Simultaneous analysis of clozapine, clomipramine and their metabolites by reversed-phase liquid chromatography

Prog. Neuro-Psychopharmacol.

ELSEVIER

& Biol. Psychiat. 2001, Vol. 25, pp. 519-533 Copyright © 2001 Elsevier Sclence Inc. Pnnted m the USA. All rights reserved 0278-5846/01/S-see front matter

PII: S 0 2 7 8 - 5 8 4 6 1 0 0 ) 0 0 1 8 4 - 6

SIMULTANEOUS ANALYSIS OF CLOZAPINE, CLOMIPRAMINE AND THEIR METABOLITES BY R E V E R S E D - P H A S E LIQUID CHROMATOGRAPHY LIONELLA PALEGO, LILIANA DELL'OSSO, DONATELLA MARAZZITI. LUCIANO BIONDI, NANNINA SARNO, ANTONIO CIAPPARELLI. ANNALISA GIROMELLA, GINO GIANNACCINI, ANTONIO LUCACCHINI and GIOVANNI B. CASSANO Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Pisa, Italy (Final form, March 2001)

Abstract

Palego, Lionella, Liliana Dell'Osso, Donatella Marazziti, Luciano Biondi, Nannina Sarno, Antonio Ciapparelli, Annalisa Giromella, Gino Giannaccini, Antonio Lucacchini and Giovanni Battista Cassano: Simultaneous analysis of clozapine, clomipramine and their metabolites by reversed-phase liquid chromatography. Prog. Neuro-Psychopharmacol. & Biol, Psychiat. 2001,25,pp. 519-538.~Y2001El~erSc~nceInc. i. The authors present here a sensitive and rapid reversedphase liquid chromatographic method which enables the simultaneous analysis in plasma of two different drugs and their metabolites: the atypical neuroleptic clozapine and the tricyclic antidepressant clomipramine. 2, Samples and the internal standard (dibenzepine) were extracted through an automated solid-phase procedure, evaporated to dryness and injected into the chromatograph, Mobile phase was a mixture of water and acetonitrile (63:37, v:v) containing TEMED and triethylamine. The total chromatographic time was of 14 min and analyte peaks were detected by means of an ultraviolet spectrophotometer preset at 254 nm. 3, Results revealed an assay sensitivity of 5 Mg/L for clozapine or norclozapine and of i0 ~g/L for clomipramine and desmethylclomipramine, Recoveries for these drugs and their metabolites were more than 60% and their coefficient of variation (within day and day-to-day) ranged from 1.3 % to 2.5 %. In spiked plasma, within day and day-to-day coefficients of variability (CV) were less than 5%, The simultaneous evaluation of these two drugs with adequate sensitivity and precision makes it particularly useful for therapeutic drug monitoring during mona- or polypharmacotherapy,

519

L. Palego et al.

520

Kevwords:

clomipramine, clozapine, their demethylated dibenzepine, reversed-phase liquid chromatography

metabolites,

Abbreviations: obsessive-compulsive disorder (OCD), photodiode array detector (PDA). reversed-phase liquid chromatography (RPLC). selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs) Introduction

The

use

of

atypical

clozapine

(@Leponex).

serotonin

reuptake

fluoxetine,

current

of as

well

as

et al,

In

clomipramine tricyclic 1980:

mood

concurrent

(McDougle

et al,

clozapine

or exacerbate

1994:

exists

al,

1993:

drug-drug

concerning

clozapine

and

analytical

Biondi

drug

of

clozapine of

et

al.

Montgomery, for

during

1996).

treatment

to clomipramine

As very

However,

few

the

by adverse information

interactions

between

and Hedenmalm,1996). rapid

and

two

it

and sensitive

clomipramine

these

OCD

disorders

et al.

may be tempered

(Spigset

with

to precipitate

1999).

of a simple,

monitoring

1995:

approach

Steinert

al,

et al.

employed

(Marks

spectrum

pharmacological

to dispose

features

commonly

symptoms

et

Frankeburg,

Cassano

positively

interaction,

clomipramine

subwith

drugs

for

the

during

treatment.

