Liquid chromatographic determination of neurotransmitter serotonin from human cerebrospinal fluid using 2-hydroxynaphtaldehyde as derivatizing reagent

Article abstract of DOI:10.14233/ajchem.2013.15023

The reagent 2-hydroxy naphthaldehyde was used for the spectrophotometric and reversed phase HPLC separation and determination of glycine, lysine, octopamine, serotonin and tyramine. The derivatives formed were examined spectrophotomatrically for the effec

Full text of DOI:10.14233/ajchem.2013.15023

Asian Journal of Chemistry; Vol. 25, No. 17 (2013), 9452-9456
http://dx.doi.org/10.14233/ajchem.2013.15023
Liquid Chromatographic Determination of Neurotransmitter Serotonin from
Human Cerebrospinal Fluid Using 2-Hydroxynaphtaldehyde as Derivatizing Reagent
*
INDRA PANJWANI , G.M. ARAIN and M.Y. KHUHAWAR
Institute of Advance Research Studies in Chemical Sciences Jamshoro, University of Sindh, Jamshoro, Pakistan
*Corresponding author: E-mail: icpanjwani@yahoo.com
(Received: 26 December 2012;
Accepted: 1 October 2013)
AJC-14203
The reagent 2-hydroxy naphthaldehyde was used for the spectrophotometric and reversed phase HPLC separation and determination of
glycine, lysine, octopamine, serotonin and tyramine. The derivatives formed were examined spectrophotomatrically for the effects of pH,
derivatizing reagent concentration and heating time in aqueous-methanolic solution. The derivatives indicated molar absorpitivity of 2.8
× 103-7.7 × 103 L mol-1 cm-1. The spectrophotometric calibration curves were observed within 1.0-20.0 µg mL-1 at λmax 420 nm. The
derivatives of 2-hydroxy naphthaldehyde were easily eluted and separated from Kromasil 100 C-18 10 µm (25 cm × 0.46 cm id) column.
The elution was carried out with methanol: water: acetonitrile and acetate buffer (0.1 M) (pH 6) (47:25:18:10 v/v/v/v) with a flow rate of
1 mL min-1 and UV detection was at 240 nm. A number of amino acids and pharmaceutical additives were checked and did not interfere
to the determination. The method was successfully applied for the determination of lysine from pharmaceutical preparation and serotonin
and glycine from human cerebrospinal fluid. The analysis was further supported by standard addition method. The recovery of lysine
from pharmaceutical preparation was 97.46 % with RSD 2.8 %. Glycine and serotonin were determined from human cerebrospinal fluid
and found 8.13-16.118 µmol L-1 and 3.92-7.44 nmol L-1 with RSD 1.6-3.3 and 1.4-3.6 %.
Key Words: HPLC, Lysine, Glycine, Serotonin, 2-Hydroxy naphthaldehyde, Cerebrospinal fluid.
in biological samples and lysine from pharmaceutical prepa-
ration.
INTRODUCTION
Neurotransmitter serotonin (5-hydroxytryptamine or
5-HT) is a monoamine neurotransmitter synthesized from the
essential amino acid tryptophan in serotonergic neurons in the
central nervous system and enterochromaffin cell in the gastro-
intestinal tract of animals including humans. Serotonin is
implicated in number of physiological and behavioural functions
such as control and regulation of affective behaviour, sleep,
vigilance, reward, motor and cognitive functions as well as
neuroendocrine functions, regulating food intake and body
temperature. Serotonin is most important for the treatment of
various disorders, particularly anxiety, depression, schizophrenia
stroke, obesity, pain, hypertension, vascular disorders, migraine
and nausea1,2. Glycine is inhibitory amino acid3 and act mainly
in the spinal cord and in the brain system via the strychnine-
sensitive glycine receptor. Glycine also exhibits a key role in
the excitatory neuro-transminion in the N-methyl-D-aspartate
receptor complex4,5. Lysine is an essential amino acid and is a
building block of protein. Lysine is important for proper growth
and lowers cholesterol. It helps to absorb the calcium6. It is
therefore of interest to develop simple, rapid, specific and
accurate method for the measurement of serotonin and glycine
Various analytical methods for the determination of 5-HT,
glycine and lysine and their metabolites have been reported;
of these the most frequently used are pre or post column
derivatization with high performance liquid chromatography
(HPLC)7,8 with electrochemical or fluorescence detection9-14
gas chromatography (GC)15,16, gas chromatography/mass
spectrometry (GCMS)17,18, capillary electrophoresis (CE)5,19-21
,
,
capillary electrophoresis laser-induced fluorescence (CE-LIF)1,
spectrophotometric22, polarographic23 and flow injection analy-
sis24. A number of derivatizing reagents have been reported
for the HPLC determination of the amino acids and amines
such as; ninhydrin25 densylchloride26, 1,3-oxadizole27, o-
pathalaldehyde28, phenyl isocynate29 and 9-fluroenyl methyl
chloroformate30.
