A novel strategy for spectrophotometric simultaneous determination of amitriptyline and nortriptyline based on derivation with a quinonoid compound in serum samples

Article abstract of DOI:10.1016/j.saa.2016.06.013

A novel and efficient strategy for the simultaneous determination of two tricyclic antidepressant (TCA) drugs [amitriptyline (AT), and its main metabolite (nortriptyline; NT)] via a combination of magnetic solid phase extraction (MSPE), and spectrophotometric techniques in serum is suggested. For this purpose, the imidazolium ionic liquid (Imz)-modified Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2-Imz) was employed as an adsorbent for theMSPE. Preconcentration (loadingdesorption) studies were performed under optimized conditions including pH, adsorbent amount, contact time, eluent volume, and desorption time. Afterward, determination of each drug was carried out by specific strategy. Acetaldehyde (AC), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil; CL) were used as chemical reagents for reaction with NT, while AT did not react with these reagents. This method is based on the condensation reaction between secondary amine group of NT and AC to afford an enamine, and subsequently reaction with CL to produce a chlorinated quinone-substituted enamine. The final product exhibited maximum absorption at 556 nm, while the AT was determined at 240 nm. The limits of detections (LODs) for NT and AT in serum sample were obtained as 0.19 and 0.90 ng mL^-1, respectively. The limits of quantifications (LOQs) were obtained to be 0.63 and 2.93 ng mL^-1 for NT and AT, respectively. A linear range was obtained to be 1 to 5 ng mL^-1. Results indicated that the suggested method is applicable for simultaneous determination of NT and AT in serum samples.

Full text of DOI:10.1016/j.saa.2016.06.013

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 168 (2016) 235–243
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A novel strategy for spectrophotometric simultaneous determination of
amitriptyline and nortriptyline based on derivation with a quinonoid
compound in serum samples
Amir Farnoudian-Habibi a, Bakhshali Massoumi b, Mehdi Jaymand a,
⁎
a
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, P.O. Box: 51656-65811, Tabriz, Islamic Republic of Iran
b
Department of Chemistry, Payame Noor University, P.O. Box: 19395-3697, Tehran, Islamic Republic of Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel and efficient strategy for the simultaneous determination of two tricyclic antidepressant (TCA) drugs
[amitriptyline (AT), and its main metabolite (nortriptyline; NT)] via a combination of magnetic solid phase ex-
traction (MSPE), and spectrophotometric techniques in serum is suggested. For this purpose, the imidazolium
ionic liquid (Imz)-modified Fe3O4@SiO2 nanoparticles (Fe3O4@SiO2-Imz) was employed as an adsorbent for
the MSPE. Preconcentration (loading–desorption) studies were performed under optimized conditions including
pH, adsorbent amount, contact time, eluent volume, and desorption time. Afterward, determination of each drug
was carried out by specific strategy. Acetaldehyde (AC), and 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil; CL)
were used as chemical reagents for reaction with NT, while AT did not react with these reagents. This method is
based on the condensation reaction between secondary amine group of NT and AC to afford an enamine, and sub-
sequently reaction with CL to produce a chlorinated quinone-substituted enamine. The final product exhibited
maximum absorption at 556 nm, while the AT was determined at 240 nm. The limits of detections (LODs) for
NT and AT in serum sample were obtained as 0.19 and 0.90 ng mL−1, respectively. The limits of quantifications
(LOQs) were obtained to be 0.63 and 2.93 ng mL−1 for NT and AT, respectively. A linear range was obtained to
be 1 to 5 ng mL−1. Results indicated that the suggested method is applicable for simultaneous determination
of NT and AT in serum samples.
Received 14 March 2016
Received in revised form 3 June 2016
Accepted 6 June 2016
Available online 07 June 2016
Keywords:
Amitriptyline
Nortriptyline
Magnetic solid phase extraction
Chloranil
Spectrophotometry
Simultaneous determination
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
The most commonly used methods for determination of AT, NT, and
other tricyclic antidepressant (TCA) drugs are spectrophotometric
Since the introduction of amitriptyline (AT) [{3-(10,11-dihydro-5H-
dibenzo[a,d]cycloheptan-5-ylidene) propyldimethylamine hydrochlo-
ride}] in 1960 this drug still is an important drug in the treatment of de-
pression, especially in developing countries [1,2]. AT is metabolized
mainly by demethylation forming nortriptyline (NT) [3-(10,11-
dihydro-5H-dibenzo[a,d]cyclohepten-5-ylidene)propyl-(methyl)-
amine hydrochloride], and by hydroxylation, lead to the formation of E-
10-hydroxy(EHAT) and Z-10-hydroxyamitriptyline (ZHAT). The NT is
further demethylated to desmethylnortriptyline (NNT), and hydroxyl-
ated to E-10-hydroxy (EHNT) and Z-10 hydroxynortriptyline (ZHNT).
The demethylation of AT and NT is mainly catalyzed by CYP2C19, with
the participation of other CYP enzymes that formed in higher drug con-
centrations. The formation of the E-10-hydroxy metabolites is depen-
dent on the activity of CYP2D6, with stereospecificity to the (−)-EHAT
and (−)-EHNT metabolites. The main metabolic pathways of AT and
NT in blood are shown in Scheme 1 [3].
method [4–7], micellar liquid chromatography (MLC) [8], voltammetry
[9], capillary gas-liquid chromatography (CGLC) [10], capillary zone
electrophoresis (CZE) [11], high-performance liquid chromatography
(HPLC) [12–14], electron-capture gas chromatography (ECGC) [15],
thin layer chromatography (TLC) [16], LC-MS [17], GC [18], liquid-liquid
microextraction (LLE)-GC [19], GC–MS [20], gas-liquid chromatography
(GLC) [21], and chemometric methods [22].
