Chromatographia 2000, 52 ,309 - 313.pdf
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Solid Phase Microextraction Coupled
with Microcolumn Liquid Chromatography
for the Analysis of Amitriptyline in Human Urine
2000 52, 3 0 9 - 3 1 3
K Jinno1 / M Kawazoe 1 M
Hayashida
1School of Materials Science, Toyohashi Univer sif/of Tech nolog~ Toyohashi 441-8580, Japan
2 Department of Legal Medicine, Nippon 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 ur ine. SPME condi tions which affect extraction
efficiency were optimized, a nd under the optimum conditions the system was a few hundred
times more sensitive than direct LC analysi s wit hout SPME. For amitripf, /hne the detecti on limit
was 3 ng mL 1 and the calibration curve was linear in the range of 5- 50 0n g mL 1. The
SPME-micro LC method has been applie d to the analysis of amitripb line in patient s urine.
Introduction
The t r icycl ic an t idepressan ts (TCAs) am i-
t r ip ty l ine and imipramine, which are
widely used in psych iat ry , are metabo l ized
in the l iver to nor t r ip ty l ine and desipra-
mine, respect ively . These d rugs are as-
sume d to exert their cl inical effects by in-
teract ion wi th the noradrenerg ic o r sero-
tonerg ic systems [1]. Ex t ract ion and i so la-
t ion of an t idepressan ts f rom hu man f lu ids
is very impor tan t fo r the tox ico log ical ,
pharmaceut ical , and forensic analysis o f
these d rug s [2, 3].
Several analy t ical methods have been
emp l o y ed fo r t h e d e t e rmi n a t i o n o f TCAs
in human f lu ids and t i ssues . Al though
l iqu id l iqu id ex t ract ion (LLE) has been
widely used fo r sample p repara t ion [3 5 ],
the method i s t ime-consuming and uses
large vo lumes o f o rgan ic so lven ts , the d is-
posal o f which causes env i ronme ntal p ro-
b lems. So l id -phase microex t ract ion
(SPME) i s a so lven t - f ree sample-prepara-
t ion technique deve loped by Pawl iszyn et
al . [6 10]. The techn ique was origina lly
developed as a so lven t- f ree p rocedure fo r
u se wi t h g as ch ro mat o g rap h y (GC) , an d
SPM E GC has been successful ly app l ied
to the analysis o f wide var iety o f com-
pounds, fo r exam ple po lycycl ic arom at ic
hydrocarbons (PAHs) , benzene, to luene,
ethy lbenzene, and o- , m- , and p-xy lene
(BTEXs) [11 1 5] . Mos t o rgan ic com -
pounds cannot , however , be analyzed by
GC because they are non-vo lat i le o r semi-
vo lat i le , and thermal ly lab i le . To analyze
such compounds SPME should be used in
combinat ion wi th LC and we have devel -
oped a dev ice to enab le SPM E to be in ter -
faced wi th LC on convent ional o r micro
co lumns, and have used the techn ique fo r
the analysis o f pest ic ides in env i ronme ntal
water and of some drugs in huma n ur ine
[16 19].
As an ex tension of p rev ious work ,
S P M E co mb i n ed wi t h mi c ro co l u mn l i -
q u i d ch ro mat o g rap h y (1 .0 mm i .d . co l -
umn) has been used for the analysis o f
ami t r ip ty l ine in human ur ine. Reducing
the d iameter o f the LC co lumn has several
advantages , includ ing reduced consump-
t ion of o rgan ic so lven t as the desorp t ion
medium and the mobi le phase; th is re-
duces po l lu t ion of the env i ronment . With
samp l e p reco n cen t r a t i o n b y S P M E t h e
volume of so lven t requ i red was on ly
30 ixL, mu ch less than f or c onven tional
LLE. Co u p l i n g o f mi c ro LC wi t h S P M E
can be regarde d as the m ost ef fect ive sys-
tem in terms of performance, economy,
and env i ronmental p ro tect ion , because o f
low so lven t consumpt ion .
