Nontricyclic Antidepressants - Journal of Analytical Toxicology

Journal of Analytical Toxicology, Vol. 31, July/August 2007 

Simultaneous Determination of Nontricyclic 

Antidepressants in Human Plasma by Solid-Phase 

Microextraction and Liquid Chromatography (SPME-LC) 

Bruno los~ Gon~;alves Silva 1, Regina Helena Costa Queiroz 2, and Maria Eug~nia Costa Queiroz 1,* 

1Departamento de Qufmica, Faculdade de Filosofia Ci~ncias e Letras de Ribeir~o Preto, Universidade de S~o Paulo, 

Ribeir~o Preto 14040-901, SP, Brasil and 2Laborat6rio de Toxicologia, Faculdade de Ci~ncias Farmac~uticas de Ribeir~o Preto, 

Universidade de S~o Paulo, Ribeir~o Preto 14040-901, SP, Brasil 

I Abstract I 

A sensitive, seledive, and reproducible solid-phase 

microextraction and liquid chromatographic (SPME-LC) method 

for simultaneous determination of mirtazapine, citalopram, 

paroxetine, fluoxetine, and sertraline in human plasma was 

developed, validated, and further applied to analyze plasma 

samples obtained from patients with depression. Important factors 

in the optimization of SPME efficiency are discussed, including the 

fiber coating, extraction time, pH, ionic strength, influence of 

plasma proteins, and desorption conditions. The limit of 

quantitation of the nontricyclic antidepressants in plasma varied 

from 25 to 50 ng/mL with a coefficient of variation lower than 5%. 

The response of the SPME-LC method for most of the drugs was 

linear over a dynamic range of 50 to 500 ng/mL, with all of them 

having correlation coefficients greater than 0.9970. The 

performance of the SPME-LC method allowed the nontricyclic 

antidepressants analyses in therapeutic levels. 

Introduction 

Depression is one of the most common of all major psychi- 

atric illnesses. The antidepressants, selective serotonin 

reuptake inhibitors (paroxetine, fluoxetine, citalopram, and 

sertraline), and an antagonist of central (x~-adrenergic autore- 

ceptors (mirtazapine) have fewer cardiovascular and anti- 

cholinergic adverse effects than the tricyclic antidepressants. 

Many nontricyclic antidepressants are potent inhibitors of 

several different isozymes of the cytochrome P450 family 

of enzymes, and when these drugs are given simultaneously, 

they may mutually influence their biotransformation. In such 

* Author to whom correspondence should be addressed: Maria Eug~'nia Costa Queiroz, 

Departamento de Quimica, Faculdade de Filosofia Ci6ncias e Letras de Ribeir~o Preto, 

Universidade de S~o Paulo, Bandeirantes Avenue, 3900, 14040-901, Ribeir~o Preto, SP, 

BrasiL E-mail: mariaeqn@ffclrp.usp.br. 

cases, the risk of overdosing and adverse effects should be con- 

sidered, and a laboratory measurement of plasma levels be- 

comes necessary. Understanding the similarities and differ- 

ences in the pharmacology of these drugs can guide the 

clinician to an optimal use of this important class of antide- 

pressants (1,2). 

Several chromatographic methods that require complex iso- 

lation procedures to improve the sensitivity and specificity of 

the assay by removing any interference from the matrix while 

concentrating the analyte, such as liquid-liquid extraction, 

have been developed for the determination of nontricyclic an- 

tidepressants in plasma and serum (3-5). Solid-phase mi- 

croextraction (SPME), which was introduced by Pawliszyn and 

co-workers in 1990, is an alternative to the previously de- 

scribed classical method, which incorporates sample extrac- 

tion, concentration, and sample introduction into a single pro- 

cedure. SPME is defined as an extraction technique where the 

volume of the extracting phase is very small in relation to the 

volume of the sample and the extraction of analytes is not ex- 

haustive. The extraction efficiency is determined by the parti- 

tioning of the analyte between the sample matrix and the ex- 

traction phase. The partitioning is controlled by the 

physiochemical properties of the analyte, the sample matrix, 

and the extraction phase (6). SPME has been successfully ap- 

plied to analyze drugs in biological samples, mainly by coupling 

with gas chromatography (SPME-GC), and this has been the 

subject of several reviews (7-10). 

