liquid is nebulized at the outlet of the capillary as the result of the vibration. Utilizing standard 360 μm OD/100 μm ID capillary, cVSSI works with a wide range of flow rates from 1 μL/min to 1 mL/min. The power consumption is as low as 130 mW. ESI-like MS spectra are obtained for small molecules, peptides and proteins. 5 orders of magnitude linear response for acetaminophen solution is achieved with a limit of detection (LOD) of 3 nM. cVSSI is also demonstrated to be compatible with LC-MS analysis. Two LC-MS applications are demonstrated with cVSSI: 1) separation and detection of a mixture of small molecules; 2) bottom-up proteomics using a protein digest. A mixture of 9 common metabolites was appropriately separated and detected using LC-cVSSI-MS. In the bottom-up experiment, 78 peptides were detected using LC-cVSSI-MS/MS with a protein coverage of 100% for cytochrome c, which is comparable with the coverage obtained using LC-ESI-MS. cVSSI offers a means of interfacing LC or other continuous flow-based applications to mass spectrometers with the salient features of voltage-free, flexibility, small footprint and low power consumption.

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

Cited by 28 publications

References 39 publications

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease

Chronic Low Dose Oral Exposure to Microcystin-LR Exacerbates Hepatic Injury in a Murine Model of Non-Alcoholic Fatty Liver Disease

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

Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI) for Voltage-Free Liquid Chromatography-Mass Spectrometry

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