ABSTRACT
A rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5HT) and norepinephrine (NE) in brain microdialysates. The assay has also been utilised for the simultaneous measurement of these neurotransmitters and cocaine in brain dialysates. The neurotransmitters and cocaine were resolved in a single 4-min run using a binary gradient elution profile. The analytes were detected using tandem mass spectrometry in the positive ion electrospray mode. The limits of detection for DA, NE, 5HT and cocaine were 200, 1000, 900 pM and 1 pg ml(-1), respectively.
Subject(s)
Biogenic Monoamines/analysis , Brain Chemistry , Chromatography, High Pressure Liquid/methods , Cocaine/analysis , Mass Spectrometry/methods , Animals , Brain Chemistry/drug effects , Chromatography, High Pressure Liquid/instrumentation , Cocaine/pharmacology , Extracellular Space/drug effects , Extracellular Space/metabolism , Linear Models , Male , Mass Spectrometry/instrumentation , Microdialysis/methods , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.
Subject(s)
1-Methyl-4-phenylpyridinium/analysis , Brain/metabolism , Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/analysis , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/chemistry , 1-Methyl-4-phenylpyridinium/chemistry , 1-Methyl-4-phenylpyridinium/toxicity , Animals , Antiparkinson Agents/pharmacology , Brain/cytology , Brain/drug effects , Brain Chemistry , Male , Mice , Reproducibility of Results , Selegiline/pharmacology , Sensitivity and Specificity , Time Factors , Tissue DistributionABSTRACT
A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.
Subject(s)
Acetylcholine/analysis , Chromatography, High Pressure Liquid/methods , Dialysis Solutions/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Anesthetics, Local/pharmacology , Animals , Brain Chemistry/drug effects , Male , Microdialysis , Neostigmine/analysis , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Sensitivity and Specificity , Tetrodotoxin/pharmacology , Time FactorsABSTRACT
A study has been undertaken to evaluate the usefulness of MALDI Q-TOF data for protein identification. The comparison of MS data of protein digests obtained on a conventional MALDI TOF instrument to the MS data from the MALDI Q-TOF reveal peptide patterns with similar intensity ratios. However, comparison of MS/MS Q-TOF data produced by nanoelectrospray versus MALDI reveals striking differences. Peptide fragment ions obtained from doubly charged precursors produced by nanoelectrospray are mainly y-type ions with some b-ions in the lower mass range. In contrast, peptide fragment ions produced from the singly charged ions originating from the MALDI source are a mixture of y-, b- and a-ions accompanied by ions resulting from neutral loss of ammonia or water. The ratio and intensity of these fragment ions is found to be strongly sequence dependent for MALDI generated ions. The singly charged peptides generated by MALDI show a preferential cleavage of the C-terminal bond of acidic residues aspartic and glutamic acid and the N-terminal bond of proline. This preferential cleavage can be explained by the mobile proton model and is present in peptides that contain both arginine and an acidic amino acid. The MALDI Q-TOF MS/MS data of 24 out of 26 proteolytic peptides produced by trypsin or Asp-N digestions were successfully used for protein identification via database searching, thus indicating the general usefulness of the data for protein identification. De novo sequencing using a mixture of 160/18O water during digestion has been explored and de novo sequences for a number of peptides have been obtained.
