ORIGINAL

Silver Nanoparticles–Polyaniline Nanocompositefor Microextraction in Packed Syringe

Habib Bagheri • Solmaz Banihashemi

Received: 9 October 2013 / Revised: 14 January 2014 / Accepted: 14 January 2014 / Published online: 31 January 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract A rapid, convenient and reliable method for

microextraction in packed syringe (MEPS) of the loop

diuretic furosemide (FUR) in urine along with high-per-

formance liquid chromatography (HPLC) was developed.

A nanocomposite based on silver nanoparticles/polyaniline

(Ag-NPs/PANI) was synthesized and used as the MEPS

packing material. This nanocomposite was prepared con-

veniently using interfacial polymerization without the need

for any templates or functional dopants. The feasibility of

the synthesized nanocomposites was examined by isolation

of FUR from diluted urine samples. After extraction, the

analyte was desorbed by 200 lL of methanol. It was then

dried and the residue was dissolved in 30 lL of methanol

and an aliquot of 25 lL was, finally, injected into the

HPLC system. Important parameters influencing the

extraction and desorption processes were optimized and 25

cycles of draw–eject gave maximum peak area, when

desorption was performed. The linearity was studied by

preconcentration of 5 mL of diluted urine sample spiked

with a standard solution of FUR in the concentration range

of 15–750 lg L-1. The coefficient of determination was

satisfactory (r2 [ 0.99) and the relative standard deviation

(RSD %) value under the optimized condition was 8.8 %.

The limit of detection and limit of quantification were 7

and 15 lg L-1, respectively.

Keywords High-performance liquid chromatography �Microextraction in packed syringe � Silver nanoparticles/

polyaniline nanocomposite � Furosemide

Introduction

Nanocomposite materials, due to their improvement in

mechanical, thermal and chemical properties, have attracted a

great deal of attention [1, 2]. Nanocomposites formed by metal

nanoparticles (NPs) dispersed in electrically conducting

polymers, such as polyaniline (PANI) or polypyrrole, have

been the focus of many research fields in the past few years [3].

This originates from their intrinsic physical properties and

potential application in advanced technologies. PANI is a

conducting polymer of particular interest, due its high stabil-

ity, low monomer cost, large conductivity range, and the

different redox states that can be synthesized. In recent years,

researchers have focused on the development of bioinert and

biocompatible polymers and nanocomposites to minimize

nonspecific adhesion and inflammatory effects but at the same

time retain their physicochemical properties [4]. This modi-

fication could be physical, chemical or biochemical in nature.

In vivo studies have shown that both conductive and non-

conductive forms of PANI, emeraldine salt and base, exhibit

good tolerance and biocompatibility [5, 6].

Metal NPs, such as silver and gold, have potential in

technological applications [7]. Polymers have been shown

to be excellent hosts for trapping NPs of metals and

semiconductors [8, 9]. This is because of their ability to act

as stabilizers or surface capping agents. When NPs are

embedded or encapsulated in a polymer, the polymer ter-

minates the growth of the particles by controlling the

nucleation and more limited particle size distribution is

achieved within the desired limits. The Ag-NPs/PANI

nanocomposite readily forms using interfacial polymeri-

zation without the need for templates or functional dopants.

High-quality nanocomposite is obtained even when com-

mon mineral acids, such as hydrochloric, sulfuric, or nitric

acid, are used as dopants [10].

