Solid-Phase Microextraction Coupled

with Microcolumn Liquid Chromatography

for the Analysis of Amitriptyline in Human Urine
2000, 52, 309- 313
K. Jinno 1. / M. Kawazoe 1 / M. Hayashi da 2
1 School of Materials Science, Toyohashi Universif/of Tech nolog~ Toyohashi 441-8580, Japan
2 Department of Legal Medicine, Ni ppon Medical School, Bunkyo-ku,Tokyo 113-8602, Japan
Key Wards
Column liquid chromatography
Solid-phase microextraction
Tricyclic antidepressants
Human urine
Summary
Solid-phase microextraction (SPME) is a solvent-free sample-preparation technique that en-
ables isolation and pre-concentration of analytes from a sample on a thin film coating a
fused-silica fiber. In this study SPME coupled with microcolumn liquid chromatography (micro
LC) has been used for the determination of four tricyclic antidepressants (amitripb, hne, imipra-
mine, nortripf,/hne, and clesipramine) in hu man urine. SPME conditions which affect extraction
efficiency were optimized, and under the optimum conditions the system was a few hundred
times more sensitive than direct LC analysis without SPME. For amitripf,/hne the detection limit
was 3 ng mL 1 and the calibration curve was linear in the range of 5- 500ng mL 1. The
SPME-micro LC method has been applied to the analysis of amitripb'line in patient's urine.
Introduction
The tricyclic antidepressants (TCAs) ami-
triptyline and imipramine, which are
widely used in psychiatry, are metabolized
in the liver to nortriptyline and desipra-
mine, respectively. These drugs are as-
sumed to exert their clinical effects by in-
teraction with the noradrenergic or sero-
tonergic systems [1]. Extraction and isola-
tion of antidepressants from human fluids
is very important for the toxicological,
pharmaceutical, and forensic analysis of
these drugs [2, 3].
Several analytical methods have been
employed for the determination of TCAs
in human fluids and tissues. Although
liquid liquid extraction (LLE) has been
widely used for sample preparation [3 5],
the method is time-consuming and uses
large volumes of organic solvents, the dis-
posal of which causes environmental pro-
blems. Solid-phase microextraction
(SPME) is a solvent-free sample-prepara-
tion technique developed by Pawliszyn et
al. [6 10]. The technique was originally
developed as a solvent-free procedure for
use with gas chromatography (GC), and
SPME GC has been successfully applied
to the analysis of wide variety of com-
pounds, for example polycyclic aromatic
hydrocarbons (PAHs), benzene, toluene,
ethylbenzene, and o-, m-, and p-xylene
(BTEXs) [11 15]. Most organic com-
pounds cannot, however, be analyzed by
GC because they are non-volatile or semi-
volatile, and thermally labile. To analyze
such compounds SPME should be used in
combination with LC and we have devel-
oped a device to enable SPME to be inter-
faced with LC on conventional or micro
columns, and have used the technique for
the analysis of pesticides in environmental
water and of some drugs in human urine
[16 19].
As an extension of previous work,
SPME combined with microcolumn li-
quid chromat ography (1.0mm i. d. col-
umn) has been used for the analysis of
amitriptyline in human urine. Reducing
the diameter of the LC column has several
advantages, including reduced consump-
tion of organic solvent as the desorption
medium and the mobile phase; this re-
duces pollution of the environment. With
sample preconcentration by SPME the
volume of solvent required was only
30 ixL, much less than for conventional
LLE. Coupling of micro LC with SPME
can be regarded as the most effective sys-
tem in terms of performance, economy,
and environmental protection, because of
low solvent consumption.
Materials and Methods
Materials
The SPME holder and fiber assemblies for
manual sampling were purchased from Su-
pelco (Bellefonte, PA, USA). The non- po-
Original Chromatographia Vol. 52, No. 5/6, September 2000 309
0009-5893/00/02 309- 05 $ 03.00/0 9 2000 Friedr. Vieweg & Sohn Verlagsgesellschaft mbH
~ N H ( C H 3 ) L C H 2 C H 2 N (C H a )2 L C H 2 C H 2 N H (C H 3 )
amitriptyline nortriptyline imipramine desipramine
(pKu=9.42) (pKa=9.70) (pK.=9.50) (pK.=10.44)
H3C
L C H 2 C H 2 N (C Ic H a )2 L C H 2 C H (C H 3 )C H 2 N (C H ~ ) 2
clomipramine trimipramine mianserine
Figure 1. The structures of the tricyclic antidepressants investigated in this study.