It is an unquestionable fact that the development of new and effi-
cient approach for extraction, preconcentration, and isolation of real
samples are required to enhance sensitivity, and selectivity of the ana-
lytical method. In this respect, solid phase extraction (SPE) can be con-
sidered as a versatile approach, mainly due to its improvements in
automation, reproducibility and high-throughput capability [23–25].
In the last decade, a great deal of research effort has focused on the im-
provement of SPE systems. A relatively new development in this field is
the use of magnetic nanoparticles (MNPs), which is the so-called mag-
netic solid phase extraction (MSPE). The MSPE has some advantages
such as excellent adsorption efficiency, rapid separation from the matrix
through external magnetic field without retaining residual
⁎
Corresponding author.
E-mail address: m_jaymand@yahoo.com (M. Jaymand).
http://dx.doi.org/10.1016/j.saa.2016.06.013
1386-1425/© 2016 Elsevier B.V. All rights reserved.

236
A. Farnoudian-Habibi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 168 (2016) 235–243
Scheme 1. Main metabolic pathways of amitriptyline and nortriptyline in blood.
magnetization after its removal, and has recently exhibited significant
advantages in separation science [26–28]. The Fe3O4@SiO2-ionic liquid
(Fe3O4@SiO2-IL) is composed of a core-shell structure with a core of
dense and globular magnetic nanoparticles, and a SiO2-IL shell that the
magnetic property of the core solved the separation difficulty, while
the shell-type mesoporous structure shortens the diffusion path, and in-
creases the effective area for adsorption [29,30].
chemicals including sodium hydroxide, potassium hydroxide, metha-
nol, and acetonitrile were of analytical grade or the highest purity avail-
able from Merck (Darmstadt, Germany), and were used as received. All
organic solvents used throughout this study were analytical grade, and
double distilled water (DDW) was used throughout the study.
2.2. Synthesis of adsorbent
This paper describes the development and validation of an efficient
analytical method by a combination of MSPE, and spectrophotometric
techniques for the simultaneous determination of AT and NT in the
serum sample. This method is based on the condensation of free second-
ary amine group of NT and AC to afford an enamine, and subsequently
reaction with CL to produce a chlorinated quinone-substituted enamine
which was measured spectrophotometrically. Meanwhile, it should be
mentioned that NT is a secondary amine and AT is a ternary amine
and both of them have the same maximum absorption at 240 nm in
UV–vis spectroscopy (overlapping) before reaction. Since AC reacted
with the primary or secondary amines, thus AT remains as not reacted
species in solution with maximum absorption at 240 nm (without
shift). This matter is the base of the selective simultaneous determina-
tion of AT and NT drugs.
The imidazolium ionic liquid (Imz)-modified Fe3O4@SiO2 nanoparti-
cles (Fe3O4@SiO2-Imz) as adsorbent was synthesized in our laboratory
as illustrated in Scheme 3 [31].
2.3. Drug loading experiments
It should be mentioned that, since both drugs have relatively similar
chemical structures (Scheme 2), all of loading and desorption processes
2. Experimental
2.1. Chemicals and reagents
Amitriptyline hydrochloride (Hetero Drugs Ltd., Hyderabad, India),
and nortriptyline hydrochloride (Pfizer Egypt, S.A.E., Cairo, Egypt)
were used as received. The chemical structures of these drugs are
shown in Scheme 2. Acetaldehyde (AC), and chloranil (CL) were provid-
ed from Sigma-Aldrich (USA), and were used without purification. All
Scheme 2. Chemical structures of amitriptyline and nortriptyline hydrochlorides.

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237
Fig. 2. The effect of the contact time on the loading efficiency of AT and NT.
(5 mL; 20/80 v/v), the vials were capped and mixed vigorously for
5 min, and then centrifuged at 5000 rpm for 10 min. The organic layer
was transferred to another tube containing 0.50 mL of acidic phosphate
buffer (25 mmol L−1 potassium dihydrogen phosphate adjusted to
pH 2.4 with phosphoric acid (85%)), mixed for 1 min, and then centri-
fuged at 5000 rpm for 10 min. The organic layer was discarded, and a
10 mL aliquot of the aqueous phase was injected for preconcentration
and quantitative determination. Final concentration of drugs in serum
Scheme 3. Synthesis of Fe3O4@SiO2-Imz nanoparticles.
are approximately equal for both of them. Loading studies performed as
follows: 10.0 mL of each drug solution (0.20 mg L−1) diluted to 350 mL
by DDW, then 0.50 g of adsorbent was added to the each flask. After-
wards, solutions pHs were adjusted in the range of 3.0 to 9.0 with
0.1 mol L−1 of HCl or NaOH solutions, and pH was optimized in 7. Beside
of pH, the adsorbent amount, and contact time were also optimized.
Meanwhile, for high efficiency in preconcentration procedure, it is nec-
essary MSPE and desorption processes are carry out efficiently. In order
to obtain the best desorption results, 2 mL of acetonitrile–KOH (5% w/v)
was added on drugs loaded adsorbent and stirred for 10 min. The resul-
tant solutions were used for simultaneous determination of drugs.
was 0.20 mg L−1
.
