Liquid chromatographic–electrospray mass spectrometric determination of 1-methyl-4-phenylpyridine (MPP⁺) in discrete regions of murine brain

Abstract

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is widely used as a neurotoxin in several models of Parkinson’s disease in mice. MPTP is metabolized to 1-methyl-4-phenylpyridinium (MPP+), which is a mitochondrial toxicant of central dopamine (DA) neurons. There are species, strain, and age differences in sensitivity to MPTP. Simultaneous measurement of the MPTP active metabolite MPP+ and dopamine (DA) in the brain would be helpful in mechanistic studies of this neurotoxin. The objective of this study was to develop a liquid chromatography–mass spectrometry (LC/MS) method for analysis of MPTP and MPP+ in brain tissue and correlate these in the same sample with changes in DA measured via HPLC coupled with electrochemical detection. Twenty-five C57BL/6J7 8-week old female mice were used in the study. Mice were given a single subcutaneous injection of MPTP (20 mg/kg) and were sacrificed 1, 2, 4, or 8 h later. Zero time control mice received an injection of 0.9% normal saline (10 ml/kg) and were killed 1 h later. Brains were rapidly harvested and quickly frozen, and microdissected brain regions were placed in 0.1 M phosphate-citric acid buffer containing 20% methanol (pH 2.5). A new LC/MS method was successfully developed that utilized selected reaction monitoring (SRM) of MPP+ m/z 170→127, 170→128, and 170→154 fragmentation for quantitation and area ratios (m/z 127)/(m/z 128) and (m/z 154)/(128) for identity confirmation. A similar SRM strategy from m/z 174 was unable to detect any significant levels of MPTP down to 0.4 ppb. According to this method, MPP+ was detected in the nucleus accumbens (NA) and the striatum (ST), with the levels in the NA being 3-times higher than those in the ST. The advantage of this approach is that the tissue buffer used in this procedure allowed concurrent measurement of striatal DA, thus enabling direct correlation between accumulation of tissue MPP+ and depletion of DA concentrations in discrete regions of the brain.

Recovery of hypothalamic tuberoinfundibular dopamine neurons from acute toxicant exposure is dependent upon protein synthesis and associated with an increase in parkin and ubiquitin carboxy-terminal hydrolase-L1 expression

“…The content of DA in supernatants was determined with high pressure liquid chromatography coupled with electrochemical detection (HPLC-ED) using a Waters 515 HPLC pump (Waters Corporation, Milford, MA) and an ESA Coulochem 5100A electrochemical detector with an oxidation potential of +0.4 V. DA content was quantified by comparing the peak heights of each sample to the peak heights of standards. MPP+ content in supernatants was determined using HPLC coupled with mass spectrometry (MS) as described previously (Lehner et al, 2011). …”

Comparison of the D2 Receptor Regulation and Neurotoxicant Susceptibility of Nigrostriatal Dopamine Neurons in Wild-Type and CB1/CB2 Receptor Knockout Mice

“…Peak heights of standards were used to determine content in samples. MPP+ was measured using HPLC-MS (Lehner et al 2011). Protein content of samples was determined by a Lowry protein assay to normalize sample content.…”

Simultaneous Measurement of Serotonin, Dopamine and Their Metabolites in Mouse Brain Extracts by High-Performance Liquid Chromatography with Mass Spectrometry Following Derivatization with Ethyl Chloroformate

“…The striatal levels of DA metabolites, DOPAC, 3-MT and HVA were also reduced in the MPTP-treated mice. DISCUSSION MPTP, as a neurotoxin in several models of Parkinson's disease in non-human primates and some rodents, such as C57BL/6 mice, is capable of selective destruction of dopaminergic neurons 22) located in substantia nigra. As DA is metabolized to DOPAC, 3-MT and HVA via monoamine oxidase and catechol-o-methyl transferase in the normal state, the damage of substantia nigral cells in MPTP-treated mice is consistently accompanied by a decrease in striatal DA and its metabolites (DOPAC, 3-MT and HVA).…”