(a) without TEA
j I50mAU
4 5 6 7
50mAU 2
r l ? _
I I i I I i i
0 10 20 30 40 50 60
Time (rain)
Figure 2. Separation of seven antidepressants by micro LC: (a) without and (b) with addition of
0.18% TEA to the mobile phase. Peaks: 1 = desipramine; 2 = nortriptyline; 3 = imipramine; 4 = ami-
triptyline; 5 = mianserin; 6 = clomipramine; and 7 = trimipramine.
lar polydimethylsiloxane coating (100 ixm
film thickness) was used as the extraction
medium in SPME. All solvents were re-
agent grade purchased from Kishida Che-
mical (Osaka, Japan); deionized water was
obtained from a Milli-Q system (Milli-
pore, Tokyo, Japan).
column (Shiseido, Tokyo, Japan). The
mobile phase was 50:50 acetonitrile water
+0.18% triethylamine (TEA); the flow
rate was 501xLmin 1. The injection vo-
lume was 1 ixL, the column temperature
was controlled at 40 ~ and the detection
wavelength was 240 nm.
Apparatus
HPLC was performed with a Nanospace
SI-1 (Shiseido, Tokyo, Japan) comprising
a pump, a UVVi s detector, a column
oven, and a degasser. Borwin chromato-
graphy software (Jasco, Tokyo, Japan)
was used for data acquisition and hand-
ling. Compounds were separated on a
150 1.0mm i. d., particle size 5 ixm, Cap-
cell PAK Cis UG 80 polymer-coated Cis
Patients
The study was approved by the Human
Ethical Committee of Nippon Medical
School. The patient, a 34 year-old female,
was found unconscious in her bedroom.
She was immediately taken to the Critical
Care Medical Center of Nippon Medical
School, Tokyo, Japan. On admission to
the hospital a urine sample was collected
for screening of drugs and she was diag-
nosed as having taken an overdose of
drugs prescribed for treatment of schizo-
phrenia.
SPME Procedure
SPME consists of two processes adsorp-
tion of analytes by the fiber coating and
the desorption from the coating into an
appropriate solvent. Samples were pre-
pared by spiking sodium borate buffer so-
lution (5 mM, pH 9; 15 mL) with standard
compounds in a 20-mL sample vial with a
cylindrical-shaped stirrer bar (4 6 mm).
After extraction by direct immersion the
SPME fiber was withdrawn into the fiber
holder and inserted into the laboratory-
made desorption device [16 19] which
was filled with acetonitrile as the deso-
rption solvent. After desorption the ana-
lytes were transferred to the sample loop
of the injector by flushing solvent through
the interface.
Results and Discussion
The stationary phase used in this study,
Capcell PAK, a polymer-coated octadecyl
silica (ODS), was selected to eliminate the
chromatographic effects of residual sila-
nol groups and to prevent tailing of the
peaks of basic compounds. The chemical
structures of the drugs investigated, ami-
triptyline, clomipramine, desipramine,
imipramine, mianserin, nortriptyline, and
trimipramine, are depicted in Figure 1
and a typical separation of the seven drugs
is shown in Figure 2a. It is apparent that
all the peaks tail, despite the use of poly-
mer-coated ODS. We assumed that the
tailing was still induced by residual sila-
nols on the stationary phase, because the
tricyclic antidepressants are basic com-
pounds. Triethylamine (TEA) was, there-
fore, added to the mobile phase to solve
this problem. Use of 0.18% TEA in the
mobile phase resulted in improved peak
shapes, as is apparent from Figure 2b;
these conditions were used in further stu-
dies. Under these conditions mianserin
was eluted as the first peak, because of the
absence of alkyl chains in its structure.
In SPME sampling extraction is based
on the distribution equilibrium between
the SPME fiber and the sample matrix
[20]. The conditions with the greatest ef-
fect on extraction efficiency are tempera-
ture and rate of stirring. Matrix pH also
affects the extraction efficiency when the
310 Chromatographia Vol. 52, No. 5/6, September 2000 Original
4. E+06
9 amitriptyline
-- #- - imipramine p
/
x nortriptyline /
3. E+06 /
-- ~--- desipramine
'fi
~2. E+06
~D
1. E+06
0. E+00 . . . . .
2 4 6 8 10
pH of mat r i x
Figure 3. The effect of pH on the efficiency of extraction of four drugs.
SPME conditions: extraction temperature 60 ~ stirring rate 1200rpm;
salt concentration 0.4 g mL 1; desorption time 30 min; desorption solvent
acetonitrile (30 ~tL). The concentration of each drug is 500ngmL 1. The
relative standard deviation of each data point was between 1.9 and 7.9%
(~= 3).
O
6. 0E+05
4. 0E+05
2. 0E+05
0. 0E+00
9 amitriptyline
-- e - - imioramine
x n o r t r i p t y l i n e
r a l t l l n e
. . . . . -X . . . . . . . . . . . . . . . . . X . . .