Normal the

the

technique

therapeutic patient

responds

and

a potential

of polypharmacotherapy

and

could be useful

are

which

some

clozapine

useful

1994:

has revealed

becoming

of

OCD

psychotic

Fuchs.

is

of

et al.

a

obsessive-compulsive

for schizophrenia usefulness

cases,

potent

psychotic

treating

1988: Piccinelli some

as

fluvoxamine.

1991:

one of the most

Moreover,

et

with

et al,

(@Anairanil),

with

effects

disorders

combination

in

highly

(Baldessarini

for

such

schizophrenia

particular,

be,

patients

the

pharmacotherapy

McElroy

antidepressants

may

(Tejera

to

sertraline

symptoms

1990;

Zohar et al,

1996),

the

drugs,

clomipramine,

and

for

negative

(Calabrese 1997),

addition

neuroleptic-refractory

predominant 1991),

in

inhibitors

paroxetine

increasingly types

antipsychotic

and most

reversed-phase published

liquid

techniques

chromatographic for

detecting

methods these

S i m u l t a n e o u s a n a ~ s i s o f c l o ~ p i n e , c l o m i p r a m i n e a n d t h e i r metabolites

compounds

(Godbillon

Volpicelli the

et

al,

analysis

of

metabolites A

and Gauron,

1993), a

single

in biological

RPLC

method

quantitative

with

and

Hating

they were

specific

here.

standard,

starting

by

clozapine

and

UV

detection of

for

the

clozapine,

using

1988;

and

to its

that

plasma as

by

has

the

previously

metabolites

simultaneous norclozapine.

in

dibenzepine

from

their

al,

limited

compound

desmethylclomipramine

developed

et

all

fluids,

analysis

clomipramine

1981;

although

521

been

internal

described

Weigmann

and

for

Hiemke

(1992). Methods

Druas

All

reagents

Distilled

were

water

of

analytical-grade

(Sigma

acetonitrile

(Riedel-de

(J,T, Backer,

Deventer,

Chemical Haen,

tetramethylethylenediamine (Milan,

Italy),

Holland)

and

Dibenzepine (Milan.

acetic

(the

internal

from

from

(Rome, obtained

provided

(Milan,

RBI

desmethylclomipramine

Italy);

(Milan, were

Italy);

gifts

from

Sigma

(Deventer,

was

from

standard)

methanol

Backer

Merck

Italy)

N,N,N',N'-

obtained

J.T.

better,

and

clozapine was generously

Institute

purchased

FRG)

was

from

acid

or

Milan,

were HPLC grade.

(TEMED)

triethylamine

Italy);

Research

Seelze,

Holland)

(RG]

Co.,

Italy). from

RBI

by Sandoz

norclozapine

was

clomipramine

and

Novartis

(Milan,

Italy), The

stock

prepared months. of

solution

in methanol

and

the

internal

stored

at

standard

-20°C

for

(1 g/L) less

From this stock solution a working standard

dibenzepine

1:50

of

was

in methanol

obtained

to yield

a

by diluting

than

6

solution

the stock

final dibenzepine

was

solution

concentration

of 20 mg/L, Stock prepared

standard in

solutions

methanol

and

of

analytes

stored

at

(I g/L)

-20°C.

were

Working

also

plasma

L. Pale~ e t a l .

522

standards stock

solutions

solution and

(drug-free

spiked

with

drug-free

as

follows:

1:4

were

plasma

of i mg/L of clozapine,

desmethylclomipramine.

diluted

plasma)

This

made to

by

yield

norclozapine, solution

(250 Ng/L);

diluting a

was

then

1:20(50

final

clomipramine serially

~g/L)

and

1:40

Model

168

(25 ~g/L).

AeDaratus A

Model

116

photodiode

liquid

array

chromatograph

detector

(PDA)

Instrument,

Milan,

system

chromatography

for

Gold

PC,

were

performed column

used

at

(5

Italy), for

20-22°C

~m

Chromatography

the

division

from

by

250

package

x

4.6

diameter)

mm

5.0)

Analysis

were

reversed-phase C18

Milan,

210A

(Beckman)

phase extraction, a

Speedvac

isocratic 1.5

trap

liter,

(Savant)

(Savant,

separation

ml/min

i00 M1

loop

(Beckman).

samples were essicated

centrifuge

condensation at

and

was

linked

RT4104)

performed

flow-rate.