H. Bagheri (&) � S. Banihashemi

Environmental and Bio-Analytical Laboratories, Department

of Chemistry, Sharif University of Technology,

P.O. Box 11365-9516, Tehran, Iran

e-mail: bagheri@sharif.edu

123

Chromatographia (2014) 77:397–403

DOI 10.1007/s10337-014-2628-6

Furosemide (FUR) is a loop diuretic drug, which pro-

duces greater diuresis than the common diuretics. It acts by

inhibiting the co-transporter of sodium, potassium and

chloride, and further causes excretion of calcium, magne-

sium and bicarbonate ions. It is used in the pharmaco-

therapy of various diseases and is considered as a doping

agent in sports. This medicine is used to treat excessive

fluid accumulation and swelling (edema) of the body

caused by heart failure, cirrhosis, chronic kidney failure,

and nephrotic syndrome [11]. Owing to its extensive use,

FUR has long attracted the attention of many analysts. A

variety of analytical methods have been proposed for the

determination of FUR in biological fluids and pharma-

ceutical samples. Several methodologies including high-

performance liquid chromatography (HPLC) [12, 13],

HPLC/MS [14], spectrophotometric and fluorometric sys-

tems [15, 16] have been developed for the determination of

FUR in biological fluids.

One of the important steps in an analytical method is the

extraction of the compounds of interest from the sample

matrix. Microextraction in packed syringe (MEPS) is a new

miniaturized version of solid-phase extraction (SPE) in

which sorbent amounts, sample volumes and desorption

solvent volumes are minimized [17]. In MEPS, the tinny

sorbent material is manually inserted inside the syringe

between two polyethylene filters (SPE frits, 20 lm pore size)

[18]. For this purpose the size of SPE frits has to be changed

to match with the used syringes. Usually, the sample is drawn

through the sorbent by an autosampler and the target analyte

is adsorbed by the solid phase. The sorbent is then washed by

water and/or acidic solution to remove the interfering

materials. Afterward, the analyte is eluted with an organic

solvent or the LC mobile phase. Although MEPS has been

extensively used in analysis of various samples, its bio-ori-

ented applications have gained more attentions [19, 20].

In this work, the Ag-NPs/PANI nanocomposite was

synthesized through a two-phase water/toluene interfacial

reaction and eventually used as a MEPS sorbent for the

extraction of FUR from urine samples.

Experimental

Reagents and Standards

Methanol, acetonitrile (HPLC grade), ethanol, acetone,

toluene, hydrochloric acid (HCl), sodium hydroxide

(NaOH), ammonium peroxydisulfate, silver nitrate, sodium

borohydride, tetraoctylammonium bromide (TOAB),

potassium dihydrogen phosphate, potassium hydrogen

phosphate and sulfuric acid were obtained from Merck

(Darmstadt, Germany). Aniline (ANI) (99 %) was pur-

chased from Fluka (Buchs, Switzerland).

For the determination of FUR (C12H11ClN2O5S), its

tablets were powdered and solubilized in 25 mL of abso-

lute methanol. This solution was sonicated for 10 min and

then centrifuged for 20 min at 5,000 rpm. Supernatants

were transferred in a flask and the solvent was evaporated

using a rotary evaporator and eventually FUR white pow-

der was obtained. The FTIR spectrum of FUR is shown in

Fig. 1. The peaks observed at 3,285 and 1,591 cm-1 are

attributed to the N–H stretching and bending bands,

respectively. The peak appeared at 1,323 cm-1 is charac-

teristic of sulfone group while the peak at 1,672 represents

the C=C and C=O bands.

The stock solution of 1,000 lg mL-1 of FUR was prepared

by dissolving 10 mg FUR in 10 mL methanol and stored at

4 �C. The standard working solutions were prepared daily by

appropriate dilution using three-distillated water.

The pH values in the optimization stage were adjusted

by the addition of 0.1 M NaOH or HCl until the desired pH

value was reached.

Human Urine Samples

Fresh human urine samples, from a healthy volunteer, were

collected and placed in graduated centrifuge tubes. These

solutions centrifuged for 10 min at 3,500 rpm and stored at

4 �C until assay. Similarly, urine samples from the same

volunteer, already taken an oral dose of 40 mg of FUR,

collected before its administration at interval times and

stored as mentioned.

Sample Preparation

Diluted urine samples (1 mL urine diluted with 4 mL

water) were adjusted at the pH value of 2 using 0.1 M HCl.

After spiking the samples with the appropriate amounts of

FUR the extraction process was performed.