SPME has been introduced for direct coupling with liquid 

chromatography (LC) or LC-mass spectrometry (MS) in a 

small volume desorption interface (10) or off-line LC desorp- 

tion (11-13) in order to analyze a wide variety of compounds, 

especially for the determination or identification of thermally 

unstable, low to nonvolatile, or very polar compounds not 

amenable to GC or GC-MS. 

In this manuscript, a simple off-line SPME-LC method was 

developed and validated for simultaneous analyses of fluoxetine, 

Reproduction (photocopyinR) of editorial content of this journal is prohibited without publisher's permission. 313

paroxetine, citalopram, mirtazapine, and sertraline in human 

plasma. SPME variables that affect the extraction efficiency 

were optimized, and the influence of the fibers coatings and 

plasma proteins were investigated. The SPME-LC was further 

applied to analyze plasma samples from elderly depressed pa- 

tients. 

Material and Methods 

Reagents and analytical standards 

Fluoxetine and paroxetine analytical standards were kindly 

donated by Lilly (S~o Paulo, Brazil) and Libbs (S~o Paulo, 

Brazil), respectively. Citalopram, mirtazapine, and sertraline 

were donated by Roche (S~o Paulo, Brazil), and clomipramine 

(internal standard) was donated by Pfizer (S~o Paulo, Brazil). 

The working standard drug solutions, based on interval con- 

centrations, were prepared by diluting the stock solutions of 

these drugs (1 mg/mL in methanol) to a proper methanol 

volume. These solutions were stable for 45 days when the tem- 

perature was kept at -20~ The water used to prepare the 

mobile phase was previously purified in a Milli-Q system (18 

mega ohm) (Millipore, S~o Paulo, Brazil). Analytical grade 

sodium chloride (Merck, $5.o Paulo, Brazil) was used after 

purification by heating at 300~ overnight. Methanol and 

acetonitrile (both high-performance liquid chromatography 

grade), hydrochloric acid, and acetic acid were obtained from 

J.T. Baker (Phillipsburg, N J). Monobasic and dibasic phosphate, 

sodium borate, and sodium acetate were purchased from Merck 

(Darmstadt, Germany). 

Plasma samples 

Plasma from healthy volunteers not subjected to pharma- 

cological treatment for at least 72 h (blank plasma) was gently 

supplied by the Hospital das Clfnicas de Ribeir~o Preto (Uni- 

versity of S~o Paulo, Brazil). This plasma was used for the 

SPME-LC method validation. 

The plasma samples were colleted from patients subjected to 

therapy with nontricyclic antidepressants for at least two 

weeks. Blood samples were drawn 12 h after the last drug ad- 

ministration. The principles embodied in the Helsinki Decla- 

ration were adhered to, and the Ethics Committees at the Uni- 

versity of S~o Paulo in Ribeir~o Preto, Brazil approved the 

study. 

SPME equipment 

The SPME holder, the fibers coated with polydimethyl- 

siloxane (PDMS, 100-pro film thickness), polydimethyl- 

siloxane/divinylbenzene (PDMS/DVB, 65-pm film thickness), 

the carbowax/templated resin (CW/TPR-100, 50-pm film thick- 

ness), and the carbowax/divinylbenzene (CW/DVB, 65 pm film 

thickness) were all obtained from Supelco (S~o Paulo, Brazil). 

The virgin fibers were conditioned by immersion into methanol 

for 30 min and dried prior to use. 

