Reprinted from: Brain and Behavior. Raju TR, Kutty BM, Sathyaprabha TN and Shanakranarayana Rao BS (eds.
),
National Institute of Mental Health and Neuro Sciences, Bangalore, India. 2004:134-141.
ESTIMATION OF NEUROTRANSMITTERS IN THE BRAIN BY
CHROMATOGRAPHIC METHODS
Deepti Nair, Ramkumar K, Srikumar BN, Raju TR and Shankaranarayana Rao BS
Chromatography refers to a group of techniques
used to separate complex mixtures on the basis of
different physical interactions between the
individual compounds and the stationary phase
of the system. The basic components in any
chromatography technique are the mobile phase
(gas or liquid), which carries the complex mixture
(sample); the stationary phase (solid or liquid),
through which the mobile phase flows; the column
holding the stationary phase; and the separated
components (eluate).
Modes of separation
a. Adsorption chromatography (liquid-solid
chromatography)
It is based on the competition between the
sample and the mobile phase for adsorptive sites
on the solid stationary phase. There is equilibrium
of solute molecules being adsorbed to the solid
surface and desorbed and dissolved in the mobile
phase. Those molecules, which are most soluble in
the mobile phase, move fastest, whereas those,
which are least soluble, move slowest. The
stationary phase can be either acidic polar (eg, silica
gel), basic polar (eg, alumina) or nonpolar (eg,
charcoal). Commonly, the non-polar organic
eluents for example hexane, dicholoromethane,
and ethyl actetate are used. It can be used to
separate compounds, which are highly soluble in
organic solvents (eg., Fat-soluble vitamins).
b. Partition chromatography
Partition chromatography is also referred to as
liquid-liquid chromatography. Separation of solute
is based on the relative solubility in an organic
(nonpolar) solvent and an aqueous (polar) solvent.
Modern partition chromatography uses pseudo-
liquid stationary phases that are chemically bonded
to the support or high-molecular-weight polymers
that are insoluble in the mobile phase. Partition
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systems are called normal phase when the mobile
phase is less polar than the stationary solvent and
are termed reverse phase when the mobile solvent
is more polar. When the elution strength of the
mobile phase is constant throughout the
separation, it is called isocratic elution and when
varied is called as gradient elution.
In liquid chromatography, the resolution is
proportional to the column length (i.e., the number
of theoretical end plates/unit length). Increasing
the surface area leads to an increase in the number
of theoretical plates. An increase in the surface area
can be achieved by decreasing the particle size but
comes with the disadvantage of increased
resistance to flow, which impairs resolution because
of backpressure. In the recent past, new stationary
phases with small particle size that can withstand
high pressures and offer better resolution have been
developed. This has lead to the development of high
performance liquid chromatography (HPLC).
High Performance Liquid Chromatography
(HPLC)
The term High Performance Liquid
Chromatography (HPLC) was coined to describe
the separation of molecules under high pressure
in a stainless steel column filled with a matrix and
is used for the separation and determination of
organic and inorganic solutes (mol.wt.< 1000)
(Figure 1).
Fundamentals of HPLC
1. Selectivity: It is the ability of a column to
separate two components depending on its
affinity and retention.
2. Capacity factor: It is the ability of a column to
retain a particular compound.
3. Resolution factor: It is the resolving power of a
column to separate two structurally closely
related compounds.
 Figure 1. Basic components of the High Performance Liquid Chromatography (Bender, 1972).
Reverse phase High Performance Liquid
Chromatography
Reverse phase High Performance Liquid
Chromatography (rpHPLC) is a form of partition
chromatography in which the chemically bonded
phase is hydrophobic (nonpolar) and the starting
mobile phase is more polar than the stationary
phase.
The HPLC consists of these basic components:
pump, column, sample injector, detector and
recorder.
Pump: A pump forces the mobile phase through
the column at a much greater velocity. The pump
can be pneumatic syringe type, reciprocating or
hydraulic amplifier. The most widely used pump
is the mechanical reciprocating pump, which is
now used as a multihead pump with two or more
reciprocating pistons. During pumping, the pistons
operate put of phase (180º for two heads, 120º for
three) to provide constant flow.