2.5. Analytical procedure
For quantitative determination the molar ratio of NT to reagents (AC
and CL) was selected as 1:1, to prevent the byproduct generation. Brief-
ly, 10 mL mixture of NT and AT (0.20 mg L−1) was prepared, and the
final volume was adjusted at 300 mL using DDW, then preconcentration
process was carried out under optimum condition. After
preconcentration, 1 mL AC 0.1% (v/v) (in acetonitrile) was added on
preconcentrated mixture, and stirred for about 15 min until enamine
(NT-AC) formation. Then, 1 mL of CL solution 1 mmol L−1 (in acetoni-
trile) was added to the reaction mixture, and stirred for about 40 min
to development of color. Subsequently spectrophotometrically mea-
surements indicated that the maximum absorption at 240 nm is related
to AT, and maximum absorption at 556 nm corresponded to the final
product (chlorinated quinone-substituted enamine; NT-AC-CL).
2.4. Serum sample preparation
To 1.0 mL of serum, the internal standard, AT and NT (0.16 mg L−1),
and 0.50 mL of 0.6 mol L−1 sodium carbonate-sodium bicarbonate buff-
er (pH 9.8) were added. After addition of ethyl acetate/n-heptane
To the best of our knowledge, this strategy has not yet been reported
for simultaneous determination of AT and NT. Moreover, in comparison
with previous studies the present investigation showed that the free
Fig. 1. The effect of the solution pH on the loading efficiency of AT and NT.
Fig. 3. The effect of the adsorbent amount on the loading efficiency of AT and NT.

238
A. Farnoudian-Habibi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 168 (2016) 235–243
Table 1
Adsorption isotherms parameters of analytes onto adsorbent.
Freundlich isotherm equation
logqe = logKF + 1 / nlogCe
AT
NT
n
KF
R
2.12
2.10
43.45 (mg g−1
0.9908
)
44.86 (mg g−1
0.9911
)
amines give more sensitive assays than to their corresponding hydro-
chloride salts [32].
2.6. Instrumentation
Fig. 5. Desorption time of AT and NT from Fe3O4@SiO2-Imz nanoparticles.
Ultraviolet–visible (UV–vis) absorption spectra of the samples were
measured using a Shimadzu 1601 PC UV–vis spectrophotometer
(Shimadzu, Kyoto, Japan).
attract to IL and loadings of drugs decreased due to decreasing of
interactions.
3. Results and discussion
3.1.2. Effect of the contact time
3.1. Parameters affecting the drug loading
The contact time between drugs and adsorbent is one of the most
important parameters that can affect the performance of loading pro-
cess. The effect of contact time on the loading processes of the drugs
was also investigated, and the results obtained are summarized in Fig.
2. The results indicated that, 30 min under optimum condition (pH 7)
is suitable for high loading efficiency.
3.1.1. Effect of pH
Solution pH is an important parameter that affects the adsorption
process of drug molecules. The solution pH would affect both aqueous
chemistry, and surface binding-sites of the adsorbent. The effect of the
solution pH on the adsorption of the drugs onto Fe3O4@SiO2-Imz nano-
particles was assessed at different pH values, in the range of 3 to 9, with
a stirring time of 30 min. In these experiments conditions, the initial
concentrations of drugs and adsorbent amount were set at
0.20 mg L−1 and 0.50 g, respectively, for all batch tests. Fig. 1, presents
the effect of the solution pH on the loading efficiency of drugs under
mentioned condition. After vigorous shaking for about 30 min (accord-
ing to Fig. 2), pH 7 was chosen as optimum.
In explanation of this fact it should be pointed out that, since pka NT
and AT are 9 and 9.5, respectively. Thus, in lower pHs analytes are cat-
ionic (hydrochloride salt), and the loading efficiency is decreased [33].
In the other words, low adsorption for AT and NT is expected due to re-
pulsion forces between the cationic drugs and cationic parts of adsor-
bent, weak π–π interaction as well as weak attraction forces between
the cationic drugs and counter-ions of adsorbent (Cl−) [34]. However,
with increasing of pH the Cl− was replaced by OH− group and the IL
is more activated [35]. On the other hand, the drugs were converted
to free amine forms. Hence repulsion and attraction forces are reduced,
while π–π interactions are increased significantly. In higher pHs, OH−
3.1.3. Effect of the adsorbent amount
The effect of the adsorbent quantity in drugs loading was investigat-
ed by adding various amounts of the adsorbent in the range of 0.1 to
1.0
g into the beaker containing 10 mL of drugs solutions
(0.20 mg L−1) at pH 7 for all batch experiments. The suspensions
were stirred vigorously for 30 min, and then supernatant was analyzed
for the remained drugs. As shown in Fig. 3, the results indicated that the
addition of 0.50 g of adsorbent lead to maximum loadings of drugs.
3.1.4. Equilibrium studies
The equilibrium isotherm of a specific adsorbent represents its ad-
sorptive characteristics and is very important to the design of adsorp-
tion process. Developing an appropriate isotherm model for
adsorption is essential to the design and optimization of adsorption pro-
cess. Several isotherm models such as Langmuir, Freundlich, Redlich–
Peterson, Dubinin–Radushkevich, Sips, and Temkin have been devel-
oped for evaluating the equilibrium adsorption of compounds from so-
lutions [31]. In this respect, the Freundlich equations is one of the most
Fig. 6. The effect of the solvent volume on desorption of AT, and NT from Fe3O4@SiO2-Imz
Fig. 4. Freundlich isotherm of AT and NT onto Fe3O4@SiO2-Imz nanoparticles.
nanoparticles.