Instrumentation 

The LC system used was a Pro Star Varian (model 230, CA) 

314 


liquid chromatography equipped with a 20-pL Rheodyne 

injector (Varian). Signals were monitored by a diode-array 

detector (Varian, model 330) and UV detector (Varian, model 

310) set at 230 nm. The separation of antidepressants was 

performed using a LiChrospher | 60 RP-select B (C]8) column 

(Merck) at room temperature (25~ with a mobile phase of 

an acetate buffer solution (0.25 mol/L, pH 4.5)/acetoni- 

trile/methanol (60:37:3, v/v/v), in the isocratic mode at a flow 

rate of 1.0 mL/min. The acetate buffer solution was prepared 

with 3.73 g of sodium acetate and 4.42 rnL of acetic acid (18 

tool/L) diluted in 500 mL of water (purified in a Milli-Q 

system). The mobile phase was filtered and degassed prior to 

use. 

SPME procedure 

In a conic glass vial (5 mL, Alltech, Paran~i, Brazil) sealed 

with a screw cap with silicone septum, 50 pL internal standard 

(10.0 pg/mL clomipramine) and 4 mE of buffer solution 

were added to 1 mL of the aqueous sample, which was spiked 

with the drugs standard solutions, resulting in a concentration 

level of 500 ng/mL. The sample was vortex mixed for 10 s 

before extraction. The vial was heated on a hotplate using 

a heater block (Supelco). The fiber was then immersed 

in the sample, and the extraction was performed under stirring 

[magnetic stir bar, polytetrafluoro ethylene (PTFE), trian- 

gular]. The drugs were desorbed from the fiber by exposure 

in a few microliters of the acetonitrile using a glass vial 

(V-shape, 1 mL, Alltech). After the organic extract evapora- 

tion, 100 pL of the mobile phase was added into the vial 

and vortex mixed. Fifty microliters of this extract was injected 

into the LC with IN and diode-array detectors (LC-UV-DAD 

system). 

Optimization of the SPME conditions 

The optimization of the SPME conditions was performed 

5OOOO 

40000. 

30.~- 

~IXI) 

10.1~ 

O- 

9 ~ 

9 C ~ 

~ne ~v 

v nut.re //* 

Ca~ITPR PDMS 'Ca~Ix/DVB ' PDMSI/DVB 

Figure 1. Evaluation of the coating on the SPME efficiency. SPME condi- 

tions: aqueous sample (1 mL) spiked with antidepressants (500 ng/mL), di- 

luted with 4 mL of borate buffer solution (0.05 molL, pH 9.0), T = 60~ 

t = 45 min. Desorption in acetonitrile during 15 rain at 60~ Triplicate 

analyses were performed for all experiments and the mean values were 

used to plot the results.


according to the SPME procedure described. IYiplicate analyses 

were performed for all experiments, and the mean values were 

used to plot the results. 

The choice of SPME fibers was the first step. PDMS, 

PDMS/DVB, CW/TPR-100, and CW/DVB coatings were evalu- 

ated to choose the appropriate coating for this application. 

After selecting the best fiber, the other SPME variables were op- 

timized with it. 

The influence of the matrix pH on the antidepressant ex- 

traction was investigated. For this purpose, four pH values: 4.5 

[acetate buffer (0.25 tool/L) prepared with 4.42 mL of acetic 

acid (18 tool/L) and 3.73 g of sodium acetate, diluted in 500 

mL], 6.9 [phosphate buffer (0.01 tool/L) prepared with 0.38 g 

of monobasic sodium phosphate and 0.49 g of dibasic sodium 

phosphate, diluted in 500 mL], 9.0 [borate buffer (0.05 mol/L) 

prepared with 4.0 g of sodium borate and 14.5 mL of hy- 

drochloric acid (0.03 tool/L) diluted in 500 mL], and 10.0 [bo- 

rate buffer (0.05 tool/L) prepared with 3.4 g of sodium borate 

and 1.25 mL of hydrochloric acid (1.0 tool/L) diluted in 500 

mL] were investigated. 