Column: The stationary phase is packed into
long stainless steel columns. Usually, HPLC is run
at ambient temperature, although columns can be
placed in an oven and heated to enhance the rate
of partition. Fine, uniform column packing results
in much less band broadening but requires
pressure to force the mobile phase through. The
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packing can also can be pellicular (an inert core
with a porous layer), inert and small particles or
macroporous particles. The most common material
used for column packing is silica gel. It is very stable
and can be used as solid packing in liquid - solid
chromatography or coated with a solvent, which
serves as the stationary phase (liquid-liquid).
Reversed phase HPLC is now very popular. The
stationary phase is made up of nonpolar molecules
(eg, octadecyl C-18 hydrocarbon) bonded to silica
gel particles. For this type of column packing, the
mobile phase commonly used is acetonitrile,
methanol, water or any combination of solvents.
Reverse phase columns are stable in the pH range
of 2-7 and at elevated temperatures.
Reversed phase column can be used to separate
ionic, non-ionic and ionizable samples. A buffer is
used to produce the desired ionic characteristics
and pH for the separation of the analyte. Column
packings vary in size (3-20mm), with the smaller
particles used mostly for analytical separations and
the larger ones for preparative separations.
Sample injector: A small syringe can be used
to introduce the sample into the path of the mobile
phase that carries it into the column. The best and
most widely used method is the loop injector. The
sample is introduced into a fixed-volume loop.
When the loop is switched, the sample is placed
into the path of the flowing mobile phase and is
flushed into the column. Loop injectors have high
reproducibility and are used at high pressures.
Detectors: Modern HPLC detectors monitor the
eluate as it leaves the column and ideally, produce
an electronic signal proportional to the
concentration of each separated component.
Spectrophometers that detect absorbances of visible
or ultraviolet light are commonly used.
Fluorescence detectors are also used, because many
biologic substances fluoresce strongly. Another
common HPLC detector is the amperometric or
electrochemical detector. These devices measure
current produced when the analyte of interest is
either oxidized or reduced at some fixed potential
set between a pair of electrodes.
Recorder: It is used to record detector signal versus
the time taken for the mobile phase to pass through
the instrument starting sample injection. The graph is
called a chromatogram. The retention time is used to
identify compounds when compared with standard
retention times run under identical conditions. Peak
area is proportional to the concentration of the
compounds that produced the peaks.
ESTIMATION OF AMINO ACIDS IN THE
BRAIN
There are several methods that were employed
in the past to estimate the levels of different
aminoacids in the brain. The commonly used two
methods are described below.
1. Estimation of glutamate and GABA levels by
multiple development paper chromatography
Principle
This method follows the principle of different
partition coefficients that can be obtained from a
stationary cellulose phase with a mobile solvent
phase for different aminoacids, which aid in their
separation. Extraction and quantification are done
by reacting the aminoacids with ninhydrin
(triketohydrindene hydrate). Ninhydrin reacts with
an aminoacid to produce CO2, NH2 and its lower
aldehyde and ultimately yields a chromophore
known as Ruhemann’s purple with an absorbance
maximum around 515nm.
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Preparation of reagents
1. Solvent: butanol: acetic acid: water (12:3:5):‚
To 60 ml of butanol, 15 ml of acetic acid and 25
ml distilled water are added.
2. 0.25% Ninhydrin: 200 mg of Ninhydrin is
dissolved in 99 ml of acetone. To this solution
1ml of pyridine is added.
3. 0.005% CuSO4 solution: 5 mg of cupric
sulphate is dissolved in 10 ml 75% alcohol.
Standards
a. 2µM glutamate: 2.942 mg of glutamate is
dissolved in 10 ml of distilled water.
b. 2µM GABA: 2.062 mg of GABA is dissolved in
10 ml of distilled water.
Assay procedure
For the estimation of amino acids, the original
method developed by Sadasivudu and Murthy
(1978) is adapted with some modifications in our
laboratory as described below (Nagaraja and
Desiraju, 1993, Shailesh Kumar and Desiraju T,
1990, 1992; Shankaranarayana Rao et al. 1998;
Sunanda et al 2000).