Materials and Methods
Materials
The SP ME holder and f iber assembl ies fo r
man ual sam pl ing were purchased f rom Su-
pelco (Bel lefon te , PA, USA). The
n o n p o
Orig inal Chro mat ograp hia Vol . 52 , No . 5 /6 , Sep tember 2000 309
0009-5893/00/02 309- 05 $ 03.00/0 9 2000 Friedr . Vieweg & Sohn Verlag sgesellsch aft mb H
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~ 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 NC Ic H a ) 2 L C H 2 C H C H 3 )C H 2 N C H ~ )
clomipramine trimipramine mianserine
Figure. The structures of the tricyclicantidepressants investigated n this study.
(a) without TEA
j I50mAU
5 6 7
5 mAU 2
r l _
i i i
0 10 20 30 40 50 60
Time (rain)
Figure . 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 inj ection vo-
lume was 1 ixL, the colum n temperature
was controlled at 40 ~ and the detection
wavelength was 240 nm.
pparatus
HPLC was performed with a Nanospace
SI-1 (Shiseido, Tokyo, Japan) comprising
a pump, a UVVis detector, a column
oven, and a degasser. Borwin chromato-
graphy software (Jasco, Tokyo, Japan)
was used for data acquisition and ha nd-
ling. Compounds were separated on a
150 • 1.0mm i. d., particl e size 5 ixm, Cap-
cell PAK Cis UG 80 polymer-coated Cis
atients
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 treat ment 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 st andard
compounds in a 20-mL sample vial with a
cylindr ical-shaped stirrer ba r (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 i njector by flushing solvent through
the interface.
Results an d 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. I t is apparent t hat
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 st ationary phase, because the
tricyclic antidepressants are basic com-
pounds. T riethylamine (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 i n 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
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4 . E + 0 6
9 amitriptyline
- - #- - imipramine p
x nortriptyline /
3 . E + 0 6
-- ~--- desipramine
'fi
~ 2 . E + 0 6
~D
1 . E 0 6
0 . E + 0 0 . . . . .
2 4 6 8 10
p H o f m a t r ix
F igur e 3 . T he e f f ec t o f pH on the e f f ic i ency o f ex t r ac t i on o f f ou r d r ugs .
SPM E cond i t ions : ex t rac t i on temper a tu r e 60 ~ s t i rr i ng r a t e 1200r pm;
sa l t concen t r a t i on 0 .4 g mL 1 ; deso r p t ion t ime 30 min ; deso r p t ion so lven t
ace ton i t r il e (30 ~ tL ). T he co ncen t r a t i on o f each d r ug i s 500 ngm L 1 . T he
r e l a t i ve s t andar d d ev ia t i on o f each da t a po in t was be tween 1 .9 and 7 .9%
(~= 3) .
O
6 . 0 E + 0 5
4 . 0 E + 0 5
2 . 0 E + 0 5
0 . 0 E + 0 0
9 amitriptyline
-- e- - mioramine
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
2 0 4 0 6 0 8 0
E x t r a c t i o n t e m p e r a t u r e ( ~
F igur e 4 . T he e f f ec t o f t emper a tu r e on the e f fi c iency o f ex t r ac t i on o f f ou r
d r ugs . SPM E cond i t i ons : ex t r ac t i on t ime 60 min ; pH o f mat r ix 9 ; s t i r ri ng
r a t e 1200r pm; sa l t concen t r a t i on0 .4 gm L 1 ; deso r p t ion t ime 30m in ;des-
o r p t ion so lven t ace ton i t ri l e ( 30 ~ tL ). T he concen t r a t i on o f each d r ug i s
500 ngm L 1 . T he r e l a t ive s tandar d dev ia t i on o f each da t a po in t was be -
tween 4.8 and 9.4% (n = 3) .
8 . 0 E + 0 6
9 amitriptyline
-- o . - i m i p r a m i n e /
x
nortriptytline
/
6 . 0 E + 0 6 . - /
"fi
O
4 . 0 E + 0 6
2 . 0 E + 0 6
: : : : :
4 1 . . . . . . . . . . . 4 . . . . .