The ionic strength of the matrix solution (addition of 10% 

NaCl), the extraction temperature (15~ 30~ 45~ and 

60~ and the equilibrium time (15, 30, 45, and 60 rain) effects 

on the antidepressants extraction efficiency were also investi- 

gated. Different desorption times (5, 15, 30, 45, and 60 rain) 

were evaluated at 60~ 

High molecular weight compounds, such as proteins, can be 

adsorbed irreversibly to the fiber, decreasing the extraction ef- 

ficiency of the analytes. In order to preserve the fiber coating 

or increase the lifetime, the plasma matrix was substituted 

with aqueous samples during the optimization process. Once 

the conditions were optimized, the plasma samples were used 

during analytical method validation. 

The fiber was submitted to a cleanup process between ex- 

tractions to assure efficient protein removal by exposing the 

fiber to a water/methanol solution (50:50, v/v) under stirring at 

1200 xg for 15 min. 

4000O 

2~000 

I0~0- 

O" 

9 Mrta=~l 

9 Qtalopmm [ "\, 

ParoKeline / .'..--*--L\ 

9 Ruox~ne / --'/ ...... ~"* 

ql Sertmline l .... J~'/ 

9 ...~:~" .,."" "4 

. - ~ ..~7: "~ 

..... . ......... 

,i': .'. "/ .... S"::"" ....A ...... 

Figure 2. Effect of pH on the SPME efficiency. SPME conditions: aqueous 

sample (1 mL) spiked with antidepressants (500 ng/mL), diluted with 4 mL 

of buffer solution, T = 60~ t = 45 min. Desorption in acetonitrile during 

15 rain at 60~ Triplicate analyses were performed for all experiments, 

and the mean values were used to plot the results. 

r~ 

Results and Discussion 

Optimization of the SPME conditions 

The properties (physical and chemical) of the coating are 

crucial for the partition process. TWo distinct types of SPME 

fiber coatings were evaluated: PDMS, homogeneous pure 

polymer, which extracts analytes via absorption; and the second 

type that includes PDMS/DVB, CW/DVB, and CW/TPR mixed 

coatings, in which the primary extracting phase is a porous 

solid. The heterogeneous coatings extract analytes via adsorp- 

tion rather than absorption (6). 

Two simple rules should be applied for the selection of the 

coating in a first attempt for a new SPME method. The polarity 

of the coating should match the polarity of the analyte, and the 

coating should be resistant to chemical (pH, salts, and addi- 

tives) and physical (high temperature) conditions (7). Based on 

these rules and extraction efficiency results (Figure 1), the 

PDMS/DVB fiber coating, among those investigated (PDMS, 

PDMS/DVB, CW/TPR-100, and CW/DVB), was selected to opti- 

mize other SPME variables. 

The addition of 10% NaCI reduced the amount of antide- 

pressants extracted (data not shown), and probably the salt it- 

self interacted with the drugs in solution through electrostatic 

interactions (14,15), thereby reducing the ability of the drugs 

adsorb to the fiber coating. 

The matrix dilution procedure with buffer solution mini- 

mizes the effect of the protein in the SPME process. This pro- 

cedure decreases the matrix viscosity and increases the diffu- 

sion coefficients, allowing for a higher total amount extracted 

for a given extraction time. The pH of the extraction mixture 

is important for drugs possessing a pH-dependent dissociable 

group. It is only the undissociated form of the drug that will be 

extracted by the fiber coating (14,15). In a buffered extraction 

mixture, more drugs can be extracted by the coating than in an 

extraction mixture where the pH is not buffered. In an un- 

buffered extraction mixture, the ratio of undissociated to dis- 

sociated forms of the drugs can vary. Therefore, one does not 

achieve the continual transfer of the drug from the dissociated 

to the undissociated form, and then to the fiber coating (14,15). 

Matrix pH can be adjusted to optimize the SPME conditions for 

basic drugs. Thus, the sensitivity of the SPME-LC method was 

significantly improved by diluting the samples with the borate 

buffer solution in a pH 9.0, in which drugs (pKa values from 7.1 

to 9.9) were partially or totally in the nonionic form that en- 

abled them to be extracted by the SPME fiber (Figure 2). Pre- 

cautions must be taken when the direct-immersion SPME is 

used because extreme pH values (lower than 2 and higher than 

10) can damage the coating, making it difficult to implement 

large pH changes. The robustness of the PDMS/DVB fiber was 

confirmed with over 40 extractions with a minimum loss of ex- 

traction efficiency (data not shown). Changes in the recovery 

due to changes in the fiber were compensated for by the use of 

an internal standard quantitation procedure. 