2-Hydroxy napthaldehyde has some resemblance to 2,3-
naphthaldicarboxaldehyde and does not require the addition
of mercaptoethanol to block the second carbonyl group.
2-Hydroxy naphthaldehyde has been used as derivatizing
reagent for the HPLC determination of 4-aminobutyric acid31,
glutamine32, tryptophan, histadine and methionine33,34 and
dopamine35. Present work examines the use of 2-hydroxy

Vol. 25, No. 17 (2013)
LC Determination of Neurotransmitter Serotonin from Human Cerebrospinal Fluid 9453
naphthaldehyde for the simultaneous separation of glycine,
lysine, octopamine, tyramine and serotonin and determination
of serotonin and glycine from cerebrospinal fluid samples and
lysine from pharmaceutical preparation. The experimental
conditions are optimized for derivatization and HPLC separa-
tion. Linearity, limit of detection (LOD), limit of quantitation
and repeatability of quantitation is examined.
at 75 ºC. The remaining procedure was followed as spectro-
photometric procedure. The solution was mixed well and 20 µL
was injected on the column Kromasil 100 C-18 10 µm (25 cm
× 0.46 cm id) and derivatives were eluted with mixture of
methanol: water: aceto-nitrile and acetate buffer (0.1 M) pH 6
(47:25:18:10 v/v/v/v) with a flow rate of 1 mL/min and UV
detection was at 240 nm.
HPLC determination of serotonin and glycine from
cerebrospinal fluid (CSF) samples: Cerebrospinal fluid
samples (2-5 mL) were added equal volumes of methanol and
centrifuged at 12000 rpm for 15 min. The supernatant layer
was collected and reagents were added as HPLC analytical
procedure. The solution was heated on water bath in nitrogen
atmospheres near to dryness and contents were dissolved in
methanol and final volume was adjusted to 1 mL with methanol.
The solution 20 µL was injected on the column and derivatives
were eluted as HPLC procedure. The concentration of serotonin
and glycine in cerebrospinal fluid was evaluated from an external
calibration curve, prepared from standard solution following
the same procedure using regression equation Y = ax + b.
EXPERIMENTAL
A Hitachi 655A liquid chromatograph connected with
variable wavelength UV monitor and Rheodyne 7125
injector was used. The responses of the UV detector were
connected with computer with CSW-32 (Data Apex Ltd ©
2001www.dataapex.com) software. A Kromasil 100 C-18 10
µm (25 cm × 0.46 cm id) was used through out the study. The
spectrophotometric studies were carried out on double beam
Hitachi 220 spectrophotometer (Hitachi (Pvt.) Ltd., Tokyo,
Japan) with dual quartz cuvettes.
Serotonin creatinine sulfate, octopamine, tyramine, metha-
nol (E. Merck, Germany) and acetonitrile (Fluka Switzerland)
were used. Glycine, serine, leucine, valine, arginine (E. Merck
Germany), phenylalanine, histadine, histamine, methionine,
tyrosine, lysine, isolucine (Fluka Switzerland) were used.
Sodium acetate, acetic acid, sodium bicarbonate, sodium
carbonate, boric acid, borax, ammonium chloride, ammonia
(23 %), acetonitrile, hydrochloric acid (37 %) (E. Merck
Germany) were used. Freshly prepared doubly distilled water
from all glass was used for HPLC studies. The buffer solutions
in the pH range 1-10 at unit interval were prepared from the
following: potassium chloride (1 M)-hydrochloric acid (0.1 M),
pH 1-2, sodium acetate-acetic acid (1 M) pH 3-6, ammonium
acetate-acetic acid (1 M) pH 7, boric acid-borax (1 M) pH 8-
9 and ammonium chloride-ammonia solution (1 M) pH 10.