Instrumentation

A Knauer (Berlin, Germany) HPLC system including a

K-1001 HPLC pump, a K-1001 solvent organizer, an on-

line degasser, a dynamic mixing chamber and a UV

detector model K-2501 was used for separation and

determination of analyte. The separation was performed on

the Waters C18 (4.6 9 250 mm) column (particle size:

3–5 lm). The solvents used as mobile phase were metha-

nol–phosphate buffer (KH2PO4/K2HPO4) (pH 5.5; 5 mM)

(30:70, v/v) at flow rate of 1 mL min-1. The UV detection

was performed at 234 nm wavelength.

FTIR spectrum was recorded by an ABB Bomem

MB100 (Quebec, Canada). A Varian (Australia) model

AA-220 atomic absorption spectrometer was used. The pH

398 H. Bagheri, S. Banihashemi

123

of solutions was measured by a pH-meter E520 (Metrohm

Herisau, Switzerland). The SEM images were obtained by

a Cambridge Stereoscan 360 SEM Instrument (England).

Preparation of Ag-NPs/PANI Nanocomposite

The Ag-NPs were prepared according to the previously

described methods [21, 22]. Typically, 3.7 mL of an aqueous

AgNO3 solution (0.03 M) was added to 10 mL of a toluene

solution containing TOAB (0.05 M) to form a two-phase

system. The system was maintained under vigorous stirring

until all the silver ions were transferred into the organic phase.

While the stirring was continued, aqueous sodium borohy-

dride (3.1 mL of a 0.4 M) was slowly added. The system was

maintained under stirring for 20 min and then the organic

phase was extracted. The Ag-NPs/PANI nanocomposite was

obtained in the following manner: a 3.2-mmol amount of ANI

was dissolved in 10 mL of a toluene solution of silver NPs

obtained as described earlier. This solution with 10 mL of

1 M H2SO4 aqueous solution containing 0.8 mmol of

ammonium peroxydisulfate was transferred to a beaker,

generating an interface between the two layers. Also, PANI

was prepared according to the above-mentioned procedure

without addition of Ag NPs.

MEPS Condition

For this study, 1-mL syringes were used. An amount of 2 mg

of the prepared sorbent was manually inserted inside the

syringe between two polyethylene filters (SPE frits, 20 lm

pore size). For this purpose, the radial size of SPE frits has to

be changed to be adopted with the used syringes. Prior to the

first time use, the sorbent was manually conditioned by rinsing

with methanol, acetonitrile and water. Afterward, the spiked

urine sample (5 mL) was drawn on to the syringe up and down

several times using a variable speed stirring motor which

attached to a circular plate. Samples must be drawn with

proportional speed to decrease the extraction time and to

obtain good percolation between sample and solid support. In

this work the speed of stirring motor was adjusted at

80 lL s-1. The solid-phase sorbent was then washed once by

1 mL of water to remove the proteins and other interfering

materials. Then, the syringe was dried under nitrogen flow for

about 30 s and the analyte was then desorbed by 200 lL

methanol. The desorption step was performed by solvent

aspiration into the syringe and then dispiration into the

desorption glass vial. Next, the desorption solvent was evap-

orated under N2 flow until complete solvent drying. Finally,

30 lL methanol was added to the desorption vial and then

25 lL of desorbed solution was injected into the HPLC sys-

tem. The MEPS sorbent was cleaned with 5 9 100 lL of the

desorption solution (methanol) followed by 5 9 100 lL of

the washing solution (water) after each run.