After the dissection of different brain regions,
each region is homogenized in 80% double distilled
ethanol (for every 100mg of the brain tissue, 2ml
of 80% alcohol is used). Homogenates are
transferred to polypropylene tubes and centrifuged
at 1200rpm for 10 min. 1ml of the supernatant is
then transferred into small test tubes and
evaporated to dryness at 70 oC in an oven. The
residue is reconstituted in 100 ml distilled water
and 10 ml is used for spotting on Whatman No.1
Chromatography paper. Standard solutions of
glutamate and GABA at a concentration of 2 mM
are also spotted using an Eppendorf micropipette;
the spots are dried with a hair drier. The
chromatograms are then stitched at the sides and
placed in a chromatography chamber containing
butanol: acetic acid: water (65: 15: 25, V/V) as
solvent. When the solvent front reached the top of
the papers, the papers are removed and dried. A
second run is performed similarly, after which the
papers are dried, sprayed with ninhydrin (0.25%
in acetone with 1% pyridine) and placed in an oven
at 100 oC for 4 min. The portions which carry
glutamate and GABA spots corresponding with the
standard, are cut and eluted with 0.005% CuSO4
in 75% ethanol. Their absorbance is read against a
blank in a LKB- 4050 spectrophotometer (Fig.M26)
at 515nm and the levels are expressed as mmoles/
gram wet weight tissue.
Calculations: The levels of glutamate and GABA
are calculated by using the following formula;
Unknown OD Standard in mg (3µg) 100
A= X X
Standard OD Volume spotted (10µl) x
where,
A = Aminoacid content in umoles/gram wet weight tissue
1000 = Conversion factor for gram wet weight tissue
X = weight of the tissue in grams
2. Estimation of Amino Acids by HPLC Using
Fluorescence Detector with Pre-Column
Derivatization
Principle
Fluorescence detectors are probably the most
sensitive among the existing modern HPLC
detectors. When compounds having specific
functional groups are excited by shorter wavelength
energy and they emit light of higher wavelength
radiation, called fluorescence. Usually, the emission
is measured at right angles to the excitation. Roughly
about 15% of all compounds have a natural
fluorescence. Fluorescence intensity depends on
both the excitation and emission wavelength,
allowing selective detection of some components
while suppressing the emission of others.
Most of the amino acids, upon reacting with o-
phthaldehyde (OPA) at an alkaline pH give rise to
fluorescent derivatives which could be separated
on a C18 column by reverse phase high-pressure
liquid chromatography. Chemical derivitization of
a compound or a mixture of compounds prior to
analysis is generally done for the following reasons:
1. Making a compound suitable for the analysis
2. Improving the analytical efficiency for the
compound
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3. Improving the sensitivity of detection of the
compound
The ability of OPA to react rapidly and
completely at room temperature with primary
amines led to its use as a pre-column derivatization
reagent for amino acids. The resulting highly
fluorescent 1-alkylthio-2-alkylisoindole derivatives
are less polar than their respective amino acids and
can be separated on C-18 reversed-phase HPLC
Reverse phase separations are routinely carried out
in the pH range of 6.0 to 7.5 for optimal
fluorescence. Sensitivities in the femtomole to
picomole range are achievable.
Tissue Preparation and Estimation of Amino acid
Neurotransmitters
The following procedure has been used and
standardized in our laboratory. The brain is rapidly
removed and cooled in an ice-cold buffer. The
different brain regions are micro dissected, weighed
and dropped into chilled 2ml of sodium acetate
buffer with 20% methanol in polycarbonate test
tubes. The tissues are then homogenized in an ice-
cold condition for 2 min. The homogenized tissue
is centrifuged for 30 min at ~5000 rpm. The
supernatant is filtered with 0.2-µm pore size
cellulose acetate membrane and stored in a deep
freezer (-800 C) till it is used for the analysis. Tissue
samples are subjected for HPLC. The
neurotransmitters are separated on a Supelco C-
18 column (15 X 0.46 ID and 3 µm). The
chromatograms are analyzed using Winchrom
data station.
Preparation of the OPA reagent
5mg of o-pthalaldehyde is taken in a 1.5 ml
eppendorf vial. 400µl of methanol, 100µl of borate
buffer and 10ml of β- Mercaptoethanol is added
and vertexed thoroughly. This reagent mixture has
to be protected from light source and stored in the
refrigerated condition.
Preparation of the amino acid standards
The standards for each amino acid are prepared
individually by dissolving a known quantity of
amino acid in warm distilled water to make 10mM
concentration and stored in refrigerated condition.
From this stock, working standards are prepared
by diluting 100mL of stock solution to 4ml buffer
(composition is same as the mobile phase). 20ml of
the working standard is taken for the
derivatization.