The equilibration time is defined as the time after which the 

amount of analyte extracted remains constant, and it corre- 

sponds to the amount extracted after infinite time within the 

limits of experimental error (14). Figure 3 shows the time ex- 

traction profiles (15 to 60 rain) at different temperature values 

315

(15~ to 60~ Increased SPME extraction efficiency with in- 

creased time and temperature for SPME extraction was ob- 

served. The conditions selected among those investigated were 

45 rain and 60~ In order to save analytical time, without too 

much loss of the extraction efficiency, the following extractions 

were not performed in equilibrium partition conditions. The 

extraction time and mass transfer conditions were strictly con- 

trolled to assure good precision because small variations in the 

extraction time could affect the amount of analyte extracted by 

the fiber. When equilibrium times are excessively long, shorter 

extraction times can be used (14). 

The time extraction enhance neither reduced the amount ex- 

7,~. lle~/'AZ4RI~ 


tracted nor replaced drugs in the surface coating; therefore, the 

drugs competition by active sites on the coating did not affect 

the amount of analytes extracted (Figure 3). 

For solid sorbents such as PDMS/DVB, the linear range is 

narrower because of a limited number of adsorption sites on 

the surface. The theory of analyte extraction by porous SPME 

coatings was developed on the basis of the Langmuir adsorp- 

tion isotherm (6). According to this theory, the number of ef- 

fective surface sites where adsorption can take place is limited. 

When all such sites are occupied, no more analyte can be ex- 

tracted. They are therefore most appropriate for the moni- 

toring of either low concentration samples or higher concen- 

~1 ~o~oo .j CIT.4LQRRAM 

~ + 

2~oo / 9 

lO 

Extraction time (minl 10 2~) ~) ~) 50 60 

5,500 

m m 

_ 5,000 ...r 

80,:)00 

70,3(}0 - 

~o 

50,3(00 

40,3(]0 

30.300 

20,3~0 

10~0 

0 10 

110,000 

Ex'bact]on time (rain.) 

E:d'raction time (rain) Extraction time (rain) 

FLUOXEnNE 

Cl /-'/" 

[ ~,r,~ ~ / 

[-~-m~ .-"> 

/ .I" +...---+ . 

9 ~ . ~ . ~ . ~ . ~ 

Exizac~on lime (min) 

Figure 3. Effect of the time and temperature on the SPME efficiency. SPME conditions: aqueous sample (1 mL) spiked with antidepressants (500 ng/mL), diluted 

with 4 mL of borate buffer solution (pH 9.0). Desorption in acetonitrile over 15 min at 60~ Triplicate analyses were performed for all experiments, and the 

mean values were used to plot the results. 

316


trations for shorter periods of time (6,14). The SPME desorp- 

tion conditions were selected not only to obtain the highest 

quantitative desorption (maximum detector response), but 

also to minimize the carryover. 

Acetonitrile and the mobile phase were 

investigated as desorption solvent, where 

acetonitrile showed the best results (data 

not shown). The solubility of the analytes 

is better in acetonitrile. Desorption time 

was varied from 5 to 60 min. It was found 

that the peak areas increased from 5 to 15 

rain desorption time, but remained 

nearly constant for desorption time of 15 

to 60 rain, which corresponded to the 

complete desorption of the drugs from 

the SPME fiber, as no detectable carry- 

over was observed. A desorption process 

was performed at the best extraction tem- 

perature of 60~ 

The magnetic stirring improves the 

mass transfer of the analytes from the 

plasma sample to the fiber coating, thus 

reducing the effect of static layer zone 

produced close to the fiber as a result of 

slow diffusional transport of analyte 

through the stationary layer of liquid ma- 

trix surrounding the fiber (14). Thus, the 

samples during extractions were stirred 

at a maximum rate of 1200 x g. 