The cerebrospinal fluid (CSF) samples were collected
from Neurosurgical ward Liaquat University of Medicine and
Health Sciences, (LUMHS) Jamshoro with the verbal permission
from patient/parents of patient. A part of cerebrospinal fluid
was collected during surgical procedure ventriculopritoneal
shunt with the verbal consent of the surgeon. The diagnosis of
the patients was obtained from the records of the hospital with
the permission of the duty doctor. The patient/attendant of the
patient and duty doctor were informed the objectives of the
research and they accepted to participate in the project.
Spectrophotometric procedure: Solution (0.1-1.0 mL)
containing 10-200 µg glycine, lysine, tyramine, octopamine
or serotonin was transferred separately to 10 mL volumetric
flask and were added 1 mL acetate buffer pH 6 and 2 mL
reagent HN (0.1 % w/v in methanol). The mixture was heated
for 20 min on water bath at 75 ºC and the solution after cooling
at room temperature was adjusted to the mark with methanol.
The solutions were mixed well and absorbance was recorded
against the reagent blank on Hitachi 220 spectrophotometer
within 220-500 nm.
HPLC determination of serotonin and glycine from cere-
brospinal fluid samples using standard addition technique
The cerebrospinal fluid sample (2-5 mL) was treated as
HPLC determination from cerebrospinal fluid. Supernatant
liquid was separated and divided into two equal portions (A and
B). The fraction B was added glycine 8 or 5 µg and serotonin
8, 6 or 5 µg and both the solution A and B were treated as
HPLC determination from cerebrospinal fluid. The final
volume was adjusted up to 1.0 mL with methanol. The amount
of glycine and serotonin in fraction A was evaluated from
standard calibration curve and in fraction B was calculated
from average increase in the response with added standard.
Analysis of lysine from pharmaceutical preparation:
Five tablets of lysine (Holland Barrett, Netherland) containing
1000 mg/tablet lysine were crushed to powder and an amount
1.5018 g corresponding to one tablet was dissolved in water.
The solution was filtered and volume made up to 100 mL with
deionized distilled water. The solution (1.0 mL) was further
diluted to 100 mL and solutions 0.6 mL and 1.0 mL were
taken and HPLC procedure was followed as described. The
quantitation was made from external calibration curve pre-
pared from standard solutions using linear regression equation
Y= ax + b.
Analysis of lysine from pharmaceutical preparation
by standard addition: Five lysine tablets (Holland Barrett,
Netherlands) were processed and aqueous solution were
prepared as analysis of pharmaceutical preparation. Three
solutions of 1 mL each from final prepared solution were trans-
ferred to 10 mL volumetric flasks. Two solutions were added
lysine standards corresponding to 2.0 and 4.0 µg mL-1 at final
concentration.All the solutions were processed as HPLC proce-
dure as described. The quantitation was made from linear cali-
bration and from the average increase in response (n = 5) with
added standards.
HPLC procedure: Solution (0.1-1.0 mL) containing
8-100 µg glycine, lysine, tyramine, octopamine and serotonin
was transferred to 5 mL volumetric flask, was added 0.5 mL
of acetate buffer pH 6, 1 mL reagent 2-hydroxy naphthal-
dehyde (0.1 % w/v in methanol) and was warmed for 20 min
RESULTS AND DISCUSSION
The reagent 2-hydroxy-1-naphthaldehyde (HN) reacts
with primary amino group containing compounds to form

9454 Panjwani et al.
Asian J. Chem.
highly stable Schiff bases31-35. The reaction of glycine and
serotonin with 2-hydroxy naphthaldehyde were examined in
aqueous methanolic solution (Fig. 1). The reactions initially
were monitored spectrophotometrically to optimize the reaction
condition. The conditions, which gave maximum responses
(absorbance at particular wave length) was considered as optimal.
