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J Biomol Tech. 2007 February; 18(1): 39.
PMCID: PMC2291838

P114-T HPLC/MS of Pesticides and Metabolites in Brain Tissue using Microwave Sample Preparation and MS Specific Experiments

Abstract

Parkinson’s disease (PD) is a neurodegenerative disease that is difficult to diagnose because there are no specific diagnostic biomarkers. It cannot be diagnosed solely on pathology. A reliable animal model may assist in identification of novel biomarkers. Paraquat (PQ) has been identified both epidemiologically and experimentally as a possible risk factor for PD in humans and mice. It has been demonstrated to preferentially kill dopaminergic neurons in the substantia nigra in mice and rats and its effects have also been found to be persistent with an apparent half-life of ~ 28 days. Its neurotoxic affects have been increased with concomitant doses of MPTP or other pesticides. One of the keys to determine it’s efficacy as a model for PD is to measure its concentration and disposition effectively in the brain tissue of the animal.

In order to measure the concentration of PQ, a mass spectrometric measurement was developed that was sensitive enough to measure low concentrations in small regions of the brain. Tissue samples are typically 10–15 mg in size and the concentrations within the sample are approximately 0.1 to 1.0 ng/mg. In addition to the measurement of PQ, MPTP and its metabolite MPP+ also had to be measured for this work. The MS method had to be able both to identify and quantify each of these analytes, unambiguously. The key was good sample preparation. Being able to separate the analyte from the brain tissue was not straightforward. A microwave accelerated solvent extraction (MASE) method was used to prepare these samples for mass spectrometric analysis. This was coupled with a centrifuge filtration cleanup of extract. We have also used microwave assistance for extraction of chlorinated pesticides (PCBs) and other similar compounds from matrices such as serum and plasma, but the brain tissue extraction was one of the more difficult methods.


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