Based upon the data, it was concluded 

Clfalopram 

Tree mini 

LS. 

A 

'F 

that the best experimental conditions among those investi- 

gated for the nontricyclic antidepressants assays were as fol- 

lows: direct extraction mode using PDMS/DVB fiber (65-1Jm 

film thickness), 1.0 mL of sample plasma matrix in a conic 

A 3o 

' i' l' ~ . . . . . . . . . . . . . . . 

, , , , , , , , I , , r i v , i , , i i r i i i , i 

| 3 4 II II 7 I g 10 11 12131415 e 1 2 3 4 5 il 7 II g t4 1112 

The (rain) Time (mlnl 

Figure 4. SPME-LC chromatograms: blank plasma sample (A) and blank plasma sample spiked with 

antidepressants, resulting in 500 nglml (plasma level)(B). 

15- 

5- 

4- 

3- 

2- 

1- 

Plm~xetme 

J i i i , r , i i | , , , J , 

o ! 3 4 S e $ | 10 $1 12 I] 14 15 

Tme (rim) 

),&. 

B , C 

, 

1 2 5 4 S 

! 

I.$. 

LS 

II r II II III It 12 13 14 IS 

Figure 5. SPME-LC analysis of plasma samples from elderly depressed patients receiving therapeutic dosages. Drug concentrations found were 357 ng/mL of the 

citalopram (A), 289 ng/mL of the paroxetine (B), and 270 nglmL of the sertraline (C). 

B 

317

glass vial sealed with a screw caps with silicone septum, mod- 

ified with 4 mL borate buffer (pH 9.0), at an extraction tem- 

perature of 60~ using heater block, under magnetic stirring 

(stir bar, PTFE, triangular) at 1200 x g, for 45 rain, and fol- 

lowed by the drugs off-line desorption upon an exposure of the 

fiber to acetonitrile at 60~ during 15 min using a glass vial (V- 

shape). 

Validation of the method 

The use of internal standards for quantification is routine for 

many methods, and it can give satisfac- 

tory results for microextraction as well. 

Clomipramine, the internal standard se- 

lected, is closely related to the analytes of 

interest, particularly in terms of partition 

coefficient for the extraction phase. If the 

internal standard is extracted with a sig- 

nificantly different extent than the ana- 

lyte, error in the analysis will be either 

under- or over-stated (6). 

The specificity (selectivity) of the 

SPME-LC method is demonstrated by 

representative chromatograms from 

drug-free plasma sample (blank plasma), 

this same plasma sample spiked with an- 

tidepressants (resulting in 500 ng/mL) 

(Figure 4), and plasma samples obtained 

from patients subjected to therapy with 

nontricyclic antidepressants (Figure 5). 

Additional drug-free human plasma from 

several individuals were tested and 

showed no significant interference at the 

retention times of the analytes, which 

showed the ability of the method to mea- 

sure unequivocally the drugs in the pres- 

ence of endogenous plasma components. 

The precision (interassays coefficients) 

for the investigated drugs in the plasma 

were determined by spiking the plasma 

(blank) with different concentrations of 

each analyte, and submitting the spiked 

samples to the described procedure (Table 

I). The coefficient of variation (CV) was 

lower than 12%, indicating that the 

SPME-LC proposed method would be ac- 

ceptable for the quantitation of plasma 

concentration in clinical use. 

The limit of quantification (LOQ) of 

the investigated antidepressants in the 

plasma varied from 25 to 50 ng/mL 

(Table II). This LOQ was determined as 

the lowest concentration on the calibra- 

tion curve in which the precision (CV) 

was lower than 5% (Table I), and the 

signal-to-noise ratio was approximately 

5. The limit of detection (LOD) of the in- 

vestigated antidepressants in the plasma 

varied from 10 to 25 ng/mL (Table II), 

318 


based on a signal-to-noise ratio of about 3. 