Mobile

a

by

The

injection For

to dryness to

preset

phase

(Waters

Italy),

chromatograph was also fitted with a manual sample valve

Beckman

(version

Symmetry

Millipore,

(Beckman

the

chromatography. a

of

a

Beckman

managed

software

with

particles

all

both

and

solidthrough

refrigerated

at

pumping

-80°C. mobile

composition

The phase

was

per

370 ml of acetonitrile and 630 ml of distilled water,

containing adjusted

0.5 %

(v:v) TEMED

and 0,01%

to pH 6.5 with acetic

and Hiemke,

1992).

acid

The monitoring

(v:v)

triethylamine,

(modified

wavelenght

from Weigmann on the PDA was

254 nm, Experimental Procedure Blood tubes

was withdrawn

from

out-patients psychotic accordingly at

the

in

the morning

fasting drug-free (5 men

features to DSM

and (n=8)

5 women) or

OCD

IV diagnostic

"Dipartimento

di

into

lithium

healthy volunteers with

mood

(n=l)

or

criteria,

Psichiatria,

and

heparin from i0

disorders anorexia 1994,

with (n=l),

recruited

Neurobiologia,

Simultaneousanalysisofclo~pine, clomipramineandtheir metabolites Farmacologia patients the

e Biotecnologie"

gived

study,

their

The

patients,

(mean ± S,E.M.), doses

of

of

whose

(Table

and

only

(Table

collected

12-15

hours

i).

until

the

solid-phase

plasma

sample

reversed

were

mixed

and

wad

its

at

The

this

by

cartridges of

were

water:

washed

Weigmann

of

internal through Italy),

samples

5% methanol

The

plasma

conditioned

with

(I ml) in water

1 ml of methanol.

was

evaporate

to

dryness

for 45 min at 45°C through

was

and

1 ml

correct

then,

with

For

(Waters).

to

at

slightly

Milan,

order

in

weeks. a

then passed

added

were

centrifuged

20 ~g/L

Waters,

to a refrigerated

residue of

and

taking

and stored

two

the

the

receiving

metabolites.

of

(Oasis,

after

blood

employed

described

years

were

and

processor

eluate

~i

intake

in

constant

were

collected within

50 Ml

1 ml

samples

linked

I00

1.98

days

2

automated

cartridges

centrifuge

phase.

±

patient's

last

and

with

Oasis

and

final

-80°C,

of

was

that

drugs were eluted with

The

30,2 12

All

introduced

patients

authors

clozapine

sample

methanol

centrifugating

of Pisa,

be

while

performed the

from

for recovery.

loaded

the

cartridges

a

Five

(I Mg dibenzepine)

standard

of

(v:v):

was

phase on

internal 1 ml

for

solution

mounted levels

assay,

procedure (1992)

than

ml

Plasma

extraction,

Hiemke

to

was

more

I0

after

at 3,000 x g for i0 min,

standard

I)

clomipramine,

clozapine

modified

age

the pharmacotherapy,

clozapine

-20°C

consent

were at the steady state receiving

clozapine

beginning both

of the U n i v e r s i t y

informed

523

condensation

dissolved

suspension

in

were

600

by

a Speedvac trap preset

~i

injected

of

mobile

into

the

chromatograph. Statistical Standard

Analysis calibration

four different compounds) software

curves

concentrations

were

obtained

(Beckman)

from

the

for (25,

by

analytes 50,

means

250 and of

least-squares

the

in

plasma

I000 ~g/L Gold

linear

at of

system

regression

L. P a l e g o

524

of

the

peak

height

(dibenzepine)

Plasma L e v e l s

ratios

versus

of

~ al.

the

analyte/internal

standard

the known amount of the analytes.

in P a t i e n t s Taking C l o z a p i n e

(CL) and C l o m i p r a m i n e

(CMI).

