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



~ 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

310 Chro matogra phia Vol. 52, No. 5/6, September 2000 Original



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



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-

312 Chromat ographi a Vol. 52, No. 5/6, September 2000 Original



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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