The linearity of the SPME-LC method was determined using 

drug-free plasma spiked with the antidepressants from 25 to 

500 ng/mL for citalopram and fluoxetine, and from 50 to 500 

ng/mL for other drugs. These intervals were linear with the cor- 

relation coefficients better than 0.997 and CVs of the points of 

the calibration curve lower than 12% in all cases (Table I). 

These results show that the developed method permits the de- 

termination of antidepressants in plasma over a wide range of 

concentrations. Although no therapeutic window has been 

clearly defined for selective serotonin reuptake inhibitors, most 

Table I. SPME-LC Interassays Precision and Extraction Efficiency (Recovery) 

Antidepressants 

Recovery (%) 

Concentration Measured CV n = 5 

Evaluated Concentration (%) (ng/mt) 

(ng/mL) (ng/mL) n -- 5 (500 ng/mL) 

Mirtazapine 50* 53,3 3.8 

200 184.2 8.3 

300 279.5 11.9 

500 473.7 7.3 

Citalopram 25* 24.1 + 2.9 5.0 

200 193.5 _+ 9.6 6.9 

300 304.1 + 11.2 3.3 

500 496.4 15.7 7.9 

Paroxetine 50* 54.1 _+ 5.5 2.7 

200 187.9 _+ 21.2 11.8 

300 285.3 + 20.8 8.3 

500 478.5 + 33.1 14.8 

Fluoxetine 25* 27.2 3.6 5.1 

200 194.4 7.0 4.9 

300 290.0 _+ 10.6 4.0 

500 488.8 17.3 8.5 

Sertraline 50* 49.0 4.7 

200 190.8 3.2 

300 286.8 6.0 

500 479.9 5.6 

* LOQ = concentration corresponding to the limit of the quantitation. 

Table II. Linearity, LOD, and LOQ for SPME-LC Method 

Regression Line 

LOQ* (500 ng/mL) 

Antidepressants Slope Intercept r 2 Value 

Mirtazapine 0.0029 _+ 0.0001 0.0704 _+ 0.0619 0.9999 

Citalopram 0.0090 + 0.0001 0.1004 + 0.2203 0.9999 

Paroxetine 0.0017 _+ 0.0001 -0.3864 + 0.1871 0.9970 

Fluoxetine 0.0033 _+ 0.0001 -0.1745 0.0931 0.9998 

Sertraline 0.0016 + 0.0001 -0.0671 _+ 0.0338 0.9999 

* LOQ = Concentration that correspond to the limit of quantitation; signal/noise = 5. 

* LOD = Concentration that correspond to the limit of detection; signal/noise = 3. 

17.1 

8.08 

11.9_+1.7 

10.2 

8.12 

LOD* 

(n~/mL) 

25.0 

10.0 

25.0 

10.0 

25.0 

LOQ 

(ng/mL) 

50.0 

25.0 

50.0 

25.0 

50.0


reports showed that the plasma concentrations ranged be- 

tween 50 to 200 ng/mL (16). The SPME-LC method showed 

LOQ values and linearity very close to those described in the lit- 

erature for classical methods (LLE-LC and SPE-LC) used for 

the analysis of the same antidepressants in biological samples 

(3-5,11,18). 

The recovery rates were assessed by replicate analysis (n = 5) 

of plasma samples spiked with analytical standards that re- 

sulted in a concentration level of the 500 ng/mL (Table I). 

SPME of drugs in highly complex matrices, such as plasma 

samples, usually show low recovery rates, as is well docu- 

mented in the literature (19,20). SPME is not an exhaustive 

process; it is based on the partitioning of analytes between a 

very small phase immobilized on an SPME fiber and the matrix. 

Low recovery does not necessary imply insufficient either sen- 

sitivity or precision of the method (15). In this work, the linear 

calibrations were calculated with data that resulted from 

plasma samples spiked with drugs after SPME-LC procedures. 

It is important that calibration samples have the same com- 

position as test samples. If the matrix in test samples differs 

from that of calibration samples, partitioning will not be the 

same and the calibrations will not be valid. 

The proposed SPME-LC method was compared with the tra- 

ditional methodology of liquid-liquid extraction (LLE-LC) (21). 