Derivatization mixture for injection
20µl of supernatant of the tissue or standard
100µl of sodium tetra borate buffer (pH 9.5)
870µl of mobile phase
10µl of OPA reaction mixture
The derivatization reaction are carried out in
an amber colored vial and this reaction mixture
incubated for 2 min at room temperature. After
completion of the incubation period, 20ml is
injected to HPLC for analysis, which gave the 100-
pico-mole concentration in the case of standard
injection.
The concentration of amino acids is estimated
by isocratic separation. The column is saturated
with the mobile phase before the analysis. The flow
rate of mobile phase is maintained at 0.9 ml/min
so as to generate a backpressure of around 120-
125 kg/cm 2 . The excitation wavelength and
emission wavelength are set at 330 nm and 450
nm, respectively. The best response is obtained
when the excitation and emission slit width is kept
at 5nm. The chromatograms of amino acids is
stored and analyzed after the completion of the
experiment. The samples are analyzed using an
external standard.
Figure 2. The rpHPLC chromatogram profile of the
aspartate, glutamate, serine, glutamine, glycine,
taurine and GABA.
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The rpHPLC chromatogram profile of the amino
acid neurotransmitters viz. aspartate, glutamate,
serine, glutamine, glycine, taurine and GABA are
given in Figure 2. The concentrations of aminoacids
is calculated based on the area under these
respective peaks.
ESTIMATION OF BIOGENIC AMINES AND
THEIR METABOLITES BY HPLC
The levels of biogenic amines and their
metabolites are estimated using several
chromatographic procedures. Two of the following
methods are widely used in the recent past.
1. Estimation of Biogenic Amines by HPLC with
Fluorimetric Detection
The levels of noradrenaline (NA), dopamine
(DA) and 5- hydroxy tryptamine (5-HT) are
estimated by high performance liquid
chromatography with Fluorimetric detection
(HPLC - FD) as developed by Lakshmana and
Raju (1997) and adopted with some
modifications (Lakshmana et al. 1998;
Shankaranarayana Rao et al 1998; Shelke et al.
1997; Sunanda et al 2000; Vijayakumar and
Meti, 1998).
Preparation of Reagents
1. 0.1M perchloric acid (PCA, mol. Wt = 100.46)
is prepared by dissolving 10.8 ml of
commercially available 60% PCA in 1 liter of
quartz distilled water (QDW).
2. 0.02M Sodium acetate (Mol wt = 82.03) is
prepared by dissolving 1.64g of sodium acetate
in 1 litre QDW.
3. 0.1375% W/V Heptane sulfonic acid (HSA,
Mol.wt = 202.24) is prepared by dissolving
1.375g of HSA in 1 litre QDW.
4. 16% V/V methanol is prepared by mixing 160
ml of methanol in 840ml of QDW.
5. 0.1mM ethylene diamine tetra acetic acid
(EDTA ; mol, wt = 372.2) is prepared by
dissolving 37.2 mg of EDTA, disodium salt in 1
litre of QDW.
Chromatographic conditions
The mobile phase consists of sodium acetate
(0.02M), methanol (16%, V/V), heptane sulfonic
acid (0.1375%), EDTA (0.2mM) and dibutylamine
(0.01%, V/V). The pH is adjusted to 3.92 ± 0.01
with orthophosphoric acid and filtered through
0.45m membrane filter and degassed. The flow rate
is set to 0.9ml per minute so as to yield a pressure
of 125-130 kg/cm 2. The column is washed first
with quartz-distilled water and then with 80%
methanol. After- wards, it is equilibrated with the
sodium acetate buffer, for atleast 24 hours before
injecting standards/samples.
Sample preparation
Following decapitation the heads are collected
into ice cold 0.1M PCA. Immediately the brains
are removed and the desired brain regions are
dissected. The tissues are weighed and
homogenized in 1ml of PCA (0.1M). After
centrifugation at 14,000rpm for 15 min at 4oC, the
supernatant is filtered through 0.45 µm membranes
and 100 ml of the filtrate is injected into the HPLC
pump. After separation, NA, DA, Isoproterenol
(IP) and 5-HT are detected at the excitation
wavelength of 280 nm and an emission wavelength
of 315nm, while keeping the slit width 10/10.
All separations are isocratic and are carried out
at room temperature. The peaks of the amines are
plotted by a recorder on a chart paper running at
a speed of 2.5 mm/minute. The biogenic amine
peaks are identified by comparing the retention
period of the peaks with that of external standards.