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239
Fig. 8. Optimization of the concentration of chloranil.
Scheme 4. Synthesis of chlorinated quinone-substituted enamine.
The plot of (logqe) versus (logCe) gives a straight line with a correla-
tion coefficient of 0.9908 and 0.9911 for AT and NT, respectively. KF and
1/n were determined from the intercept and slope of the plot, respec-
tively. It is well accepted that under a constant temperature, the values
increased with decreasing adsorption energy, which implied that the
larger of n value, and the stronger adsorption intensity. In general,
values n N 1 illustrate that adsorbate is favorably adsorbed on an adsor-
bent, while n b 1 indicate that adsorbate is unfavorably adsorbed on an
adsorbent. The high values of n and KF suggest that the drugs are
commonly used models, and the experimental results of this study were
fitted with this model. The isotherm curves were investigated in order
to obtain adsorbent capacity for both species (AT and NT). This proce-
dure was carried out by adding 0.50 g of adsorbent in 10 mL of DDW
at pH 7. Afterward, 10 mL of drugs with different concentrations (all ad-
justed at pH 7) were added, and final volume was set in 100 mL with
DDW (pH was controlled remain at 7).
The samples with concentrations range of 40 to 1500 mg L−1 and
pH 7 were shaken vigorously at room temperature. The samples were
sealed during the experiments to prevent evaporation and change in
volume. The Freundlich equation is given as:
=n
1
qe ¼ KFCe
and the linear form of the Freundlich isotherm is:
logqe ¼ logKF þ 1=n logCe
where KF [(mg g−1) (mg L−1)n] is the Freundlich adsorption equilibri-
um constant which is an indicator of the adsorption capacity, and n is
a characteristic coefficient relating to adsorption intensity. The calculat-
ed parameters for Freundlich isotherm and the correlation coefficients
(r) are listed in Table 1.
Fig. 9. A: UV–vis spectra of NT-AT mixture [red spectrum is mixture of NT-AT; blue
spectrum is mixture of NT-AT in the presence of AC]. B: UV–vis spectra of NT in the
presence of AC, and subsequently the reaction of intermediate enamine with CL. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Fig. 7. Optimization of the concentration of acetaldehyde.

Table 2
Effects of some common interferents on the determination of AT and NT (0.2 ng mL−1).
Coexisting substance
Concentration in serum (mg L−1
)
Variation (% ΔA)a
AT
NT
Cadaverine (CAD)
Putrescine (PUT)
Spermidine (SPD)
Histamine (HIS)
Tryptamine (TRP)
2-Phenylethylamine (PEA)
Tyramine (TYR)
0.020
0.075
0.015
0.050
0.010
0.0003
0.001
100.00
−0.2
+0.3
−0.1
−2.5
−1.5
−0.05
−0.1
−0.4
−3.2
−5.5
−3.0
−6.0
−5.0
−0.4
−2.0
−2.7
L-Leucin
Acetaldehyde
Furaldehyde
Acrolein
Benzaldehyde
Hexanal
0.90
3.50
0.60
0.10
+1.0
−5.0
−4.50
−5.0
−0.01
+0.005
+0.2
+0.002
−0.5
−3.0
1 × 10−4
30.00
100.00
−0.02
−3.50
−4.20
Sacarose
Glucose
+0.8
a
ΔA: Absorption variation.

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241
Table 3
Linearity parameters, limit of detection and limit of quantification in determination of NT and AT with proposed method.
Sample
Data pointsa
Slope
Y-intercept
r2b
Range (ng mL−1
)
LODc (ng mL−1
)
LOQc (ng mL−1
)
Standard
AT
NT
AT
NT
12
12
10
5
0.213
0.333
0.205
0.314
0.008
−0.011
0.112
0.999
0.999
0.998
0.998
1–5
0.5–2.5
1–5
0.62
0.12
0.90
0.19
2.05
0.40
2.93
0.63
Serum
0.021
1–2.5
a
Data points are the number of concentrations included in the calibration curve.
r, Regression coefficient of the calibration curve. Each sample analysis was repeated five times.
The limit of detection and limit of quantification, were calculated as 3Sb / m and 10Sb / m, respectively, where Sb and m are the standard deviation (SD) of the blank and the slope of
b
c
the calibration curve, respectively.
3.2. Preconcentration studies
3.4. Optimization of derivation condition
3.2.1. Drugs desorption
For optimization of derivation condition, some affecting factors such
as concentrations of AC and CL, and reaction time were evaluated. The
color of the reaction was described as a function of the concentrations
of AC and CL reagents. The results indicated that the color development
is dependent on concentrations of both reagents. As shown in Figs. 7
and 8, the highest color intensity was obtained when the concentration
of AC was 0.1% (v/v), and the optimum concentration of CL was obtain-
ed as 1 mmol L−1, which were selected for further experiments. The re-
sults obtained from optimization of the reaction time indicated that the
complete color development was attained after 40 min at room temper-
ature (Fig. 9).
Drugs desorption from the adsorbent was carried out by washing the
AT and NT loaded Fe3O4@SiO2-Imz nanoparticles using 2 mL of acetoni-
trile–KOH (5% w/v) that give the desorption efficiency of 99 and 97% for
AT and NT, respectively. In addition, 10 and 8 min were found to be suf-
ficient for desorption of NT and AT from adsorbent, respectively. Thus,
for improvement of results 10 min was chosen as optimum desorption
time (Fig. 5). When strong basic eluent added on loaded adsorbent,
drugs convert to their basic forms and their interactions with adsorbent
were decreased. On the other hand, organic solvent caused to separa-
tion of drugs from the adsorbent surface.