Results and Discussion

Synthesis and Characterization

Interfacial polymerization was performed in an aqueous/

organic biphasic system with ANI dissolved in an organic

solvent and the oxidant, ammonium peroxydisulfate,

Fig. 1 FTIR spectrum of FUR extracted from tablet

Silver Nanoparticles–Polyaniline for MEPS 399

123

dissolved in an aqueous acid solution. After 3–5 min, Ag-

NPs/PANI nanocomposite with a green color formed at the

interface and then gradually the diffusion process occurred

towards the aqueous phase. As the reaction proceeded, the

color of the organic phase became somehow darker and

eventually stopped changing, which was an indication of

reaction completion. After 24 h, the entire water phase was

filled homogeneously with dark-green Ag-NPs/PANI,

while the organic layer changed into red-orange, which

could be due to the formation of ANI oligomers. The

aqueous phase was then collected and filtered and the

precipitate washed with water and ethanol to obtain a clear

filtrate solution. Then, it was placed in a solution of 0.1 M

ammonium hydroxide for 3 h and filtered. Finally, the

nanocomposite was washed with water and ethanol and

dried at 50 �C [3, 10]. The same procedure was applied to

the preparation of PANI without the usage of Ag-NPs. The

presence of Ag in the prepared nanocomposite was

examined by atomic absorption spectroscopy. After dis-

solving the Ag-NPs/PANI nanocomposite in nitric acid and

subsequent heating, the absorption signal at 328.1 nm

confirmed the presence of Ag in the nanocomposite net-

work. The SEM micrographs of Ag-NPs/PANI and PANI

are shown in Fig. 2a and b, indicating that the silver NPs

could be acting as nucleation centers for the polymerization

process. Once the polymerization is initiated the polymer

formation expands around the Ag-NPs. As the reaction

continues, the excess of ANI present at the interface begins

to polymerize, growing around the formed polymer/silver

structures. Finally, as illustrated in Fig. 2b the NPs are

homogeneously embedded within the polymer structure.

Extraction Capability of Ag-NPs/PANI

PANI has already shown to be a capable sorbent and proved

to have comparable efficiency with some well-established

commercially available sorbents [23, 24]. This capability

more probably arise from features such as high surface area,

possibility of p–p interaction and hydrogen bonding in

combination with polar functional groups. In order to eval-

uate the prepared silver-doped nanocomposite, two sets of

syringes were prepared using Ag-NPs/PANI and PANI. As

Fig. 3 shows, the Ag-NPs/PANI nanocomposite exhibits a

higher extraction capability compared to the PANI.

Optimization

After successful preparation and preliminary evaluation of

Ag-NPs/PANI, it was necessary to optimize the MEPS

condition to achieve the highest possible extraction

Fig. 2 SEM images of a PANI and b Ag-NPs/PANI nanocomposites

Fig. 3 Chromatograms obtained after the diluted urine sample spiked

with FUR extracted by a Ag-NPs/PANI and b PANI nanocomposite

400 H. Bagheri, S. Banihashemi

123

recovery and reduce the carry over effect. Influential

parameters including desorption solvent, volume of

desorption solvent, draw–eject cycles and pH effect were

therefore considered in this investigation.

Desorption Condition

Selecting the most appropriate solvent and volume is quite

essential for optimization of the desorption process. FUR

freely dissolves in acetone and methanol, and is sparingly

soluble in ethanol. Different solvents including acetonitrile,

acetone, methanol and ethanol were examined. Among

them, methanol showed a better performance, as illustrated

in Fig. 4a. Accordingly, methanol was chosen as appro-

priate elution solvent for further experiments. The suitable

elution volume for the quantitative recovery of the analyte

was also evaluated using different volumes of methanol

(100–300 lL). The study was repeated in triplicate for each

of extraction. The obtained results indicate that increasing

the desorption solvent volume causes an increase in the

analyte response up to 200 lL, but after that the analyte

response remained approximately constant. Therefore, an

elution volume of 200 lL was chosen as an optimum value

for the further extractions (Fig. 4b).

Draw–Eject Cycles

In MEPS, it is possible to draw the sample through the

sorbent located inside the syringe, once or several times

(draw–eject). The multiple pulling/pushing of the sample

by the syringe increases the extraction recovery [25, 26]. In

this study, the influence of extraction cycles (draw–eject)

on the extraction efficiency was evaluated. It was shown

that the maximum extraction yield for analyte was

achieved after 25 pump cycles with a speed of 80 lL s-1.

After this point the analyte response remained rather con-

stant and no enhancement in response was observed.

