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.