Journal of Chromatography A, 1190 (2008) 52–56

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Microwave-assisted extraction of tricyclic antidepressants from human serum

followed by high performance liquid chromatography determination
M. Woźniakiewicz a , R. Wietecha-Posłuszny a,∗ , A. Garbacik a , P. Kościelniak a,b
a
Laboratory for Forensic Chemistry, Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
b
Institute of Forensic Research, Westerplatte 9, 31-033 Kraków, Poland

a r t i c l e i n f o a b s t r a c t

Article history: A microwave-assisted extraction (MAE) method has been developed and optimized for the extraction
Received 27 November 2007 of six tricyclic antidepressants (TCAD; nordoxepin, nortriptyline, imipramine, amitriptyline, doxepin,
Received in revised form 21 February 2008
dezypramine) from human serum. Optimal parameters of MAE (solvent and extraction temperature) for
Accepted 6 March 2008
water solution of these drugs were defined. The microwave-assisted procedure developed was validated
Available online 18 March 2008
by extraction of serum samples at two concentration levels and then successfully applied to the analysis of
reference material. Limit of quantification, precision and recovery were found for the studied compounds
Keywords:
in the ranges 0.04–0.15 ␮g/mL, 1.57–4.34% (RSD) and 94–105%, respectively.
Microwave-assisted extraction
Tricyclic antidepressants © 2008 Elsevier B.V. All rights reserved.
Biological samples
HPLC
Validation

1. Introduction has been successfully applied in the extraction of various ana-

Sample preparation is one of the most important steps of the
analytical process. Analysis of complex matrices such as biologi-
cal samples requires this particular approach. Progress in sample
preparation covers the solid phase extraction (SPE) with its many
variations, ultrasound, micro-dialysis and supercritical fluid extrac-
tion. Recently, research over improved sample preparation methods
have focused on the microwave technology [1].
Microwave technology employs, destructive methods for sam-
ple preparation. The most frequently applied are digestion and
mineralization [2,3]. Abu-Samara et al. [4] were the first researchers
ever to use a domestic microwave oven in a laboratory, performing a
trace analysis of metals in biological samples. Nowadays these ana-
lytical approaches are well documented in literature. It has been
demonstrated that the application of microwave energy to digest
biological samples ensures high quality results [5,6].
The extraction of organic compounds by microwave irradi-
ation appeared within the work of Ganzler et al. [7] in 1986.
The authors presented the microwave-assisted extraction of crude
fat and antinutrients from food and, in the second application,
pesticides from soil. The reported results were satisfactory and
comparable with those obtained with conventional methods.
Since then the microwave-assisted extraction (MAE) technique
∗ Corresponding author. Tel.: +48 12 6632257; fax: +48 12 6340515.
E-mail address: wietecha@chemia.uj.edu.pl (R. Wietecha-Posłuszny).
lytes from such environmental matrices as medicinal plants [8],
sediments and soils, etc. [9–14]. Remarkable contribution into
the microwave-assisted process (MAPTM ) applied in the sample
preparation brought Paré et al. [15–23]. They patented several
technologies, e.g. for extraction of oils from plant and animal bio-
logical matter by exposure to microwave energy [16,23]. Hewimon
et al. [24] reported a method for microwave extraction of antiox-
iative anthraquinones from roots of medicinally important plant
Morinda citrifolia. The results obtained for MAE were also com-
pared with maceration, soxlet extraction and ultrasonic-assisted
extraction (UAE). The authors concluded that MAE gave the highest
extraction yields and required the shortest time when comparing
with the other tested methods. Moreover, it was found that the
anthraquinones recovery was within the range 65–96%, so it was
much better than maceration and UAE.
MAE has been rarely applied in the field of drug isolation
from pharmaceuticals and body fluids [1,25–29]. Franke et al. [1]
employed MAE for isolation of lidocaine, methadone, diazepam,
nordiazepam, propoxyphene, norpropoxyhene from blood and
serum. When analyzing spiked samples it was found that the
extraction yield obtained was lower, than when conventional
liquid–liquid extraction (LLE) was applied. However the analysis
of forensic samples revealed higher concentration values deter-
mined after MAE then after the liquid–liquid extraction. It could
be explained by dissolving drug–protein bonds by microwave irra-
diation, however it remains as the hypothesis. In the papers by
Fernández et al. [25,26] MAE was used to extract opiates, cocaine

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.03.013
 M. Woźniakiewicz et al. / J. Chromatogr. A 1190 (2008) 52–56 53