Dose

CL

NCL

(~L)

(~grL)

1.72

122

33

0.27

1.40

30

10

0.33

40

1.17

149

20

0.13

169

5.20

297

92

0.31

389

5.80

395

86

0.21

481

Cn*

Dose

CMI*

ratio

Sum

(~gIL)

CMI

DCMI

ratio

(~g/L)

(~grL)

Sum

(~L)

155

2.40

0.96

100

32

0.32

132

98

121

1.23

0.12

0.73

203

14

0.07

217

67

71

1.05

138

1.95

3.12

41

32

0.78

73

222

165

0.74

387

1.76

0.60

13

21

1.60

34

141

115

0.81

256

139

1.11

262

197

176

0.89

47.9 ±

0.51 ±

195 ±

145±

129 ±

0.94 ±

274 ±

13 °

0.16 °

47 °

29 °

19 °

0.09 °

47 °

3.05

0.83

2.5 ±

1.25 ±

123

0.56 °

0.47 °

147

±

38 °

219

373

*Doses are mg per day of clozapine or c l o m i p r a m i n e / K g body weight, °Data in bold represent the mean ± S,E,M, of the correspondent column values. NCL = N o r c l o z a p i n e and DCMI = N-desmethylclomipramine, Results

In Table standard

2, r e t e n t i o n

are

potentially interference

interferring was

had been tested, retention < 0,5%

times

reported,

observed

of all

together

analytes

with

psychotropic for

any

drug

and

those drugs, or

internal of

other Minimal

metabolite

Within and between run v a r i a n c e

(CV) of

that the

time of the a n a l y t e s and the internal s t a n d a r d was

(data not shown),

Simultaneousana~sisofclo~pine, clomipramineandtheir metaboli~s 2

Tabl~

Potential

Time

Interferences

Drug Dibenzepine

Retention time 2.80

Diazepam

3.29

Norelozapine

3.50

Desmethyldiazepam

3.92

Paroxetine

5.19

Haloperidol

5.72

Fluvoxamine

6.49

Nortriptyline

6.64

Imipramine

6.74

Clozapine

7.12

Norfluoxetine

8.67

Fluoxetine

Drugs

were

mg/L)

and

detection

The

9.81

Desmethylclomipramine

10.91

Clorpromazine

11.02

Clomipramine

13.82

injected evaluated

as

extracted

through

the

spiked

plasma

reported

method

samples with

(I

U.V

at 254 nm.

analytical

recovery

spiked with

two different

~g/L)

the

and

efficiencies

were

ranging

1.3

from

dibenzepine,

from

for norclozapine, and

525

was

measured

concentrations

internal

standard

constant

(I

(coefficient

to

2.5)

and

93

to 96 %

averaged for

in drug-free

plasma

of analytes

(50-250

mg/L). of from

clozapine,

Extraction

variation, 89 from

to 95

CV

92%

for

to

99%

from 77 to 93% for N - d e s m e t h y l c l o m i p r a m i n e

from 72 to 80% for c l o m i p r a m i n e

526

L. Palego et al.

Detection

Limit

Figure

i

drug-free

shows

a

control

typical

plasma

dibenzepine

(DBZ,

background,

without

chromatogram

spiked

img/L).

with

The

the

internal

baseline

contaminant

obtained shows

a

from

a

standard very

low

of about

4:1)

plasma peaks.

0,03 .....

"

i

0,025 ?