The pH was varied within the range 1-10 at unit interval. The
optimum responses were obtained within pH 5-7 with maximum
pH 6 and acetate buffer pH 6 was selected (Fig. 2). Heating
time at 75 ºC was varied from 5-35 min with an interval of
5 min. The maximum response was measured by heating
tyramine and lysine for 10 min, glycine 15 min, octopamine
and serotonin for 20 min, thus the warming time for deriva-
tization was fixed at 20 min (Fig. 3). The amount of derivatizing
reagent HN was varied between 1-4 mL at an interval of 1 mL
at a concentration of 0.1 % (w/v in methanol) and optimal
response was obtained with 2 mL (Fig. 4). The derivatives
once formed were highly stable and did not show any change
in absorbance up to 12 h.
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Tria.
Gly.
Lys.
Octop.
Sero.
0
5
10 15 20 25 30 35 40
Heating time (min)
Fig. 3. Effect of heating time on the 2-hydroxy-1-naphthaldehyde derivatives
1.2
1.0
Tria.
0.8
Gly.
Lys.
0.6
O
Octop.
O
N
O
OH
0.4
0.2
0
Sero.
+
H2N
OH
OH
OH
Glycine
2-Hydroxy-1-
naphthaldehyde
[(2-Hydroxy-naphthalen-1-
ylmethylene)amino]acetic acid
Gly-HN
0
1
2
3
4
(a)
Reagent (mL)
OH
OH
Fig. 4. Effect of volume of the reagent on the 2-hydroxy-1-naphthaldehyde
derivatives
O
N
H2N
+
OH
OH
2.8 × 103 and 7.7 × 103 L mol-1 cm-1 and obeyed Lambert-
Beer’s law with 1.25-10 and 0.8-10 µg L-1 with coefficient of
determination (r2) 0.9964 and 0.9973, respectively (Table-1).
To increase the selectivity of the determination at optimized
spectrophotometric conditions HPLC was investigated. Serotonin
derivative of 2-hydroxy-1-naphthaldehyde eluted from Kromasil
100C-18 column and separated from the excess of the reagent
2-hydroxy-1-naphthaldehyde. Glycine, lysine, octopamine and
tyramine also completely separated simultaneously when
eluted with methanol: water: acetonitrile: and acetate buffer
(0.1 M) pH 6 (47:25:18:10 v/v/v/v) with flow rate of 1 mL
min-1 and UV detection was at 240 nm (Fig. 5). The spectro-
photometric detector response could be measured at 420 nm,
but because of better UV sensitivity at 240 nm, was selected.
Repeatability for the elution of serotonin, glycine, octopamine,
tyramine and lysine derivatives in terms of average peak height
and retention time (n = 5) were observed with RSD within
1.8-3.4 %.
N
N
H
H
2-Hydroxy-1-
naphthaldehyde
Serotonin
1-Vinyl-naphthalen-2-ol-3-(2-amino-ethyl)-
H1-indol-5-ol
Sero-HN
(b)
Fig. 1. Chemical reactions of 2-hydroxy-1-naphthaldehyde (HN) with (a)
glycine (Gly-HN) and (b) serotonin (Sero-HN)
0.45
Tria.
0.40
0.35
Gly.
Lys.
0.30
Octop.
Sero.
0.25
0.20
0.15
0.10
0.05
0
Linear calibration curves for glycine, lysine, octopamine,
serotonin and tyramine were obtained with 1.25-10, 1-10, 0.8-
6, 0.8-10 and 0.8-4.7 µg mL-1 with coefficient of determination
(r2) 0.9964, 0.9987, 0.9979, 0.9973 and 0.9966, respectively.