Table 1 ogy and Therapeutic Monitoring, Collegium Medicum UJ (Kraków,

Chemical proprieties (dissociation constant, pKa and partition coefficient
Poland).
octanol/water, log P) and therapeutic and toxic concentrations of analyzed TDACs
[30–33]
2.2. Standards
Drug pKa log P Concentration in serum (␮g/mL)

Therapeutic Toxic
Drug stock solution (10 mg/mL) were prepared in methanol and
Amitriptyline 9.4 4.95 0.05–0.20 >0.5 stored in a refrigerator (+4 ◦ C). Spiking solutions was prepared by
Clomipramine 9.38 5.2 0.02–0.25 >0.4 appropriate diluting stock solutions with water. Working standard
Desipramine 10.2 1.4a 0.01–0.5 >1.0
drug mixtures were prepared by adding a suitable amount of spik-
Doxepin + Nordoxepin 9.0 2.4a
9,75 –b 0.03–0.4 >1.0 ing solution to water or serum. Each drug (amitryptyline (Ami),
Imipramine 9.5 2.5a 0.05–0.15 >1.0 desipramine (Des), doxepin (Dox), imipramine (Imi), nordoxepine
Nordoxepine 9.75 (Nord), nortryptiline (Nort) and clomipramine, as the internal stan-
Nortriptyline 9.7 1.7a 0.02–0.2 >0.5
dard, IS) was tested in the concentration range 0.3125–5 ␮g/mL
a
Octanol/buffer pH 7.4. for water samples and 0.125–1 ␮g/mL for serum samples of these
b
Data not found. drugs.

and its metabolites from human urine and plasma. The results from
the research, reduced solvent consumption and time suggested
MAE to be an alternative method to those more frequently used,
such as LLE and SPE. In another application the microwave energy
was used to extract felodipine and one of its degradation products
from tablets [27]. The developed method was robust and relatively
rapid, as it allowed the sample treatment to be reduced to a mini-
mum, as the tablets grinding step was excluded. Labbozzetta et al.
[28] elaborated a simple and fast open-vessel focused microwave-
assisted extraction (FMAE) method followed by LC analysis for
the determination of naproxen in suppositories (a model phar-
maceutical formulation in this study). The major benefits from
the method was the reduced solvent consumption and no further
sample cleaning-up steps required before the HPLC analysis. The
authors concluded that the described procedure could provide the
alternative fast and accurate method for the rapid screening of
naproxen-based suppositories aimed at the detection of counterfeit
and substandard drugs.
In the present study tricyclic antidepressants (TCADs) were cho-
sen as model substances. TCADs belong to the group of psychotropic
drugs which are frequently involved in clinical and forensic anal-
yses, because of their narrow therapeutic index (see Table 1).
Furthermore, for the analytical approach, these drugs are often cho-
sen in comparative experiments concerning the development of
basic drugs analysis in body tissues [34].
The aim of this work was to develop and optimize an efficient,
fast and reproducible method for extraction of tricyclic antide-
pressants using the microwave-assisted extraction. The optimal
MAE method was compared with the liquid–liquid extraction and
then the method for determination of TCADs using HPLC in human
serum was validated.
2. Experimental
2.1. Reagents and materials
Acetonitrile and methanol of HPLC-gradient grade were sup-
plied by Merck (Darmstadt, Germany). An 85% ortophosphoric
acid, 30% sodium hydroxide water solution, n-hexane, toluene,
acetone, ethyl acetate, cyclohexane and isoamyl alcohol, all of ana-
lytical grade, were purchased from POCH (Gliwice, Poland). Drug
standards and diethylamine were purchased from Sigma–Aldrich
(St. Louis, MO, USA). Deionized water (<1.0 ␮S/cm) was use
throughout. Courtesy of the local blood bank (Kraków, Poland)
they provided the human serum, so the investigation could
be carried out. The reference serum containing imipramine
(0.500 ± 0.101 ␮g/mL) from Abbott XSYSTEMS Tricyclic Antide-
pressants Controls, bottle H (Abbott Park, IL, USA) and control
serum samples were obtained from the Department of Toxicol-
2.3. Apparatus and conditions
High-performance liquid chromatography Merck-Hitachi
LaChrom Systems consisted of a L-7100 pump and L-7455 pro-
grammable diode array detector module coupled to PC with
D-7000 HSM software (Darmstadt, Germany). The mobile phase
was prepared by mixing acetonitrile and phosphoric buffer (1:1,
v/v). Phosphoric buffer (pH 2.36) was prepared by adding 1.4 mL
of 85% phosphoric acid and 1 mL of diethylamine to a volumetric
flask (1 L) and filling up with deionized water to the mark. Before
analysis the mobile phase was filtered through a FILTRAK cellulose
filter (Wiesenbad, Germany) and degassed in ultrasonic bath made
by Polsonic (Warszawa, Poland) for 15 min. In each analysis 10 ␮L
of sample was injected by the autosampler and the separation
was carried out using a PerkinElmer Spheri-5 C18 column, 100 mm
long with 4.6 mm I.D., particles 5 ␮m (Waltham, MA, USA) with
flow rate 1 mL/min. The column was put into a thermostat and
measurements were made at 50 ◦ C. Chromatograms were taken
at 254 nm and calibration curves were calculated on peak-height
ratios (drug/IS).
A MARS X microwave-assisted extraction unit by CEM
(Matthews, NC, USA), equipped with 14 pressurized 100 mL vol-
ume GreenChem® PTFE vessels also by CEM (Matthews, NC, USA)
was used for the extraction of analytes from biological materials.
An internal system allowed power and temperature to be controlled
up to 1200 W and 240 ◦ C, respectively.
2.4. Sample preparation
The water samples used during the optimization, were prepared
by adding spiking solution to 25 mL flask and diluted with deion-
ized water to the mark of volumetric flask. The concentration of
each drug was 0.2 ␮g/mL. Theoretical concentration (ctheor ) after
the extraction should be 3.3 ␮g/mL for MAE and 3.2 ␮g/mL for LLE.
Drug-free human serum was stored at +4 ◦ C. The spiked serum
samples were prepared by adding an appropriate volume of spiking
solution to the 1 mL of serum. Then samples were put into the ultra-
sonic bath for 15 min in order to balance the matrix. The reference
material of serum was prepared according to the recommendations
given by the manufacturer, and finally spiked with IS.
2.5. Extraction procedure
2.5.1. LLE
1 mL of spiked water sample was placed in a 20 mL glass extrac-
tion tube; 3 mL of 0.6 M sodium hydroxide were added, the content
was mixed before further addition of 5 mL of particular extraction
solvent (see Table 2) and closed with PTFE caps. The content was
then gently agitated for 10 min, then all vials were centrifuged for
54 M. Woźniakiewicz et al. / J. Chromatogr. A 1190 (2008) 52–56