0 . 0 E +0 0 " - . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0 5 0 1 0 0 1 5 0 2 0 0 2 5 0
E x t r a c t i o n t i m e ( m i n )
F igur e 5 . T he e f f ec t o f ex t r ac t i on t ime on the e f f ic i ency o f ex t r ac t i on o f f ou r d r ugs. SPM E cond i t i ons :
ex t r ac t i on t emper a tu r e 40 ~ pH o f mat r ix 9; s ti r ri ng r a te 1200 r pm; sa l t concen t r a t i on no add i t ion ;
deso r p t ion t ime 30 min ; d eso r p t ion so lven t ace ton i t ri l e ( 30 ~ tL ). T he concen t r a t i on o f each d r ug i s
500 ng mL 1 . T he r e l a t i ve s tandar d dev ia t i on o f each da t a p o in t was be tween 3 .9 and 10 .1% (n = 3 ).
a n a l y t e is a p o l a r c o m p o u n d . O t h e r
S P M E c o n d i t i o n s s u c h a s e x t ra c t i o n t i m e ,
s a l t c o n c e n t r a t i o n ( t o i n d u c e a s a l t i n g o u t
e f f e c t w h i c h c a n e n h a n c e e x t r a c t i o n e f fi -
c i e n c y i n c o n v e n t i o n a l S P M E ) , a n d d e s o -
r p t i o n t i m e w e r e o p t i m i z e d f o r s e l e c t e d
T C A s s u c h a s a m i t r i p t y l i n e , i m i p r a m i n e ,
n o r t r i p t y l i n e , a n d d e s i p r a m i n e . S a m p l e s
c o n t a i n i n g 5 0 0 n g m L 1 w e r e p r e p a r e d b y
s p i k i n g s o d i u m b o r a t e b u f f e r s o l u t i o n
( 5 m M , p H 9 ; 1 5 m L ) w i t h s t a n d a r d s o l u-
t i o n s ( 3 0 i xL ), b e c a u s e o f t h e s i g n i f i c a n t e f -
f e c t o f m a t r i x p H o n e x t r a c t i o n e f f ic i e n c y ,
a s is a p p a r e n t f r o m F i g u r e 3 . B e c a u s e
t h e se d r u g s a r e b a s i c c o m p o u n d s t h e y a r e
e f f i c ie n t l y e x t r a c t e d a t h i g h p H .
T h e e f fe c t o f e x tr a c t i o n t e m p e r a t u r e o n
e f f i c ie n c y w a s t h e n s t u d i e d ; t h e r e s u l t s a r e
d e p i c t e d i n F i g u r e 4 . W i t h t h e e x c e p t i o n
o f d e s i p ra m i n e a l l t h e T C A s i n v e s t ig a t e d
i n t h i s s t u d y b e h a v e d s i m i l a r l y . T h e e x -
t r a c t i o n t e m p e r a t u r e w a s , t h e r e f o r e , s e t a t
4 0 ~
W h e n t h e s t i r r i n g r a t e w a s o p t i m i z e d
t h e b e s t r e s u l ts w e r e o b t a i n e d a t 1 2 0 0 r p m ,
t h e m a x i m u m s p e e d av a i la b l e .
T o e n h a n c e e x t r a c t i o n e f f i c i e n c y i n
S P M E s a l t ( s o d i u m c h l o r i d e ) is u s u a l l y
a d d e d t o t h e s a m p l e m a t r i x . T h e r e s u l t s
o b t a i n e d d i d n o t , h o w e v e r , s h o w t h e p o s i -
t i v e b e n e f i t e x p e c t e d f r o m s a l t a d d i t i o n
p e a k a r e a s d e c r e a s e d w i t h i n c r e a s i n g
a m o u n t o f s a lt a d d e d . B e c a u s e m a t r i x p H
w a s a d j u s t e d t o 9 , a p p r o x i m a t e l y h a l f t h e
s a m p l e s w e r e i n t h e i o n i c f o r m i n t h e m a -
t r ix , a n d t h u s i o n i c s t r e n g t h s i g n i f i c a n tl y
i n f l u e n c e s e x t r a c t i o n e f f ic i e n c y . I f t h e i o -
n i c s t r e n g t h o f t h e m a t r i x i s in c r e a s e d , i n -
t e r a c t i o n w i t h t h e i o n s i s a l s o i n c r e a s e d ,
a n d s o d i s t r i b u t i o n b e t w e e n t h e fi b e r c o a t -
i n g a n d t h e s a m p l e m a t r i x m i g h t b e i n h i b -
i t e d . T h e s e r e s u l ts i m p l i e d t h a t n o s a l t
s h o u l d b e a d d e d .