Detection limits were measured as three times back ground
noise and were obtained 0.42, 0.33, 0.26, 0.26 and 0.26 µg
mL-1 corresponding to 8.33, 6.66, 5.33, 5.33 and 5.33 ng
injection-1 (20 µL) of glycine, lysine, octopamine, serotonin
and tyramine, respectively (Table-2). Limit of quantitative
measured as signal to noise ratio 10:1 were obtained 1.25,
0
1
2
3
4
5
6
7
8
9
10
pH
Fig. 2. Effect of pH on the absorbance of 2-hydroxy-1-naphthaldehyde
derivatives
At optimized conditions derivatives indicated reasonable
sensitivity with molar absorpitivities within 2.8 × 103-7.7 ×
103 L mol-1 cm-1 at λmax 420 nm. However glycine and serotonin
with 2-hydroxy-1-naphthaldehyde indicated molar absorpitivity

Vol. 25, No. 17 (2013)
LC Determination of Neurotransmitter Serotonin from Human Cerebrospinal Fluid 9455
TABLE-1
QUANTITATIVE SPECTROPHOTOMETRIC DATA OF AMINO ACIDS DERIVATIVES OF 2-HYDROXY-NAPHTHALDEHYDE
pH of max:
derivatization
Effect of heating
time (min)
Calibration range (µg
mL-1)
Molar absorpitivity
(L mol-1 cm-1)
Amino acids
r2
λmax (nm)
Tyramine
6
6
6
6
6
10
15
10
20
20
420
420
420
420
420
3-15
2.5-20
4-16
1-12
1-8
4.3 × 103
2.8 × 103
3.7 × 103
5.9 × 103
7.7 × 103
0.9958
0.9976
0.9974
0.9927
0.9963
Glycine
Lysine
Octopamine
Serotonin
10
8
mination of glycine and serotonin and lysine. Similarly pharma-
ceutical additives methylparabin, propylparabin, gum acacia,
manitol, lactose, fructose, glucose, lactose, sodium chloride,
sodium laryl sulphate, methyl hydroxypropyl cellulose,
magnesium stearate and stearic acid were added at least twice
the concentration of analyte did not affect the determination
of lysine, glycine and serotonin. Five test solutions within the
calibration range of lysine, glycine and serotonine were analyzed
and relative errors were obtained 2.3-5.0 %.
6
18.8
3
8.4
1
5.0
4
9.3
6
4
2
0
2
5
6.2
12.6
Lysine tablets used as dietary source were analyzed for
the contents of the lysine in the tablets. The amount found
was 974.6 mg/tablet with RSD 2.8 %. The value agreed with
the labeled value of 1000 mg/tablet. The lysine tablet solution
was spiked with, 2.0 and 4.0 µg mL-1 standard solutions and a
similar chromatogram was obtained with an increase in the
response (peak height/peak area) corresponding to added stan-
dards with recovery calculated, 97.0 and 98.2 % with RSD
1.8 % and 2.4 % (n = 5) (Table-4). Five human cerebrospinal
fluid samples were collected from neurosurgical ward of
Liaquat University of Medicine and Health Sciences (LUMHS),
patient suffering from meningitis, cerebral malaria, headache
fever and fits and were analyzed for glycine and serotonin
content and the amounts found were 8.13-16.12 µmol L-1 of
glycine with RSD 1.6-3.3 % and 3.92-7.44 nmol L-1 of serotonin
with RSD of 1.4-3.6 % (Fig. 6). The maximum amount of
glycine 16.12 µmol L-1 was observed from the patient suffering
from cerebral malaria and minimum 8.13 µmol L-1 from the
patient suffering from meningitis. However the maximum
amount of serotonin 7.44 nmol L-1 was observed from the
patient suffering from headache fever and minimum 3.92 nmol
L-1 was found from the patient suffering from meningitis. Cere-
brospinal fluid samples (1, 3 and 5) were added calculated
amounts 8 and 5 µg mL-1 of glycine and 8, 6 and 5 µg mL-1 of
serotonin, respectively and the results obtained correlated with
observed values by calibration with RSD 1.4-3.1 and 1.7-2.7 %,
0
5
10
15
20
Retention time (min)
