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Sensitive and selective liquid chromatography/tandem mass spectrometry methods for quantitative analysis of
Mei-Yi Zhang1, Natasha Kagan, Mei-Li A Sung
1Chemical and Screening Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA. zhangm@wyeth.com
Abstract:
The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson's disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP+), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP+ in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP+ in brain extracts with 0.4M perchloric acid (HClO4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP+ using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP+ established the limits of detection (LOD) (S/N=3) at 0.34pg/mg tissue and 0.007pg/mg tissue (5microl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D.<6%), and sensitive over a range of 0.001-1ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP+ concentrations in vivo. Two reference agents, R-(-) Deprenyl and MK-801, known to alter the concentration of MPP+ in MPTP treated mice were evaluated.
Insights
Researchers developed sensitive methods to measure neurotoxin MPP+ in mouse brains, aiding Parkinson
Area of Science:
- Neuroscience
- Biochemistry
- Analytical Chemistry
Background:
- 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces nigrostriatal pathway degeneration, mimicking Parkinson's disease (PD).
- Quantifying 1-methyl-4-phenyl pyridinium (MPP+), the toxic metabolite of MPTP, is crucial for developing PD therapeutics.
- Accurate measurement of MPP+ in brain tissue is essential for preclinical drug evaluation.
Purpose of the Study:
- To develop and validate sensitive and specific quantitative assays for MPP+ in mouse striatal tissue.
- To compare the performance of reversed-phase and cation-exchange liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for MPP+ determination.
- To apply the developed methods for in vivo monitoring of MPP+ concentrations.
Main Methods:
- Quantitative analysis of MPP+ in mouse striatal tissue using liquid chromatography/tandem mass spectrometry (LC/MS/MS).
- Separation techniques included reversed-phase and cation-exchange chromatography with volatile buffers.
- Optimization of sample preparation (pH neutralization) and electrospray ionization (ESI) conditions.
Main Results:
- Developed sensitive LC/MS/MS methods with limits of detection as low as 0.007 pg/mg tissue (cation exchange).
- Both reversed-phase and cation-exchange methods demonstrated selectivity, precision (<6% R.S.D.), and sensitivity over a wide concentration range.
- Cation-exchange chromatography offered superior sensitivity and pH tolerance compared to reversed-phase chromatography.
Conclusions:
- Sensitive and specific LC/MS/MS methods for MPP+ quantification in mouse brain tissue were successfully developed.
- The cation-exchange LC/MS/MS method is particularly suitable for MPP+ analysis due to its enhanced sensitivity and robustness.
- These validated methods enable effective in vivo monitoring of MPP+ levels and evaluation of potential Parkinson's disease treatments.
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