Fig. 9A, shows the UV–vis spectra of the NT-AT mixture. As seen in
red spectrum both analytes have maximum absorption at about
240 nm. The UV–vis spectrum of this mixture at 15 min after addition
of AC was recorded (blue spectrum). As seen in this spectrum the absor-
bance intensity at 240 nm is decreased significantly (approximately it is
halved), due to reaction of NT with AC. Thus this absorbance is related to
the AT which is unable to react with AC. On the other hand, the interme-
diate (NT-AC) has a maximum absorption at 360 nm as seen in blue
spectrum.
Fig. 9B, explain the reaction of NT with AC and subsequent reaction
of produced intermediate enamine with CL versus time. The absorbance
in 240 nm is related to the pure NT. In contrast, the maximum absorp-
tions at 360 and 556 nm are corresponded to the NT-AC and NT-AC-CL
compounds, respectively. As seen in Fig. 9B, with the increasing of reac-
tion time NT completely react with AC and the obtained intermediate
enamine was subsequently reacted with CL to afford final product
which has maximum absorption at 556 nm.
3.2.2. Volume effect
For achievement to preconcentration factor we were investigated
volume effect of the sample. Volume variations for the samples were
chosen between 50 to 400 mL. As shown in Fig. 6, the results exhibited
that in volume of 300 mL, still the loading percentages are 95 and 96%
for AT and NT, respectively. However, as seen in this figure at higher vol-
umes loadings efficiency were decreased. So, 300 mL was chosen as op-
timum volume for next steps of experiment.
3.3. Determination of drugs
As mentioned, this paper describes the development and validation
of a spectrophotometric method for the simultaneous determination of
AT and NT. This method is based on the reaction of enamine formed
from the interaction of the secondary amine group in the NT and AC
with CL to give chlorinated quinone-substituted enamine, which was
measured spectrophotometrically at 556 nm. It is important to note
that AT is a ternary amine, since AC react only with primary or second-
ary amines. Thus, AT remain as not reacted species in solution, and it
was determined at 240 nm (not shift). Therefore, after addition of AC
and CL the reaction is completed, and we will observe two maximum
in UV–vis spectrum at 240 and 556 nm for AT and chlorinated qui-
none-substituted enamine (related to NT), respectively. By this strategy,
simultaneous determination of two homologous amines (i.e., AT and
NT) with the same maximum absorption in UV–vis at 240 nm before re-
action (overlapping) was happened (Scheme 4).
3.5. Interference studies
The investigation of interfaces effects on determination process is
necessary to demonstration of method specificity. In this respect,
some main amines and aldehydes that are exist in serum and can be af-
fect the results were tested. As shown in Table 2, histamine has maxi-
mum effect of interfere (−6.0%) in this study, and overall results
indicated good specificity for the analytes. It is important to note that,
the loading capacity of the adsorbent (Fe3O4@SiO2-Imz nanoparticles)
is almost high, in part due to its nanostructured morphology and large
surface area. Thus, these interferences did not significantly decrease
Table 4
Precision and recovery of NT and AT in serum samples.a
Sample
AT
Added (ng mL−1
)
Founded (ng mL−1
)
Intra-day (%RSD)
Inter-day (%RSD)
Recovery (%)
50
100
200
50
100
200
48
98
195
49
99
196
3.8
3.1
2.2
4.1
2.8
1.1
5.1
3.2
3.3
4.2
3.1
1.5
96
98
97.5
98
99
NT
98
a
Each sample analysis was repeated five times.

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Table 5
Performance comparison of the designed method with other techniques in determination of NT and AT.
Method
LOD
NT
Reference
AT
MSPE-spectrophotometry
SPE-HPLC/UV
LC-MS/MS
TLC-densitometry, HPLC
Single-drop microextraction-GC
LLE–GC/flame ionization
LC–UV
HPLC-fluorescence
LLE-HPLC
0.12 ng mL−1
0.08 ng mL−1
0.62 ng mL−1
This study
[38]
[39]
[40]
[41]
[19]
[42]
[43]
[44]
0.04 ng mL−1
20 μg L−1
300 μg mL−1
20 μg L−1
–
0.02 μg mL−1
0.01 μg mL−1
0.01 μg mL−1
0.005 μg mL−1
0.72 mg mL−1
–
–
–
2 ng mL−1
0.58 μg L−1
5 ng mL−1
HPLC-DAD
5 ng mL−1
[3]
the loading efficiency of the AT or NT. It should be pointed out that ac-
cording to Table 2, the tolerance limit was defined as the concentrations
which give an error of ≤−6% in the determination of NT and AT. Thus,
these species do not significantly interfere with the proposed method.
method and express as intra-day and inter-day. According to Table 4,
the percentage relative error for intra-day accuracy was less than 3.8%,
with recovery of 96 to 98% for AT and 4.1% with recovery of 98 to 99%
for NT indicating good accuracy. The percentage relative standard devi-
ation for the intra-day precision did not exceed 3.8% for AT and 4.1% for
NT, indicating good repeatability. For inter-day accuracy, the percentage
relative error was less than 5.1% and 4.2% for AT and NT, respectively
with recovery of 96 to 98% for AT and 98 to 99% for NT indicating
good reproducibility. The accuracy results showed that this method is
suitable for routine analysis of the drugs in quality control laboratories.