Fig. 5. HPLC separation of derivatives of HN, (1) Glycine, (2) Lysine, (3)
Tyramine, (4) Octopamine (5) Serotonin (6) HN. Column Kromasil
100 C-18, 10 µm (25 cm × 0.46 cm id), eluant methanol: water:
acetonitrile: and acetate buffer (0.1 M) pH 6 (47:25:18:10 v/v/v/v)
with flow rate of 1 mL min-1 and UV detection was at 240 nm
TABLE-2
QUANTITATIVE HPLC DATA OF AMINO-ACIDS USING 2-
HYDROXYNAPHTHALDEHYDE AS DERIVATIZING REAGENT
Calibration
range (µgmL-1)
Detection limit ng
injection-1 (20 µL)
Amino acid
r2
Glysine
Lysine
1.25-10
1-10
8.33
6.66
5.33
5.33
5.33
0.9964
0.9987
0.9979
0.9973
0.9966
Octopamine
Serotonin
Tyramine
0.8-6
0.8-10
0.8-4.7
1.0, 0.8, 0.8, 0.8 µg mL-1 for glysine, lysine, octopamine,
serotonin and tyramine, respectively. The amino acids cystine
methonine, phenylalanine, serine, aspergine, tyrosine, cysteine,
arginine, aspartic acid, valine, isolusine and glutamic acid when
added at the same concentration did not interfere in the deter-
TABLE-3
HPLC ANALYTICAL RESULTS OF AMINO ACIDS FROM CSF SAMPLES USING HN AS DERIVATIZING REAGENT
Determination of glycine
(µmol L-1) (RSD n = 3)
Determination of serotonin
(nmol L-1) (RSD n = 3)
Subject (M/F)
Age (years)
Disease
F
35
18
17
46
2
Meningitis
Meningitis
15.452 (3.2)
8.125 (2.4)
16.118 (2.8)
13.721(1.6)
12.122 (3.3)
4.60 (3.6)
3.92 (2.4)
4.43 (2.8)
7.44 (1.4)
5.51 (3.2)
M
M
M
M
Cerebral Malaria
Headache fever
Fits
By standard addition technique
Determination of glycine
(µmol L-1)
Determination of serotonin
(nmol L-1)
Subject (M/F)
Age (years)
Disease
F
35
17
2
Meningitis
Cerebral malaria
Fits
14.896 (96.4)* (3.1) ** (8)***
15.763 (97.8)* (1.4) ** (5)***
11.796 (97.31)* (2.4)** (5)***
4.51 (98.07%)* (2.1)** (8)***
4.29 (96.92%)* (1.7)** (6)***
5.454(98.96%)*(2.7)** (5)***
M
M
* = recovery %, ** = RSD (n = 3), *** = (Std. addition µg mL-1),

9456 Panjwani et al.
Asian J. Chem.
TABLE-4
ANALYSIS OF LYSINE FROM PHARMACEUTICAL PREPARATION USING
2-HYDROXY NAPHTHALDEHYDE AS DERIVATIZING REAGENT
Amino
acids
Amount reported
(mg tablet-1)
Amount found Recovery
(mg tablet-1)
RSD %
(n = 5)
R. Error % for unknown
test solution (n = 5)
Product
Tablet lysine (Holland Barret, Netherland)
(%)
Lysine
1000
974.6
97.46
2.8
0.14-2.3
3
18.8
successfully applied for the determination of serotonin, glycine
and lysine from human cerebrospinal fluid and lysine from
pharmaceutical preparation.
2.5
2.0
REFERENCES
1. D.A. Roman, A.S. Carretero, C.C. Blanco and A.F. Gutierrez, Biomed.
Chromatogr., 18, 422 (2004).
1.5
1.0
0.5
0
2. T. Yoshitake, J. Kehr, K. Todoroki, H. Nohta and M. Yamaguchi,
Biomed. Chromatogr., 20, 276 (2006).
3. G.E. Nilson and P.L. Lutz, Am. J. Physiol., 261, R32 (1991).
4. D.M. Lambert, M. Geurts, G.K. Seriba, J.H. Poupaert and P. Dumont,
J. Pharm. Belg., 50, 194 (1995).
1
5.1
2
9.3
5.
A. Zinella, S. Sotgia, E. Pisanu, B. Scanu, M. Sanna, M.F. Usai, R. Chessa,
L. Deiana and C. Carru, Anal. Bioanal. Chem., 398, 1973 (2010).
6. M. Dousa, J. Brichac, P. Gibala and P. Lehnert, J. Pharm. Biomed. Anal.,
54, 972 (2011).
7. B.W. Boyd, S.R. Witowski and R.T. Kennedy, Anal. Chem., 72, 865
(2000).
0
5
10
15
20
25
8. D.V. Augusti, F. Carazza, R. Augusti, W.A. Tao and R.G. Cooks, Anal.
Chem., 74, 3458 (2002).
Retention time (min)
9. R. Kvetnansky, K. Pacak, D. Tokarev, J. Jelokova, D. Jezova and M. Rusnak,
Neurochem. Res., 22, 995 (1997).
(a)
3
18.8
10. M.H. Mills, D.C. Finlay and P.R. Hadad, J. Chromatogr., 564, 93 (1991).
11. A.T. Wood and M.R. Hall, J. Chromatogr. B: Biomed. Sci. Appl., 744,
221 (2000).