W h e n d e s o r p t i o n t i m e w a s o p t i m i z e d
t h e r e s u l ts c l e a r l y i n d i c a t e d t h a t o n l y a
s m a l l c a r r y - o v e r e f fe c t w a s o b s e r v e d f o r a
d e s o r p t i o n t i m e b e t w e e n 3 0 a n d 9 0 m i n .
T h e d e s o r p t i o n t i m e w a s , t h e r e f o r e , f i x e d
a t 3 0 m i n i n f u r t h e r e x p e r i m e n t s .
F i n a l l y , t h e e x t r a c t i o n t i m e w a s o p t i -
m i z e d . T h e r e s u l t s a r e s u m m a r i z e d i n F i g -
u r e 5 . O n t h e b a s i s o f th e s e r e s u l t s i t s e e m s
t h a t e x t r a c t i o n f o r 3 h i s s u f fi c i e n t f o r
e q u i l i b r a t i o n o n a l l d r u g s b e t w e e n f i b e r
c o a t i n g a n d t h e s a m p l e m a t r i x ; n o s u b -
s t a n ti a l i m p r o v e m e n t w o u l d b e e x p e c t e d
f o r e x t r a c t i o n t i m e s l o n g e r t h a n 3 h .
U n d e r t h e se o p t i m i z e d c o n d i t i o n s
( s u m m a r i z e d i n T a b l e I ), S P M E m i c r o
O r i g i n a l C h r o m a t o g r a p h i a V o l . 5 2, N o . 5 /6 , S e p t e m b e r 2 0 00 3 1 1
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Table I. Optimized SPME conditions for four
tricyclicantidepressants 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 lmAU
b) SPME/LC
I20 mAU
3
4
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) patient s urine
I 2mA U
(b) 100 ng m U 1amitriptyline
I 2mAU
0 10 20 30 40 50
Time (min)
Figure 7. Chromatograms obtained, under optimized conditions, from:
(a) patient s urine diluted 15-foldwith 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 th at of direct micro
LC analysis without SPME, as is appar ent
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 am itriptyline, imipra-
mine, and nortripty line were 3, 9, and
12ngmL-1, respectively; the highest
LOD, 40 ng mL 1, was obtai ned for desi-
pramine. These values are a factor of 10
lower than co ncentrati ons inducing symp-
toms of intoxication in man [211. Good
linearity was obtain ed for each drug in the
range 5 to 1000 ng mL 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-
tenti on time indicated by an arrow seen in
Figure 7a. A standard solution of
100 ng mL 1 amitriptyline gave the chro-
matogram shown in Figure 7b; a larger
peak at the same retenti on 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 re tention time
of 20 rain in the uri ne chromat ogram was
identified as amit riptyline and because the
peak area in Figure 7a corresponds to
33.3 ng mL 1, its concentr ati on was deter-
mined as ca 500 ng mL 1 (33.3 ng mL 1 •
15).
onclusion
Analysis of amitriptyline in human urine
by SPME micro LC has been investigated
and several factors affecting extraction ef-
ficiency were optimized. Un der optim um
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 metho d has also been
shown to be a useful tool for cl inical 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-
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p h o r e s i s C E ) , t h u s e n a b l i n g f u r t h e r r e -
d u c t i o n o f s o l v en t c o n s u m p t i o n . T h e s e i n -
v e s t i g a t i o n s a r e c u r r e n t l y i n p r o g r e s s i n
o u r l a b o r a t o r y .
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Revised ma nuscrip ts received:
Ma r 14 and A pr 7 , 2000
Accepted: M ay 2, 2000
O r i g i n a l C h r o m a t o g r a p h i a V o l . 5 2 , N o . 5 /6 , S e p t e m b e r 2 0 0 0 3 3