In addition, the performance of the designed method in comparison
with other techniques is presented in Table 5. This table indicates that
the presented approach could be considered as an efficient method in
comparison with other techniques such as LC-MS/MS, LC–UV, HPLC-
fluorescence, and LLE–GC/flame ionization.
3.6. Validation of the proposed method
The method was validated in term of sensitivity, linearity range, ac-
curacy, precision, LOD and LOQ values according to the ICH guidelines.
3.6.1. Calibration curves
Calibration curves were constructed under mentioned optimized
conditions by plotting absorbance versus concentrations of analytes on
each of three consecutive days. A linear relations was obtained between
absorbance and concentrations of drugs in the range of 1 to 5 ng mL−1
for AT and 1 to 2.5 and 0.5–2.5 ng mL−1 for NT in serum and standard
samples, respectively. Linear regression was obtained with a correlation
coefficient of 0.998 and 0.999 for both of analytes in serum and standard
samples, respectively. The absorbance increased linearly with increas-
ing concentration of the drugs in linear range. As shown in Figs. 10
and 11, under the mentioned optimized conditions, the calibration
curves for the investigated drugs in both standard (Fig. 10), and serum
(Fig. 11) samples were constructed, and the results obtained are sum-
marized in Table 3.
4. Conclusion
A novel strategy for the validated separation and simultaneous de-
termination of two antidepressant drugs (amitriptyline and nortripty-
line) in the tricyclic antidepressant (TCA) group was demonstrated.
The presented method is simple, rapid, and reliable for determination
of these drugs in their pharmaceutical dosage forms without interfer-
ence from common excipients. The influences of main interfering spe-
cies in serum sample were investigated. It was found that these
species do not significantly interfere with the proposed method. On
the basis of five replicate measurements, the LODs for NT and AT were
obtained to be 0.12 and 0.62 ng mL−1, respectively. These LOD values
are lower than those of the obtained values for many of the previously
reported spectrophotometric methods for the investigated drugs. The
proposed method is of great value in determination of the mentioned
drugs, because of their improved simplicity and sensitivity, low cost,
and free from dependence on expensive instruments and hazardous an-
alytical reagents.
3.6.2. LOD and LOQ
LOD and LOQ are important parameters in bioanalytical processes.
These parameters are determined according to the following equations.
3σ
LOD ¼ 3 ꢀ
s
10σ
s
LOD ¼
where σ is the standard deviation of the blank signals, and s is the slope
of the calibration curve. The results obtained are presented in Table 3.
These analytical results indicated that AC and CL gave relatively high
sensitivity, in part due to their excellent reactivity with NT as secondary
free amine [36,37].
Acknowledgement
We express our gratitude to Research Center for Pharmaceutical
Nanotechnology, Tabriz University of Medical Sciences for supporting
this project.
3.6.3. Accuracy and precision of the method
References
The accuracy and precision of the method were evaluated at three
concentrations (50, 100 and 200 ng mL−1) of NT and AT under optimum
conditions in five replicates on the same day (intra-day) and on three
consecutive days (inter-day). Accuracy is expressed as percentage re-
covery and presents the closeness between the true value and the
found value. The accuracy of proposed method was evaluated in term
of recovery and RSD (%) between the measured and true concentrations
of the drugs. Precision is concern with the variation in relative standard
deviation. The precision of this approach indicate repeatability of
[1] C. Leucht, M. Huhn, S. Leucht, Amitriptyline versus placebo for major depressive dis-
order, Cochrane Database Syst. Rev. 12 (2012) CD009138.
[2] F. López-Muñoz, C. Alamo, G. Juckel, H.J. Assion, Half a century of antidepressant
drugs: on the clinical introduction of monoamine oxidase inhibitors, tricyclics, and
tetracyclics. Part I: monoamine oxidase inhibitors, J. Clin. Psychopharmacol. 27
(2007) 555–559.
[3] R. Linden, M.V. Antunes, A.L. Ziulkoski, M. Wingert, P. Tonello, M. Tzvetkovb, A.A.
Soutoc, Determination of amitriptyline and its main metabolites in human plasma
samples using HPLC-DAD application to the determination of metabolic ratios
after single oral dose of amitriptyline, J. Braz. Chem. Soc. 19 (2008) 35–41.

A. Farnoudian-Habibi et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 168 (2016) 235–243
243
[4] G.G. Mohamed, F.A. Nour-El-Dien, S.M. Khalil, N.A. Mohamed, Spectrophotometric
determination of trazodone, amineptine and amitriptyline hydrochlorides through
ion-pair formation with molybdenum and thiocyanate, Spectrochim. Acta A 65
(2006) 1221–1226.
[5] F.A. Nour-El-Dien, G.G. Mohamed, N.A. Mohamed, Spectrophotometric determina-
tion of trazodone, amineptine and amitriptyline hydrochlorides through ion-pair
formation using methyl orange and bromocresol green reagents, Spectrochim.
Acta A 65 (2006) 20–26.
[6] D.H. Manjunatha, J. Seetharamappa, P.B. Kandagal, S.S. Kalanur, New extractive
spectrophotometric methods for the determination of nortriptyline hydrochloride
in pure form and pharmaceutical dosages, J. Anal. Chem. 64 (2009) 462–466.
