Simultaneous Measurement of Multiple Neurotransmitters

Within the Same Microdialysis Sample by LC-MS/MS

Carter, G. and Mitchell S.N.*
Eli Lilly and Company, Erl Wood Manor, Windlesham, Surrey, GU20 6PH
(*mitchell_stephen@lilly.com)
Introduction
Analytical methods for assaying microdialysate samples tend to be specific for a
single class of neurotransmitter and require the whole sample. Microdialysis
samples are also complex mixtures containing high concentrations of salts that
require chromatographic separation to allow measurements of individual
neurotransmitters, as well as co-eluting interferences can affect the overall
accuracy of the measurements. LC-MS/MS provides a direct measurement of
specific analytes based on their molecular mass and chemical structure.
However, the development of methods which retain and separate polar analytes,
and are suitable for MS, has been a severe technical challenge for the detection
of neurotransmitters. In order to try and resolve polar analytes from co-eluting
components in the sample matrix, and minimise ion suppression, a chemical
derivatisation procedure was explored using dansyl chloride [1].
Dansyl chloride reacts with compounds containing phenolic hydroxyl, and
primary or secondary amine groups. The resultant adducts have a larger
molecular weight, are less polar and more readily retained on the LC system,
away from the low mass to charge range where interferences are generally
higher. Detection of particular product ion fragments then allows a direct
measurement of specific analytes. Moreover, the concomitant detection of drug
(non-dansylated) by LC-MS/MS within the same dialysis sample can allow a
qualitative assessment to be made of the relationship between central exposure
and the evoked response (with the caveat that at typical flow rates used,
recovery will be less than 100%), as well as a retrospective comparison of
relative drug exposure within and between experiments.
Data will be reported showing the utility of this particular assay for measuring
multiple neurotransmitters and their response in the rat hippocampus or mPFCx
to systemic drug administration (e.g. an mGluR5 positive allosteric modulator and
S-(+)-ketamine).
Methods
Dansylation procedure
To each dialysis sample was added: Bis-Tris buffer (pH10), deuterated standards
and 0.1% dansyl chloride. The samples were then mixed and heated for 30 min
at 65C. Samples were then dried under N
2
and re-suspended in 50l mobile
phase (50:50 mobile phase A and B). After a short centrifugation, 40l was
removed and injected onto the LC.
The LC-MS/MS system consisted of an AB Sciex API4000 operated in positive
and negative turbo ion spray mode. A 35ms dwell time was set on the majority of
analytes with MS conditions optimised individually by infusion of the dansylated
derivative. The LC method used a 19.5 min gradient (5-95% acetonitrile),
including a washout and re-equilibration step, and a 2.6m C18 high resolution
HPLC column. Data was acquired using Analyst 1.4.2.
Microdialysis
Male Wistar rats (300-360g; Charles River, U.K.) were surgically anaesthetised
with isoflurane and microdialysis probes (MAB 4.7.4) implanted into the medial
prefrontal cortex (mFFCx) or ventral hippocampus. The following day animals
were connected with a harness and tether to a counter-balanced arm and liquid
swivel. The probes were perfused with artificial CSF (1 or 1.5l/min). Animals
were allowed 90 min to acclimatise to their cage. Typically samples were then
collected every 20 or 30 min for 2 h before administration of drug or vehicle.
Following collection, samples were immediately frozen on dry ice and stored at -
80C pending analysis by LC-MS/MS.
Results and Discussion
The LC-MS/MS dansyl chloride derivatisation method allowed the separation and
detection of a wide range of neurotransmitters and metabolites. The LC method
also allowed detection of (non-dansylated) ACh and drug. The method
compliments other derivatisation techniques that have also been reported for
analysis of multiple neurotransmitter systems in microdialysis and CSF samples
[2,3]. Absolute levels of each analyte in microdialysate samples of either brain
area were in the range reported using standard analytical techniques. Moreover,
the response profile following potassium stimulation (by inclusion in the aCSF
perfusing the probe), or administration of drugs, revealed that the observed
levels were physiologically and pharmacologically relevant.
Two examples showing the utility of detecting multiple neurotransmitter systems
included measuring the response to systemic administration of an mGluR5
positive allosteric modulator (LSN2463359; [N-(1-methylethyl)-5-(pyridin-4-
ylethynyl)pyridine-2-carboxamide]) [4] and the psychostimulant S-(+)-ketamine.
Firstly, injection of LSN2463359 (10 mg/kg p.o.) caused significant increase in
extracellular levels of NA, 5-HT, DA, histamine, GABA and ACh, as well as the
metabolites DOPAC, HVA, MHPG, DHPG and 5-HIAA, compared to the vehicle
control. Extracellular levels of glutamate and glycine were not affected by the
drug. Measurement of LSN2463359 in the dialysis sample suggested that the
extracellular concentration exceeded the in vitro EC50 by 10 fold, implying
appropriate occupancy of the target for the duration of the sampling period.
The administration of ketamine in rodents has been used to model the cognitive
deficits and negative symptoms associated with schizophrenia [5,6]. In the
mPFCx, injection of S-(+)-ketamine (10 mg/kg s.c.) produced a time-dependent
and exposure-dependent increase in extracellular levels of ACh, histamine, NA,
DA, 5-HT, reaching a peak 40 min after injection and returning to baseline 2-3hr
after injection. Glutamate levels also increased, however these changes were
progressive and not correlated with the extracellular concentration of the drug.
The ability to measure multiple neurotransmitter levels in the same dialysis
sample provides a more integrated measure of the extracellular environment
under baseline and evoked conditions. Where possible, the concomitant
measurement of drug also allows an estimation of free concentration and a
correlation to be made between exposure in the extracellular compartment and
the evoked response.
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