5.0
4.0
12. T. Yoshitake, J. Kehr, K.T. Todoroki and M. Yamaguchi, Biomed.
Chromatogr., 20, 267 (2006).
13. K.E. Hubbard, A. Wells, T.S. Owens, M. Tagen, C.H. Fraga and C.F.
Stewart, Biomed. Chromatogr., 24, 626 (2010).
1
5.1
3.0
2.0
14. A.C.C.L. Martins and M.B.A. Gloria, Food Chem., 118, 529 (2010).
15. P. Husek, J. Chromatogr. B: Biomed. Sci. Appl., 615, 1 (1993).
16. K.-R. Kim, J.-H. Kim, C.-H. Oh and T.J. Mabry, J. Chromatogr. A,
605, 241 (1992).
2
9.3
17. J.W. Commissiong, J. Neurochem., 41, 1313 (1983).
18. L.U. Sneddon, A.C. Taylor, F.A. Huntingford and D.G. Watson, J. Exp.
Biol., 203, 537 (2000).
1.0
0
19. P.L. Chang, T.C. Chiu and H.T. Chang, Electrophoresis, 27, 1922 (2006).
20. C. Parata, P. Bonnafous, N. Fraysse, M. Treilhou, V. Poinsot and F.
Conderc, Electrophoresis, 22, 4129 (2001).
21. V. Poinsot, P. Gavard, B. Feurer and F. Couderc, Electrophoresis, 31,
105 (2010).
0
5
10
15
20
25
22. A.M. EL-Brashy and S.M.AL-Ghannam, Pharm.World Sci., 17, 54 (1995).
23. P.J. Vandeberg and D.C. Johnson, Anal. Chem., 65, 2713 (1993).
24. C. Mayer, A. Franer, T. Schalkhammar and F. Pitterner, Anal. Biochem.,
268, 110 (1999).
Retention time (min)
(b)
Fig. 6. HPLC response of (a) HPLC response from cerebrospinal fluid
samples (b) HPLC response from cerebrospinal fluid samples after
spiking with Glycine 8.0 (µg mL-1) serotonin 6.0 (µg mL-1).
Chromatographic conditions as Fig. 5
25. M. Rubenstein, S. Chen-Klang, S. Stein and S. Undenfriend, Anal.
Biochem., 95, 117 (1979).
26. M.A. Sanz, G. Castillo and A. Hernandez, J. Chromatogr. A, 719, 195
(1996).
27. A. Alegria, R. Barbera, R. Farre, M. Ferres, M.J. Lagarda and J.C.
Lopez, J. Chromatogr. A, 721, 83 (1996).
respectively. The results of glycine in cerebrospinal fluid were
similar to those already described5,36 and value of serotonin in
cerebrospinal fluid.
28. R. Soto-Otero, E. Mendez-Alvarez, J. Galan-valiente, E. Aguilar-Veiga
and G. Sierra-Marcuno, Biomed. Chromatogr., 8, 114 (1994).
29. L.E. Lavi, J.S. Holcenberg, D.E. Code and J. Jolivet, J. Chromatogr.
Biomed. Appl., 50, 155 (1986).
Conclusion
30. D. Fekkes, J. Chromatogr. A, 682, 3 (1996).
An analytical method has been developed for the accurate,
sensitive and selective determination of serotonin and glycine
in cerebrospinal fluid and lysine from pharmaceutical prepa-
ration. The detection limit for serotonin and glycine were 5.33
and 8.33 ng injection-1 (20 µL). A number of amines, amino
acids and pharmaceutical additives examined, did not affect
the determination of serotonin and glycine. The method was
31. M.Y. Khuhawar and A.D. Rajper, J. Chromatogr. B, 788, 413 (2003).
32. M.Y. Khuhawar and A.D. Rajper, Chromatographia, 58, 479 (2003).
33. A.M. Al-Brashy and S.M.Al-Ghannam, Analyst, 122, 147 (1997).
34. S.A. McClimlock, E.C. Purdy and S.N. Young, Anal. Chim. Acta, 166,
171 (1985).
35. A.D. Rajper, G.M. Arain, F.M.A. Rind and M.Y. Khuhawar, Asian J.
Chem., 19, 4817 (2007).
36. M. Garseth, L.R. White and J. Aasly, Neurochem. Int., 39, 111 (2001).