[7] J.M. García-Fraga, A.I. Jiménez-Abizanda, F.J. Moreno, J.J. Arias-León, Simultaneous
determination of amitriptyline, imipramine and perphenazine in pharmaceutical
preparations and blood serum by multi-wavelength spectrophotometry, Anal.
Chim. Acta 252 (1991) 107–113.
[24] J. Li, Y.B. Wang, K.Y. Li, Y.Q. Cao, S. Wu, L. Wu, Advances in different configurations of
solid-phase microextraction and their applications in food and environmental anal-
ysis, Trends Anal. Chem. 72 (2015) 141–152.
[25] M.H. Manafi, M. Allahyari, K. Pourghazi, M. Amoli-Diva, Z. Taherimaslak, Surfactant-
enhanced spectrofluorimetric determination of total aflatoxins from wheat samples
after magnetic solid-phase extraction using modified Fe3O4 nanoparticles,
Spectrochim. Acta A 146 (2015) 43–49.
[26] E. Yilmaz, R.M. Alosmanov, M. Soylak, Magnetic solid phase extraction of lead (II)
and cadmium(II) on a magnetic phosphorus-containing polymer (M-PhCP) for
their microsampling flame atomic absorption spectrometric determinations, RSC
Adv. 5 (2015) 33801–33808.
[27] B. Peng, J. Zhang, R. Lu, S. Zhang, W. Zhou, H. Gao, Dispersive micro-solid phase ex-
traction based on self-assembling, ionic liquid-coated magnetic particles for the de-
termination of clofentezine and chlorfenapyr in environmental water samples,
Analyst 138 (2013) 6834–6843.
[8] S. Furlanetto, S. Orlandini, B. Pasquini, M. Del-Bubba, S. Pinzauti, Quality by design
approach in the development of a solvent-modified micellar electrokinetic chroma-
tography method: finding the design space for the determination of amitriptyline
and its impurities, Anal. Chim. Acta 802 (2013) 113–124.
[9] J.P. Marco, K.B. Borges, C.R.T. Tarley, E.S. Ribeiro, A.C. Pereira, Development and ap-
plication of an electrochemical biosensor based on carbon paste and silica modified
with niobium oxide, alumina and DNA (SiO2/Al2O3/Nb2O5/DNA) for amitriptyline
determination, J. Electroanal. Chem. 704 (2013) 159–168.
[10] S. Ulrich, T. Isensee, U. Pester, Simultaneous determination of amitriptyline, nortrip-
tyline and four hydroxylated metabolites in serum by capillary gas-liquid chroma-
tography with nitrogen-phosphorus-selective detection, J. Chromatogr. B 685
(1996) 81–89.
[11] S.M. Wu, H.L. Wu, W.K. Ko, S.H. Chen, Determination of amitriptyline and nortripty-
line in spiked plasma by capillary zone electrophoresis with β-cyclodextrin, Anal.
Chim. Acta 413 (2000) 125–129.
[12] M.T. Hamed-Mosavian, Z. Es'haghi, N. Razavi, S. Banihashemi, Preconcentration and
determination of amitriptyline residues in waste water by ionic liquid based im-
mersed droplet microextraction and HPLC, J. Pharm. Anal. 2 (2012) 361–365.
[13] R.S. Farag, M.Z. Darwish, W.M. Fathy, H.A. Hammad, New HPLC method to detect
amitriptyline in the blood of rats on combination treatment, Int. J. Chem. Anal. Sci.
4 (2013) 120–124.
[14] D. Dadgar, A. Power, Applications of column-switching technique in biopharmaceu-
tical analysis: I. High-performance liquid chromatographic determination of ami-
triptyline and its metabolites in human plasma, J. Chromatogr. B 416 (1987)
99–109.
[28] M.E. Najafabadi, T. Khayamian, Z. Hashemian, Aptamer-conjugated magnetic nano-
particles for extraction of adenosine from urine followed by electrospray ion mobil-
ity spectrometry, J. Pharm. Biomed. Anal. 107 (2015) 244–250.
[29] W. Wang, R. Ma, Q. Wu, C. Wang, Z. Wang, Magnetic microsphere-confined
graphene for the extraction of polycyclic aromatic hydrocarbons from environmen-
tal water samples coupled with high performance liquid chromatography–fluores-
cence analysis, J. Chromatogr. A 1293 (2013) 20–27.
[30] J. Chen, X. Zhu, Ionic liquid coated magnetic core/shell Fe3O4@SiO2 nanoparticles for
the separation/analysis of linuron in food samples, Spectrochim. Acta A 137 (2015)
456–462.
[31] A. Farnoudian-Habibi, S. Kangari, B. Massoumi, M. Jaymand, Determination of
losartan potassium in the presence of hydrochlorothiazide via a combination of
magnetic solid phase extraction and fluorometry techniques in urine samples, RSC
Adv. 5 (2015) 102895–102903.
[32] M. Fekria, A. Attia, Use of charge-transfer complex formation for the spectrophoto-
metric determination of nortriptyline, Farmaco 55 (2000) 659–664.
[33] M. Davudabadi-Farahani, F. Shemirani, Supported hydrophobic ionic liquid on mag-
netic nanoparticles as a new sorbent for separation and preconcentration of lead
and cadmium in milk and water samples, Microchim. Acta 179 (2012) 219–226.
[34] P. Stepnowski, J. Nichthauser, W. Mrozik, B. Buszewski, Usefulness of π…π aromatic
interactions in the selective separation and analysis of imidazolium and pyridinium
ionic liquid cations, Anal. Bioanal. Chem. 385 (2006) 1483–1491.