:

I

0,02

,~ 0,015 < 0,01 0,005

0

5

10

15

time, m;-

Fig, i: Blank plasma extract spiked with the internal standard (img/L) DBZ: Dibenzepine

The was

detection

assessed

extract

by

limit

(signal

analyzing

and was of 5 ~g/L

of i0 ~g/L

and

i000

~g/L.

curves

they were From

were

linear

linear

to noise

ratio

replicates

for clozapine

for clomipramine

Calibration weeks

15

of

blank

plasma

and norclozapine

and

and N-desmetilclomipramine,

evaluated

repeatdly,

in the analysed

regression

range

analysis

mean

over

two

from 25 to slope

(±

Simultaneous analysisofclozapine, clomipramineandtheir S.D,) 5.1

and

intercept

± 0.3

for

clozapine

0.4 for n o r c l o z a p i n e for

clomipramine

0.9993)

(± S.D,)

values

(r= 0.9994);

(r= 0.9997);

(r=

were:

0.9992):

711

2560

3141

metabolites

1065

527

± 45.8

± 25.6

and

and

12.1

±

± 156.6 and 5.4 ± 0.2

± 97

and

8,1

± 0.I

(r=

for desmethylclomipramine.

Accuracy and P r e c i s i o n M e t h o d ' s a c c u r a c y and p r e c i s i o n were d e t e r m i n e d of plasma

from d r u g - f r e e

healthy volunteers

different

concentrations

of all

analytes

and

medium

corresponding

to

Intra-assay

concentrations. running

15

low

replicates

assay v a r i a t i o n for 15 days

was

per

established

spiked

(50 and

with

was

per

by running

plasma

evaluated

run.

two

300 Ng/L),

analyte

variation specimen

in a pool

while

by

inter-

two runs per day

(Table 3). The CV range was between

2.3 and 4,9.

I n t r a - A s s a y and I n t e r - A s s a y V a r i a t i o n of A n a l y t e Plasma L e v e l s

Inter-Assay (n = 15)

Intra-Assay (n = 15 )

Mean, mg/L (4- SD)

(CV, %)

50 4- 2

(4.5)

514- 2.3

(4.5)

298 4- 9.5

(3.2)

302 4- 13

(4.4)

51 ± 1.8

(4.6)

51 4- 2.5

(4.9)

Clozapine

Norclozapine

Mean, mg/L (± SD)

(CV,%)

302 + 8.0

(2.6)

301 ± 14.

(4.8)

Cl0mipramine

55 4- 2.4

(4.5)

49 4- 1.8

(4.6)

301 ± 5.4

(2.3)

300 4- 15

(4.3)

Desmethylelom.

49 4- 1.8

(4.6)

52 :h 2.0

(4.0)

298 4- 12

(4.2)

288 + 9.4

(3.5)

Patient Plasma L e v e l s and Chromatoarams.

Table presented

1 shows as

mg

individual per

day/Kg

and body

mean

values

weight,

of

plasma

drug

doses,

levels

for

L. P a l e ~ etal. 528

each

analyte,

metabolite Figure

the

ratio

metabolite/drug

and

the

sum

+ drug. 2

shows

taking clozapine

a

chromatogram

obtained

from

a

patient

and clomipramine,

0,03

DJ i

0,025 0,02 0,015 0,01 NCL 0,005 0 0

5

10

15

ti.~ (mi.) Fig.2.

Chromatogram

patient

in Table

i.

desmethylclomipramine:

This method

paper for

high

detection, clozapine single

rather phase,

specific

previously

their metabolites is increasingly

of schizophrenia,

time-consuming UV

determination

of

and

wavelenght

sensitive of

metabolites

The methods

although

and

determination

liquid

clomipramine sub-types

a

simultaneous

and their desmethyl

compounds~

that

the

last N-

NCL=Norclozapine.

performance and

to

CL=Clozapine:CMI=Clomipramine;DCMI=

reports

the

clomipramine by

corresponding

reported

clozapine.

in human plasma

chromatography were

analytical

using

for clomipramine,

built

for detection

combination

of

used

treatment

in the

and mood expensive and/or

of each compound,

clozapine

disorders, to

UV

change

analytical this method

of

Since assay column

of and

some it is

mobile for

can considerably

Simultaneousana~sisofclo~pine, clomipramineandtheir metabolites improve

the

study

interactions.The that

proposed

alone,

but

of

clozapine

method

by

we

and

described

Weigmann

and

introduced

clomipramine

here

is quite

Hiemke

some

(1992)

drug-drug similar

for

modifications

529

to

clozapine

in

order

to

obtain also a good detection of clomipramine, Solid-phase modified

extraction

from Weigmann and Hiemke

work of our research pak

cartridges

group

were

cartridges

significantly for

have

obtained

been

consuming, acetic

since

the drugs,

filled

with

we

instead

of

which

1 ml

a

(Haring

et

al,

1988:

1993).