Table 2 Table 3
Extractants used in the optimization study The value of function F calculated for different conditions of extraction method

Solvent number Composition Volumetric ratio Extractant F-value

I n-Hexane–isoamyl alcohol 99:1 LLE MAE45W MAE50 MAE60 MAE70

II Ethyl acetate–cyclohexane 1:1
n-Hexane–isoamyl alcohol 66.28 22.37 31.39 215.49 231.20
III n-Hexane–acetone 3:1
(99:1, v/v)
IV Toluene–isoamyl alcohol–n-hexane 76:4:20
Ethyl acetate–cyclohexane 15.11 14.46 42.13 13.43 72.23
(1:1, v/v)
Toluene–isoamyl 92.16 0.00 193.70 1.32 0.00
10 min (3500 rpm). The organic layer (4 mL) was successively trans- alcohol–n-hexane
ferred to eppendorf vials and evaporated under nitrogen stream (76:4:20, v/v/v)
with mild heating (45 ◦ C). Dry residues were dissolved in 250 ␮L n-Hexane–acetone (3:1 5.64 5.77 0.00 0.00 0.00
v/v)
of the extraction solvent and vortexed for 2 min. The re-extraction
was performed by adding 50 ␮L of 0.05% phosphoric acid with IS
(4 ␮g/mL) and vortexing for 2 min, and then centrifuged for 10 min At the beginning of the optimization of the extraction method
(4500 rpm). The lower (aqueous) layer was finally transferred to for isolation of six tricyclic antidepressants, water-based samples
HPLC-vials (55 ␮L capacity). spiked with all six TCADs were extracted in 20 separate experi-
ments using various solvents (see Table 2) and different extraction
2.5.2. MAE conditions according to Section 2.5.
1 mL of spiked water or serum samples was transferred into a The recoveries (R) of six drugs were calculated by
PTFE vessel, 3 mL of 0.6 M NaOH was added and mixed. 5 mL of cfound
particular extraction mixture (see Table 2) was added, with vessels R= · 100% (1)
ctheor
than tightly closed and placed into the microwave sample prepa-
ration system. The extraction process was carried out according to where cfound and ctheor are determined and theoretical concentra-
four different procedures. In the first attempt (MAE45W), the con- tions (␮g/mL), respectively.
stant power of microwaves (45 W) for 1 min was applied. Due to the Moreover, to determine the optimal parameters of the extraction
instrument limitations no temperature data were recorded during it was designed a function F (see Eq. (2)), based on that presented by
that experiment. Then three temperature programmed methods Guan et al. [37], however in different context. It takes into account
at 50 ◦ C (MAE50), 60 ◦ C (MAE60) and 70 ◦ C (MAE70) were evalu- that the extraction procedure should be versatile offering high
ated. The program consisted of ramping for 2 min to the desired recoveries for many analytes and the differences in the extraction
temperature and holding for 1 min. The maximum power of the yield for every compound should be reduced.
oven was limited from 1200 to 300 W to avoid sample overheating.
k2 · Y
After extraction, the vessels were left in the extraction system to F= (2)
s
cool down to room temperature. The content of each vessel were
transferred to labeled glass tubes, additionally rinsing the vessels where k is the number of analytes with a recovery value exceeding
with 1 mL of extraction mixture. Then glass tubes were closed with 60%, Y is the mean extraction recovery, calculated for all ana-
PTFE caps and centrifuged for 10 min (3500 rpm). After that the lytes, and s measures the differences between n (all) analytes
upper (organic) layer (5 mL) was successively transferred to eppen- extraction recovery and it is calculated according to the following
dorf vials and evaporated under nitrogen stream with mild heating formula:
(45 ◦ C) to dry residues. The following steps were the same as in case