X . . . . . . . . " ' ' - X
I I
20 40 60 80
Ext r act i on t emper at ur e (~
Figure 4. The effect of temperature on the efficiency of extraction of four
drugs. SPME conditions: extraction time 60 min; pH of matrix 9; stirring
rate 1200rpm; salt concentration0.4 gmL 1; desorptiontime 30min;des-
orption solvent acetonitrile (30 ~tL). The concentration of each drug is
500ngmL 1. The relative standard deviation of each data point was be-
tween 4.8 and 9.4% (n = 3).
8. 0E+06
9 amitriptyline
-- o. - i mi pr a mi ne /
x nortriptytline
/
6. 0E+06 .- /
"fi
O
4. 0E+06
2. 0E+06
: : : . . . . i : : . . . . . . .
41 . . . . . . . . . . . 4 . . . . .
0. 0E+00 " - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0 50 100 150 200 250
Ext r a c t i on t i me ( mi n)
Figure 5. The effect of extraction time on the efficiency of extraction of four drugs. SPME conditions:
extraction temperature 40 ~ pH of matrix 9; stirring rate 1200 rpm; salt concentration no addition;
desorption time 30 min; desorption solvent acetonitrile (30 ~tL). The concentration of each drug is
500 ng mL 1. The relative standard deviation of each data point was between 3.9 and 10.1% (n = 3).
anal yt e is a pol ar c ompound. Ot her
SPME condi t i ons such as ext r act i on t i me,
salt concent r at i on (to i nduce a sal t i ng out
effect whi ch can enhance ext r act i on effi-
ci ency in convent i onal SPME) , a nd deso-
r pt i on t i me wer e opt i mi zed f or sel ect ed
TCAs such as ami t r i pt yl i ne, i mi pr ami ne,
nor t r i pt yl i ne, a nd desi pr ami ne. Sampl es
cont ai ni ng 500 ng mL 1 wer e pr epar ed by
spi ki ng s odi um bor at e buf f er sol ut i on
(5 mM, p H 9; 15 mL) wi t h s t andar d sol u-
t i ons (30 ixL), because of t he si gni fi cant ef-
fect of ma t r i x p H on ext r act i on effi ci ency,
as is appar ent f r om Fi gur e 3. Because
t hese dr ugs ar e basi c c ompounds t hey ar e
effi ci ent l y ext r act ed at hi gh pH.
The effect of ext r act i on t emper at ur e on
effi ci ency was t hen st udi ed; t he resul t s ar e
depi ct ed in Fi gur e 4. Wi t h t he except i on
of desi pr ami ne all t he TCAs i nvest i gat ed
in this st udy behaved si mi l arl y. The ex-
t r act i on t emper at ur e was, t her ef or e, set at
40 ~
Whe n t he st i r r i ng rat e was opt i mi zed
t he best resul t s wer e obt ai ned at 1200 r pm,
t he ma x i mu m speed avai l abl e.
To enhance ext r act i on effi ci ency in
SPME sal t ( sodi um chl or i de) is usual l y
added t o t he sampl e mat r i x. The resul t s
obt ai ned di d not , however , show t he posi -
t i ve benef i t expect ed f r om salt addi t i on
peak ar eas decr eased wi t h i ncr easi ng
a mo u n t of salt added. Because ma t r i x p H
was adj ust ed t o 9, appr oxi mat el y hal f t he
sampl es were in t he i oni c f or m in t he ma -
trix, and t hus i oni c st r engt h si gni fi cant l y
i nfl uences ext r act i on effi ci ency. I f t he i o-
ni c st r engt h of t he mat r i x is i ncreased, in-
t er act i on wi t h t he i ons is al so i ncreased,
and so di s t r i but i on bet ween t he f i ber coat -
i ng a nd t he sampl e ma t r i x mi ght be i nhi b-
ited. These resul t s i mpl i ed t hat no salt
shoul d be added.
Whe n des or pt i on t i me was opt i mi zed
t he resul t s cl earl y i ndi cat ed t hat onl y a
smal l car r y- over effect was obser ved f or a
des or pt i on t i me bet ween 30 and 90mi n.
The des or pt i on t i me was, t her ef or e, fi xed
at 30 mi n in f ur t her exper i ment s.
Fi nal l y, t he ext r act i on t i me was opt i -
mi zed. The resul t s ar e s ummar i zed in Fi g-
ur e 5. On t he basis of t hese resul t s it seems
t hat ext r act i on f or 3 h is suffi ci ent f or
equi l i br at i on on all dr ugs bet ween f i ber
coat i ng and t he sampl e mat r i x; no sub-
st ant i al i mpr ove me nt woul d be expect ed
f or ext r act i on t i mes l onger t han 3 h.