Drug-free plasma samples were spiked with standard solutions 

in concentrations resulting in the following plasma level in- 

tervals: mirtazapine, paroxetine and sertraline (50-500 ng/mL), 

citalopram, and fluoxetine (25-500 ng/mL). These samples 

have been submitted to different sample preparation methods 

and the obtained results were compared. In an agreement with 

linear regression graphs, the correlated methods showed sat- 

isfactory linearity (Table III). Therefore, the measured con- 

centrations, according to the different methods, were equiva- 

lent. Thus, the accuracy of the developed SPME-LC method 

was confirmed. 

Based on these analytical validation results, it can be ex~ 

pected that the SPME-LC methodology developed would be 

adequate for antidepressant analysis in therapeutic levels. 

Clinical application of the method 

In order to evaluate the proposed method for clinical use, the 

described protocol was applied to the analysis of plasma sam- 

ples from elderly depressed patients (Figure 5). Peak shapes and 

resolution were very similar to those obtained using spiked 

blank plasma, and no interference was apparent. 

Drug concentrations found in these samples were 357 ng/mL 

of the citalopram (Figure 5A), 289 ng/mL of the paroxetine 

(Figure 5B), and 270 ng/mL of the sertraline (Figure 5C). The 

Table IIh Comparison Between Methods: SPME-LC and LLE-LC 

plasma samples were colleted from elderly depressed patients 

in therapy with Celexa | (50 rag/day), Aropax | (40 rag/day), and 

Zoloft | (150 rag/day), respectively. 

Clinically significant depressive symptoms are detectable in 

approximately 12% to 36% of geriatric patients with another 

nonpsychiatric general medical condition. The prevalence of 

major depression ranges from 10% to 27% in stroke patients, 

from 40% to 65% in victims of myocardial infarction, from 

30% to 40% in patients with Alzheimer's disease, and from 

20% to 25% in cancer patients. Because of the aging-related 

pharmacokinetic and pharmacodynamic changes, it is not possible 

to automatically extrapolate findings on the efficacy or tolerability 

of antidepressants from younger to older populations. 

In older patients, noncompliance and medication errors are 

disturbingly common. In clinical practice, the effort to determine 

an individual dose optimum for an antidepressant drug is 

often guided by a trial-and-error dose titration strategy. However, 

with the antidepressant drugs used in psychiatry, therapeutic 

drug monitoring is a long-established tool for finding 

the individual dose optimum, and it always increases not only 

efficacy and safety (22-24). 

Conclusions 

It was demonstrated that SPME in combination with LC-UV- 

DAD, without the requirement of a special interface, offers 

high sensitivity and enough reproducibility to permit the quan- 

tification of nontricyclic antidepressants in human plasma 

after oral administration of the antidepressant. Thus, the pro- 

posed SPME-LC method can be a useful tool to determine 

nontricyclic antidepressants in plasma concentrations in pa- 

tients receiving therapeutic dosages. The method may be also 

applied to evaluate plasma levels in urgent toxicological anal- 

yses after the accidental or suicidal intake of higher doses. In 

these cases, the plasma samples dilution should work with 

plasma blank amount. 

Acknowledgments 

Antidepressants Linear Range Linear Regression Correlation Coefficient (r) 

Mirtazapine 50.0-500.0 ng/mL y =-4.21 + 1.05x 0.9999 

Citalopram 25.0-500.0 ng/mL y = -4.92 + 0.99x 0.9988 

Paroxetine 50.0-500.0 ng/mL y = -9.45 + 1.05x 0.9999 

Fluoxetine 25.0-500.0 ng/mL y = -6.83 + 1.02x 0.9998 

Sertraline 50.0-500.0 ng/mL y = -9.78 + 0.04x 0.9987 

This work was supported by grants from FAPESP (Fundar 

de Amparo ~ Pesquisa do Estado de S~o Paulo) and CAPES 

(Coordena(~o de Aperfeir de Pessoal de Nfvel 

Superior). 

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Nontricyclic Antidepressants - Journal of Analytical Toxicology

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