n
(Y
i=1 i
− Y )2
of the liquid–liquid extraction. s= (3)
n−1
3. Results and discussion In the theoretical ideal conditions, F rises to the infinity, as k is
equal to the number of analytes, Y is 100%, which results in 0 value
3.1. Optimization of MAE conditions for s. The threshold value for k was chosen to be 60% as this value is
easily achievable in LLE and thus lower recovery values cannot be
Preliminary research aimed to determine the optimal conditions accepted. Since the number of substances which could be extracted
for isolation of basic drugs from water-based samples. To find the using the developed method was the one of the major conditions,
optimal microwave-assisted method four extraction solvents were the k value is squared in Eq. (3). Finally, s parameter converge to
tested (see Table 2). The results obtained for each extractant using zero, when the yield of extraction for all individual analytes are
microwave-assisted extraction were compared with those from similar. The calculations results are presented in Table 3, where
liquid–liquid extraction. The four mixtures, listed in Table 2 were F values were collected.The maximum value for function F was
tested. They included that almost transparent for microwaves (sol- obtained using MAE70 procedure, using a mixture of n-hexane and
vents I) and those which could be heated by microwave irradiation isoamyl alcohol (99:1, v/v, solvent I). However the analysis of recov-
(solvents II–IV) [1,14,35,36]. eries (see Fig. 1) revealed that MAE60 offers quite similar extraction
In our study two instrumental parameters were optimized: yield for all analyzed drugs. Moreover, the application of a lower
power and temperature of extraction. Experiments at the con- temperature (60 ◦ C) significantly reduced the time needed to cool
stant power of microwaves were performed using 45 W for 1 min vessels down to the room temperature after the extraction. Thus,
(MAE45W). To evaluate the effect of temperature on the extraction the MAE60 and solvent I was chosen for further investigations (see
three temperature levels were tested: 50, 60 and 70 ◦ C (see Section Fig. 2).
2.5). In the experiments, time of extraction was kept as short as pos-
sible, as it was proved that the efficient MAE may last only a minute 3.2. Validation
[1]. Some preliminary trials revealed the minimum ramping time
assuring stable final temperature should be at least 2 min. Although, The validation of a HPLC method for the determination of six
the maximum temperature was held for 1 min in the experiments TCADs in human serum using the MAE60 procedure for drugs isola-
carried out. tion was performed (see Table 4). The linearity range was expressed
 M. Woźniakiewicz et al. / J. Chromatogr. A 1190 (2008) 52–56 55

Table 4
Validation parameters for method of determination 6 TCADs in serum using the MAE60 method and solvent I

Parameter Nord Nort Des Ami Imi Dox

Linearity range (␮g/mL) 0.15–2.00 0.05–2.00 0.15–2.00 0.11–2.00 0.06–2.00 0.04–2.00

