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Brain tissue slices are a common neuroscience model that allows relatively sophisticated analysis of neuronal networks in a simplified preparation. Most experimental methodology utilizes electrophysiological tools to probe these model systems. The work here demonstrates the adaptation of low-flow push–pull perfusion sampling (LFPS) to a brain slice system. LFPS is used to sample from the hippocampus of mouse brain slices. Perfusate amino acid levels are quantified following sampling with capillary electrophoresis. Glutamate was measured from the CA1 region of the hippocampus in slices taken from a cystine-glutamate transporter deletion mutant, xCT−/−, and the background strain C57BL/6J. Sampling is performed over up to 6.5 h with standard tissue slice preparation and experimentation methods. Four amino acids were quantified to demonstrate the ability to perform LFPS and show good agreement with published literature. Perfusate glutamate levels are found to be significantly lower with xCT−/− slices (1.9(±0.5) μM) relative to controls (4.90(±1.1) μM). But, experiments with control slices show a significant decrease in glutamate over the 6 h sampling period that are not seen with xCT−/− slices. Increasing the LFPS sample collection rate during the first 90 min of sampling did not show a sampling artifact in perfusate glutamate content. Sampling immediately following slicing did not show an early increasing glutamate level that would be indicative of a significant contribution from blood or tissue damage. The data presented here show a complementarity to electrophysiological studies of tissue slices. The ability to characterize extracellular fluid chemical content with LFPS in these slices provides an alternative data stream for probing neurochemical signaling networks in brain tissue slices. [ABSTRACT FROM AUTHOR]

Microcystins are toxins produced by many cyanobacteria species, which are often released into waterways during blue-green algal blooms in freshwater and marine habitats. The consumption of microcystin-contaminated water is a public health concern as these toxins are recognized tumor promoters and are hepatotoxic to humans and animals. A method to confirm human exposures to microcystins is needed; therefore, our laboratory has developed an immunocapture liquid chromatography tandem mass spectrometry (LC-MS/MS) method targeting the conserved adda portion of microcystins for the quantitation of a prevalent and highly toxic congener of microcystin, microcystin-LR (MC-LR). An acute exposure method was initially evaluated for accuracy and precision by analyzing calibrators and quality control (QC) samples ranging from 0.500 to 75.0 ng/mL in urine. All calibrators and QC samples characterized were within 15% of theoretical concentrations. An analysis of acutely exposed mouse urine samples using this method identified MC-LR levels from 10.7 to 33.9 ng/mL. Since human exposures are anticipated to result from low-dose or chronic exposures, a high-sensitivity method was validated with 20 calibration curves and QC samples ranging from 0.0100 to 7.50 ng/mL. Relative standard deviations (RSDs) and inaccuracies of these samples were within 15%, meeting United States Food and Drug Administration (FDA) guidelines for analytical methods, and the limit of detection was 0.00455 ng/mL. In conclusion, we have developed a method which can be used to address public health concerns by precisely and accurately measuring MC-LR in urine samples.

A case of an elderly female with suspected paralytic shellfish poisoning (PSP) is presented. The patient shared a meal of recreationally-harvested shellfish with her family and soon began to experience nausea and weakness. She was taken to the local emergency department and then transported to a larger hospital in Anchorage where she was admitted to the intensive care unit with respiratory depression and shock. Her condition improved, and she was discharged from the hospital 6 days later. No others who shared the meal reported symptoms of PSP. A clam remaining from the meal was collected and analyzed for paralytic shellfish toxins (PST) by the Alaska Department of Environmental Conservation Environmental Health Laboratory; the clam tested positive for saxitoxin (STX; 277 μg/100 g), neosaxitoxin (NEO; 309 μg/100 g), multiple gonyautoxins (GTX; 576-2490 μg/100 g), decarbamoyl congeners (7.52-11.3 μg/100 g) and C-toxins (10.8-221 μg/100 g) using high-pressure liquid chromatography with post-column oxidation (AOAC Method 2011.02). Urine from the patient was submitted to Centers for Disease Control for analysis of selected PSTs and creatinine. STX (64.0 μg/g-creatinine), NEO (60.0 μg/g-creatinine) and GTX1-4 (492-4780 μg/g-creatinine) were identified in the urine using online solid phase extraction with HPLC and tandem mass spectrometry. This was the first time GTX were identified in urine of a PSP case from Alaska, highlighting the need to include all STX congeners in testing to protect the public's health through a better understand of PST toxicity, monitoring and prevention of exposures.

Paralytic shellfish toxins (PSTs), including gonyautoxins and saxitoxins, are produced by multiple species of microalgae and dinoflagellates, and are bioaccumulated by shellfish and other animals. Human exposure to PSTs typically occurs through ingestion of recreationally harvested contaminated shellfish and results in nonspecific symptomology. Confirmation of exposure to PSTs has often relied on the measurement of saxitoxin, the most toxic congener; however, gonyautoxins (GTXs), the sulfated carbamate derivatives of saxitoxin, may be present in shellfish at higher concentrations. To improve identification of PST exposures, our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography method to identify GTX1-4 in human urine with tandem mass spectrometry. The reportable range varied for each analyte, with all falling within 0.899 and 250 ng/mL in urine with precision <15% and >85% accuracy as determined for all quality control samples. This new online method quantitates GTX1-4 following exposures to PSTs, supporting the work of public health authorities., (Published 2017. This artricle is a U.S. Government work and is in the public domain in the USA.)

Tetrodotoxin (TTX) is an extremely potent paralytic toxin responsible for yearly illness and death around the world. A clinical measurement is necessary to confirm exposure because symptoms of TTX intoxication cannot be distinguished from other paralytic toxins. Our group has developed an online solid phase extraction hydrophilic interaction liquid chromatography (HILIC) method for the analysis of TTX in human urine with tandem mass spectrometry. The reportable range for the method was 2.80 - 249 ng/mL in urine with precision and accuracy within 15% as determined for all quality control samples. No isotopically-labeled internal standard is available for TTX; thus a surrogate internal standard, voglibose, was investigated to compensate for matrix effects and ionization suppression. However, upon evaluation, voglibose was ineffective for this purpose. This new online method rapidly identifies TTX, facilitating the work of public health authorities and providing support to monitoring programs worldwide., (Published by Elsevier Ltd.)

Fatty acid ethyl esters (FAEE) are known to be a direct alcohol marker and are mainly investigated in hair samples for their ability to be incorporated into this matrix from sebum. The present study used an already developed methodology to provide and confirm information about the use of FAEEs in skin surface lipids as markers of alcohol consumption. The skin surface lipids were collected with Sebutapes(®) from the foreheads of teetotalers, light drinkers, social drinkers, and alcoholics. The samples were analyzed by direct solid-phase microextraction and gas chromatography-mass spectrometry for ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate. Relative FAEE/sebum allowed an evaluation of alcohol consumption. The ranges obtained for relative FAEEs in each category were as follows, teetotalers (0-13.85 pg/mg), light drinkers (11.10-26.80 pg/mg), social drinkers (20.55-86.55 pg/mg), and alcoholics (109.00-1243.40 pg/mg). A social drinker volunteer was monitored during a period of two months. The highest m(FAEE)/m(sebum) were generally detected 7-9 days after the days of high alcohol consumption. From these results, a clear distinction of teetotalers, social drinkers, and alcoholics could be established with the methodology used.