[35] L. Chen, X. Huang, Y. Zhang, D. Yuan, A new polymeric ionic liquid-based magnetic
adsorbent for the extraction of inorganic anions in water samples, J. Chromatogr. A
1403 (2015) 37–44.
[15] P. Hartvig, S. Strandberg, B. Näslund, Determination of plasma amitriptyline by elec-
tron-capture gas chromatography after oxidation to anthraquinone, J. Chromatogr.
A 118 (1976) 65–74.
[16] D.B. Faber, C. Mulder, W.A. Manint-Veld, A thin-layer method for the determination
of amitriptyline and nortriptyline in plasma, J. Chromatogr. A 100 (1974) 55–61.
[17] Y. Shen, R.H. Zhu, H.D. Li, Y.W. Liu, P. Xu, Validated LC–MS (ESI) assay for the simul-
taneous determination of amitriptyline and its metabolite nortriptyline in rat plas-
ma: application to a pharmacokinetic comparison, J. Pharm. Biomed. Anal. 53
(2010) 735–739.
[36] D.D.H. Rosenblatt, E.P. Burrows, in: S. Patai (Ed.), In the Chemistry of Amino, Nitroso
and Nitro Compounds and Their Derivatives, Part 2, John Wiley & Sons, New York,
NY 1982, pp. 1123–1125.
[37] H.D. Becker, in: S. Patai (Ed.), In the Chemistry of the Quinonoid Compounds, Part 1,
John Wiley & Sons, New York, NY 1974, p. 399.
[38] F. Zare, M. Ghaedi, A. Daneshfar, Solid phase extraction of antidepressant drugs am-
itriptyline and nortriptyline from plasma samples using core-shell nanoparticles of
the type Fe3O4@ZrO2@N-cetylpyridinium, and their subsequent determination by
HPLC with UV detection, Microchim. Acta 182 (2015) 1893–1902.
[18] S. Khanahmadzadeh, A. Tarigh, Ultrasound-assisted combined with nano sized mo-
lecularly imprinted polymer for selective extraction and pre-concentration of ami-
triptyline in human plasma with gas chromatography-flame detection, J.
Chromatogr. B 972 (2014) 6–13.
[39] E.J.J. Berm, J. Paardekooper, E. Brummel-Mulder, E. Hak, B. Wilffert, J.G. Maring, A
simple dried blood spot method for therapeutic drug monitoring of the tricyclic an-
tidepressants amitriptyline, nortriptyline, imipramine, clomipramine, and their ac-
tive metabolites using LC-MS/MS, Talanta 134 (2015) 165–172.
[19] A. Sarafraz-Yazdi, N. Razavi, S. Raouf-Yazdinejad, Separation and determination of
amitriptyline and nortriptyline by dispersive liquid–liquid microextraction com-
bined with gas chromatography flame ionization detection, Talanta 75 (2008)
1293–1299.
[20] H. Bagheri, S. Banihashemi, F.K. Zandian, Microextraction of antidepressant drugs
into syringes packed with a nanocomposite consisting of polydopamine, silver
nanoparticles and polypyrrole, Microchim. Acta 183 (2016) 195–202.
[21] M.A. Martínez, C. Sánchez de la Torre, E. Almarza, A comparative solid-phase extrac-
tion study for the simultaneous determination of fluoxetine, amitriptyline, nortrip-
tyline, trimipramine, maprotiline, clomipramine, and trazodone in whole blood by
capillary gas—liquid chromatography with nitrogen-phosphorus detection, J. Anal.
Toxicol. 27 (2003) 353–358.
[40] O.M. El-Houssini, N.H. Zawilla, Chromatographic methods for the determination of
fluphenazine, nortriptyline and its impurity amitriptyline in bulk and pharmaceuti-
cal formulations, Microchim. Acta 182 (2015) 1893–1902.
[41] A. Sarafraz-Yazdi, N. Razavi, Separation and determination of amitriptyline and nor-
triptyline in biological samples using single-drop microextraction with GC,
Chromatographia 73 (2011) 549–557.
[42] S. Ashour, N. Kattan, Simultaneous determination of nortriptyline hydrochloride and
fluphenazine hydrochloride in microgram quantities from low dosage forms by liq-
uid chromatography–UV detection, J. Pharm. Anal. 2 (2012) 437–442.
[43] P. Almudever, J.E. Peris, T. Garrigues, O. Diez, A. Melero, M. Alós, Quantification of
nortriptyline in plasma by HPLC and fluorescence detection, J. Chromatogr. B 878
(2010) 841–844.
[22] C.K. Markopoulou, E.T. Malliou, J.E. Koundourellis, Application of two chemometric
methods for the determination of imipramine, amitriptyline and perphenazine in
content uniformity and drug dissolution studies, J. Pharm. Biomed. Anal. 37
(2005) 249–258.
[44] M. Gupta, A. Jain, K.K. Verma, Determination of amoxapine and nortriptyline in
blood plasma and serum by salt-assisted liquid-liquid microextraction and high-
performance liquid chromatography, J. Sep. Sci. 33 (2010) 3774–3780.
[23] J.W. Li, Y.L. Wang, S. Yan, X.J. Li, S.Y. Pan, Molecularly imprinted calixarene fiber for
solid-phase microextraction of four organophosphorous pesticides in fruits, Food
Chem. 192 (2016) 260–267.