SSRIs

increase

(approximately Kabra

et al,

loss

of

their from

1981).

method

compensated

Weigmann

We

chose

slightly

2 ml acid

In

order

to

separating

column

(Waters)

improving

symmetry

(data not shown), for

UV

detection

of

for this compound at 254 nm

absorbance to 215

the time-

C18

and Hiemke, in

nm)

1992;

for

the

(Walace

to preset

sensitivity

by

was

some authors suggest

200

which

for

particles,

methods

Conversely,

extraction

raise

Symmetry

clozapine report maximum average et al,

Oasis

Moreover,

methanol,

usually

silica

described

of

used was more

of sample elution

employed

where Sep-

analyte

method,

previously

of peaks and method's sensitivity Previously

the

previous

the volume

synthetic

2000),

and desmethylclomipramine

the

problems

slightly

introduction

improved

procedure

in methanol

resolve basic

by

been

(1992) and from a previous

The

clomipramine

evaporation

has

(Palego et al,

used.

recoveries sample

procedure

Volpicelli

that TCAs and

far

UV

the PDA

at

clomipramine

concentrating

region

and Dahl,

the

1967:

254

has

sample

nm: been

before

injection, Extraction recoveries values

as

were

(Godbillon Volpicelli

procedure well in

and

the

as

highly

within range

Gauron,

et al,

was

of

1981;

1993),The

day

reproducible and

those Weigmann

and

day-to-day reported and

sample

precison by

Hiemke.

others 1992:

limit of detection was of 5 ~g/L

for clozapine and norclozapine,

and of i0 ~g/L

for the other

L. Ps]e~ etaL

530

analytes, more

indicating

than

that

adequate

for

the sensitivity

the

therapeutic

of the method was drug

monitoring

of

these compounds, Blank

plasma

Chromatograms clomipramine separation, 2.5-15

revealed

obtained and

showed

interferences

retention

norclozapine

clean

range.

(1991)

and Perry et al,

those

reported

by Volpicelli

higher

norclozapine

should

be

that

column

(1991),

the

discrepancy.

these

Since

if

cotreatment

greater

with

number

of

and Gauron

(1981)

interference other

TCAs

with was

investigate

patients

and will

with

have

used and

from found

However, a

it

different

were

able

which may account of

patients

which

may

to for

taking

clarify plasma (1990),

or

this

produce

involving this

a

point,

concentrations by Godbillon

No

significant

benzodiazepines,

makes

mono-

clozapine

those observed

Mavissakalian

in

who

investigations,

phenothiazines, found,

Lovedahl

we were not able to determine

patients,

and

as

with

ratio.

C-6)

number

Further

consistent

and

while differing

and

Clomipramine and desmethylclomipramine were also quite

the

well

authors

clomipramine

interactions.

of

in

clozapine

(1993),

levels,

the

clozapine alone was too small, drug-drug

al.

(Spherisorb

measure also clozapine-N-oxide

degree

as

et

with

peaks

consistent

concentrations

considered

chromatographic

good

other

plasma

concentrations

norclozapine/clozapine ratio were

extract.

treated

a

with

Our

et al.

plasma

patients

clozapine,

without

min

a from

method

SSRIs

or

useful

to

polypharmacotherapy

with

these drugs, In conclusion, patient's

clinical

this method will response,

effects due to pharmacological facilitate

the investigation

correlations response,

between

analyte

enable

compliance

the monitoring and

interactions. of more plasma

potential

This method may

throughly levels

of

side

and

the possible therapeutic

Simultaneous analysis of clozapine, clomipramine and their metabolites

531

Conclusions The method

present which

clomipamine desmethyl

paper

enable plasma

reports levels

metabolites

a simple

the measurement as

well

in patients

and

rapid

ol either as

during

their

analytical

clozapine

or

respective

the pharmacological

treatment.

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