The external standards are always injected before
injecting the tissue samples.
Standards
Stock solutions of standards (1 mg/ml) are
prepared in 0.1N Hydrochloric acid and are stored
at -20 o C and used within two weeks of
preparation. The working standard solution is
prepared freshly in 0.1 M PCA for each experiment.
The amount of standard is 3ng per 100 ml of
injection volume for each NA, DA, IP and 5-HT.
Monoamine peaks are identified by comparing their
retention time in the sample (tissue extracts)
139
solution with that of standard solution and also
by superimposing the chromatograms of the
sample spikes with and without known amount
of the standards.
Calculations
Monoamine contents in the tissues are quantified
by comparing their peak heights with that of
known standards after correcting for the recovery
of internal standard. The fluorescence units
obtained from brain tissue samples are converted
into nanograms per gram wet tissue using the
following formula (Lakshmana and Raju, 1997).
IS AT 3 1000
X X X
IT AS 100 X
Where,
IS = Fluorescence units for 3 ngs of isoproterenol
standard.
IT = Fluorescence units for 3 ngs of isoproterenol
in tissue
AT = Fluorescence units of amine in the tissue
AS = Fluorescence units of 3 ngs of amine in
standard
3 = ngs of amine standard injected.
100 = volume of sample injected in ml
1000 = Conversion factor for gram wet weight
tissue
X = weight of tissue in grams.
Minimum detection limits
The high efficiency of the HPLC separation for
biogenic amines combined with the sensitivity of
fluorometric monitoring enable the detection at
picogram range. With these chromatographic
conditions, it is possible to detect the levels of NA,
DA, isoproterenol and 5-HT at 250 pg range, in
less than 30 min in rat brain regions in a single
chromatographic run. This sensitivity is found
adequate for the analysis of these compounds in
brain tissue extracts.
A major advantage of the present procedure
(isocratic HPLC-FD method) is the ease of sample
preparation which reduces assay time and
minimizes the chances for technical errors. The
separations are achieved at room temperature at
25±1oC and the use of special monitors for column
temperature is not required.
2. Estimation of Biogenic Amines by HPLC with
Electrochemical Detection
Many compounds that are electrochemically
active can be measured using HPLC-ECD due to
the selectivity and sensitivity. This detection is
based on the measurements of the current resulting
from oxidation/reduction reaction of the analyte
at a suitable electrode. Since the level of the current
is directly proportional to the analyte
concentration, this detector could be used for
quantification.
Principle
At an applied positive potential, catecholamines
and indoleamines are oxidized at ring hydroxyl
groups with the release of two electrons. These
electrons are transferred to the electrode and the
resultant current is directly proportional to the
number of molecules oxidized. Most of the analytes
to be successfully detected require the pH
adjustments.
Mobile phase
The mobile phase consists of sodium acetate (0.03
M), Methanol (7%), heptane sulfonic acid (50mM),
EDTA (0.1 mM) and dibutyl amine (100µl). The
solution is adjusted to pH 3.98 ± 0.01 with glacial
acetic acid and filtered through 0.45µ pore size
membrane.
Preparations of the Standards
Stock solution of biogenic amines and their
metabolites is prepared at the concentration of
1mg/ml in the sodium acetate buffer (0.015M) with
20% methanol and is stored in 0º C and used
within a week of preparation. The working
standard solutions is prepared by diluting 10ml of
stock standard solution to 10µl and from this 50µl
is diluted to 5 ml, from this 20µl is injected to the
HPLC injector port which gave the concentration
of 200pM of the each standard.
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The procedure for brain tissue preparation and
processing is carried out as mentioned above for
the amiinoacids. The monoamines and their
metabolites peaks are identified by comparing their
retention time in the sample (tissue extract) solution
with that of the standard solution. Each
monoamine in the tissue is quantified by comparing
their peak area with that of known standards as
described for aminoacids.
References
1. Lakshmana, M.K. and Raju, T.R. (1997) An isocratic
assay for neuroepinephrine, dopamine, and 5-
hydroxytryptamine using their native fluorescence by
high performance liquid chromatography with
fluorescence detection in discrete brain areas of rat.
Anal Biochem 246: 166-170.
2. Lakshmana, M.K., Shankaranarayana Rao, B.S.,
Sudha, S., Dhingra, N.K., Ravikumar, R., Govindaiah
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