Slope, a 1.46 1.94 1.09 1.07 1.29 0.91
Intercept, b −0.12 −0.08 −0.08 −0.13 −0.04 −0.04
Correlation coefficient, R2 0.9942 0.9999 0.9939 0.9993 0.9998 0.9997
LOD, ␮g/mL 0.05 0.01 0.05 0.03 0.02 0.01
LOQ, ␮g/mL 0.15 0.05 0.15 0.11 0.06 0.04
Repeatability of relative retention time, RSD% 0.30 0.26 0.45 0.33 0.32 0.35
H/HIS repeatability 0.2 ␮g/mL, (n = 5) RSD% 10.10 2.48 11.09 8.27 3.39 2.28
H/HIS repeatability 0.8 ␮g/mL, (n = 4) RSD% 3.56 4.34 1.57 2.79 3.12 2.12
RE 0.2 ␮g/mL, % 115.3 115.1 105.2 123.0 110.1 112.5
RE 0.8 ␮g/mL, % 121.8 110.9 113.4 109.2 108.9 108.2

ysis Student’s t-test, did not show any significant differences

between relative retention times for every series and for all of
TCADs.
The repeatability of the analytical signal (ratios of a peak height
for analyte to a peak height for internal standard, H/HIS ) was also
determined at two concentration levels 0.2 and 0.8 ␮g/mL in single
series including 5 and 4 samples, respectively. The accuracy (RE) at
these two concentration levels were estimated by
 − cx
RE = · 100% (6)


where cx and  is found and expected (known) concentration value,

Fig. 1. Extraction recoveries obtained using n-hexane-isoamyl alcohol (99:1, v/v)
for all examined TCADs in different extraction conditions.
as the range from LOQ to the maximum measured concentration,
with the respect of R2 > 0.993. The limit of quantification (LOQ)
and the detection limit (LOD) were defined using Eqs. (4) and (5),
respectively.
3SD0.2
LOD =
a
10SD0.2
LOQ =
a
where SD is the standard deviation of analytical signal for 5 samples
at drug concentration 0.2 ␮g/mL, and a is the slope of calibration
curve.
Repeatability of relative retention time was studied in two
series (n1 = 5 and n2 = 4) on different days. The statistical anal-
respectively.
The repeatability of analytical signal for the higher concentra-
tion of drugs (0.8 ␮g/mL) was found to be better than 0.2 ␮g/mL.
This was caused by the matrix effect which is very often during
analysis of biological materials.
The accuracy of the analytical method was also evaluated by
the analysis of 2 mL control serum containing analyzed TCADs.
This material, together with 0.5 mL of reference serum containing
imipramine was analyzed using the MAE60 procedure followed by
(4) HPLC analysis. The expected and found concentration of drugs in
the control samples (n = 2) are presented in Table 5 and in Fig. 3. A
(5) precision error was expressed, as a range between the obtained con-
centration values. The calculated results for control serum samples
were close to those of the theoretical values.
The concentration of imipramine found in the reference sam-
ple was 0.480 ␮g/mL, this value is covered by the confidence range
reported by the manufacturer (0.500 ± 0.101 ␮g/mL). The received
chromatogram for the reference sample analysis was presented in

Fig. 2. Comparison between chromatograms obtained using LLE and MAE60 extrac- Fig. 3. Chromatograms obtained for the analysis of the blank sample (A) and
tion methods. (1) nordoxepin, (2) nortryptyline, (3) desipramine, (4) amitryptyline, the control sample containing 6 TCADs (B). (1) nordoxepin, (2) nortryptyline, (3)
(5) imipramine, (6) doxepin and (IS) clomipramine. desipramine, (4) amitryptyline, (5) imipramine, (6) doxepin and (IS) clomipramine.
56 M. Woźniakiewicz et al. / J. Chromatogr. A 1190 (2008) 52–56

Table 5 Further investigations, for the application of microwave-

The concentration of drugs expected and found in the control samples
assisted extraction in the field of sample preparation in
Drug Concentration (␮g/mL) Recovery (%) toxicological analysis are planned in the near future. Those include
Expected Found analyzing other psychotropic drugs as well as hair samples.

Nortriptyline 0.50 0.48 ± 0.03 95.4 ± 6.6

Imipramine 0.50 0.47 ± 0.01 94.4 ± 2.8
Acknowledgements
Amitriptyline 0.50 0.49 ± 0.03 98.5 ± 5.1
Doxepin 0.50 0.48 ± 0.01 96.3 ± 2.0 The authors would like to thank the Department of Toxicology of
Nordoxepin 0.30 0.33 ± 0.03 110.2 ± 9.1 Therapeutic Monitoring, Collegium Medicum UJ (Krakow, Poland)
Desipramine 0.20 0.21 ± 0.01 105.6 ± 5.2
for providing control and reference samples.

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