Sample pH

FUR is a weak acid with the acidic pKa values of 3.8 and 7.5

[27]. The aqueous solubility of FUR at room temperature has

been reported to be 18.25 mg L-1. The pH-solubility profile

of FUR at 30 �C showed a minimum of 10 mg L-1 at pH 2.0

and a maximum of 21.9 mg mL-1 at pH 8.0, followed by a

marginal decrease to about 18 mg mL-1 above pH 8.0 [28,

29]. The effect of the sample pH on the retention of the

analyte was investigated by extracting the spiked samples at

a concentration level of 500 lg L-1. Different pH values in

the range of 2–8 were assayed as shown in Fig. 5. It was

observed that the extraction efficiency of Ag-NPs/PANI for

the analyte decreases with sample pH enhancement. In

overall, the extraction at pH 2 was found to be the most

suitable condition. This should be due to the chemical

structure of FUR which is rather neutral at pH 2.

Method Validation

Based on the method development observed above, meth-

anol as desorption solvent, elution volume of 200 lL, 25

Fig. 4 a Effect of desorption solvent on analyte response; desorption

was performed using 100 lL of various solvents. b Effect of elution

volume on analyte peak area; desorption was performed using various

volumes of methanol. For both experiments, extractions were

performed using 5 mL sample containing analytes at level of

500 lg L-1, with 50 cycles of draw–eject

Fig. 5 Effect of pH on the extraction efficiency. Extraction was

performed using 5 mL sample containing analyte at level of

500 lg L-1, with 25 cycles of draw–eject. Desorption was performed

using 200 lL of methanol

Silver Nanoparticles–Polyaniline for MEPS 401

123

cycles of draw–eject in a same vial and sample pH 2 were

selected for the determination of FUR in the spiked urine

samples. Some useful analytical data including a limit of

detection (LOD), limit of quantification (LOQ), relative

standard deviation percent (RSD %), for FUR using the

developed method are listed in Table 1. Under the opti-

mum conditions and at the concentration level of

500 lg L-1, the RSD % for three replicates was 8.8 %.

LOD (S/N = 3) and LOQ (S/N = 10) were 7 and

15 lg L-1, respectively. The coefficient of determination

in the concentration range of 15–750 lg L-1 was satis-

factory (r2 [ 0.99). Relative recovery, defined as the peak

area ratio of urine sample and distilled water sample spiked

with analyte at the same level [30], for the spiked urine

sample was 78 %. Absolute recovery and enrichment fac-

tor [31] of the method were 46 and 52 %, respectively. The

carryover effect was evaluated by examining an un-spiked

urine sample after performing the extraction of a urine

sample spiked with 500 lg L-1 of FUR. To eliminate the

memory effect, the MEPS sorbent was washed by methanol

and water after each extraction. The carryover was less

than 0.4 %.

Also, some important characteristic parameters of the

present work are compared with those of previously

reported (Table 1).

The developed method using the Ag-NPs/PANI nano-

composite as the packing material of MEPS was applied to

the determination of FUR in real urine samples obtained

from a healthy female volunteer. Urine was collected at

different time intervals for the quantitative determination

of FUR: 0–1, 1–2, 2–4 and 4–8 h. The concentration time

data are shown in Fig. 6. Following the extraction proce-

dure described in the experimental section, the compound

was easily detected in different interval times.

Conclusion

In this study, a microextraction technique was developed

using an Ag-NPs/PANI nanocomposite as the extracting

device for isolation of FUR from urine samples. The pro-

duction of Ag-NPs/PANI nanocomposite using interfacial

polymerization is rather simple, easy and inexpensive. The

sample matrix has no significant effect for the urine sample

analysis. Solvent desorption was performed in a microvial

and then extractant was injected into HPLC system. The

effect of various parameters including desorption condition,

draw–eject cycles and pH was investigated. This method

proved to be conveniently applicable and quite easy to

manipulate with sufficient sensitivity and good

reproducibility.

Acknowledgments The Research Council and Graduates School of

Sharif University of Technology (SUT) are thanked for supporting the

project.

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