Un d e r t hese opt i mi zed condi t i ons
( s ummar i zed in Tabl e I), SPME mi cr o
Or i gi nal Chr oma t ogr a phi a Vol . 52, No. 5/6, Sept ember 2000 311
Table I. Optimized SPME conditions for four
tricyclic antidepressants studied.
Extraction temperature (~ 40
Extraction time (rain) 180
pH of matrix 9
Salt concentration (g mL -t) 0
Stirring rate (rpm) 1200
Desorption time (min) 30
Table II. Linear calibration range, correlation coefficients (r), and limits of detection (LOD).
Drug Calibration range r 2 Point number in LOD
(ngmL 1) calibration curve (ngmL -1)
Desipramine 50- 500 0.993 4 40
Nortriptyline 20- 500 0.998 5 12
Imipramine 20-1000 0.972 6 9
Amitriptyline 5- 500 0.991 7 3
(a) LC without SPME
I l mAU
1
(b) SPME/LC
I 20 mAU
3
4
i I f I
10 20 30 40
Time (min)
Figure 6. Chromatograms obtained from the drug mixture by (a) LC with-
out SPME and (b) SPME-LC. The concentration of each drug is
500 ngmL -t . Peaks: 1 -- desipramine; 2 = nortriptyline; 3 = imipramine;
4 = amitriptyline.
(a) pat i ent ' s uri ne
I 2mAU
(b) 100 ng mU 1 ami t ri pt yl i ne
I 2mAU
I I I I
0 10 20 30 40 50
Ti me (mi n)
Figure 7. Chromatograms obtained, under optimized conditions, from:
(a) patient's urine diluted 15-fold with sodium borate buffer (5 raM, pH 9)
and (b) standard solution containing 100 ng mL-1 amitriptyline.
LC resulted in sensitivity approximately
500 times higher than that of direct micro
LC analysis without SPME, as is apparent
from Figure 6. The linear calibration
range, correlation coefficient, and limits
of detection (LOD) for each drug are sum-
marized in Table II. The LOD was calcu-
lated for a signal-to-noise ratio of 3; the
values obtained for amitriptyline, imipra-
mine, and nortriptyline were 3, 9, and
12ngmL-1, respectively; the highest
LOD, 40 ngmL 1, was obtained for desi-
pramine. These values are a factor of 10
lower than concentrations inducing symp-
toms of intoxication in man [211. Good
linearity was obtained for each drug in the
range 5 to 1000 ngmL 1.
SPME-micro LC under these opti-
mized conditions was then applied to the
patient' s urine sample. The sample was di-
luted 15-fold with sodium borate buffer
solution (5mM, pH9) to adjust matrix
pH. A peak was observed in the chroma-
togram obtained from the urine, at the re-
tention time indicated by an arrow seen in
Figure 7a. A standard solution of
100 ngmL 1 amitriptyline gave the chro-
matogram shown in Figure 7b; a larger
peak at the same retention time is clearly
apparent. The identities of both peaks
were confirmed as amitriptyline by UV
Vis spectroscopy [22]. On the basis of
these results the peak with a retention time
of 20 rain in the urine chromatogram was
identified as amitriptyline and because the
peak area in Figure 7a corresponds to
33.3 ng mL 1, its concentration was deter-
mined as ca 500 ngmL 1 (33.3 ngmL 1
15).
Conclusion
Analysis of amitriptyline in human urine
by SPME micro LC has been investigated
and several factors affecting extraction ef-
ficiency were optimized. Under optimum
conditions a good linear dynamic range
was obtained and LOD values for amitrip-
tyline, nortriptyline, and imipramine in
standard solutions were found to be at the
ng mL 1 level. The method has also been
shown to be a useful tool for clinical appli-
cation.
In SPME coupled with micro LC sol-
vent consumption for each analysis was
less than 1.5mL. The results herein also
suggest the possibility of successful cou-
pling of SPME with microscale separation
techniques such as capillary electrochro-
matography (CEC) and capillary electro-
312 Chromatographia Vol. 52, No. 5/6, September 2000 Original
phoresi s (CE), t hus enabl i ng f ur t her re-
duct i on of sol vent cons umpt i on. These i n-
vest i gat i ons are cur r ent l y i n progress i n
our l abor at or y.
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Received: Jan 12, 2000
Revised manuscripts received:
Mar 14 and Apr 7, 2000
Accepted: May 2, 2000
Or i gi nal Chr oma t ogr a phi a Vol. 52, No. 5/6, Sept ember 2000 313