Applications of a Novel Sample PreparationMethod for the Determinationof Sulfonamides in Edible Meat by CZE

Muhammad Umar Farooq, Ping Su, Yi Yang&

College of Science, Beijing University of Chemical Technology, 100029 Beijing, People’s Republic of China; E-Mail: yangyi@mail.buct.edu.cn

Received: 9 October 2008 / Revised: 15 December 2008 / Accepted: 19 January 2009Online publication: 22 March 2009

Abstract

A simple, rapid and reliable method based on SPE clean-up and CZE separation wasvalidated for the trace determination of sulfonamides (SAs) in meat. Acetonitrile was used forthe extraction of SAs (sulfamethoxazole, sulfamethazine, sulfamerazine, and sulfadime-thoxine) from the samples and 1-propanol was used for the denaturing of the proteinspresent in the sample matrix. SPE procedure was employed for the clean-up and pre-con-centration of SAs prior to CZE analysis. Complete separation was achieved by using45 mmol L-1 phosphate buffer (pH = 6.3) at an applied voltage of 20 kV. Overall obtainedrecoveries were from 83.3 to 94.5% for the SAs. The detection limit of each sulfonamideranges from 4 to 6 lg kg-1. The presented one step SPE clean-up method is highly appli-cable for the determination of the SAs at a residue level below the maximum residue limit.

Keywords

Capillary zone electrophoresisSolid phase extractionSulfonamides residues

Introduction

Sulfonamides (SAs) are analogues of the

para-aminobenzoic acid (PABA) that

inhibit the folic acid synthesis in sus-

ceptible micro-organisms. These com-

pounds are not only employed for

therapeutic and prophylactic purposes,

such as gastrointestinal and respiratory

infections, but also in the livestock

industry to promote growth. Because of

their broad spectrum of activity and low

costs [1], they are often overused or

applied without respecting safety recom-

mendations. As a result, SAs undesirable

residues remain in the animal tissues,

meat, or biofluids such as milk. As it has

been observed that most of the sulfona-

mides are suspected to be carcinogenic

and enhanced the risk of developing

bacterial resistance in human body [2],

the use of these compounds is of great

public health concern and their residues

have to be controlled. Due to their

possible side effects, the usage of SAs in

animals is regulated [3]. The maximum

residue limits (MRLs) for SAs in food-

stuffs have been restricted to a rather low

level in many nations and regions. For

example, MRLs for total SAs have been

established at the level of 100 lg kg-1 [4]

in European Union countries.

The availability of rapid and reliable

cost-effective screening methods for

antimicrobial compounds is often the

most important aspect of implementing

adequate drug residue monitoring

programs. Microbiological [5–7], immu-

noassays [8] and sulfonamide antibody-

based biosensors [9] have been used as

screening methods of SAs in food. These

methods usually save time, provide sim-

plicity and remove the need of laborious

sample pretreatment steps, but they

provide only semiquantitative measure-

ments and sometimes negatively affected

by matrix components [10].

Several analytical techniques for the

quantitative detection of SAs residues

have been reported. Most widely used

technique is liquid chromatography

(HPLC) with UV, fluorescence, electro-

chemical detectors, MS detectors and

chemiluminescence [11–19]. Gas chro-

matography-mass spectrometry (GC-MS)

2009, 69, 1107–1111

DOI: 10.1365/s10337-009-1025-z0009-5893/09/05 � 2009 Vieweg+Teubner | GWV Fachverlage GmbH

Full Short Communication Chromatographia 2009, 69, May (No. 9/10) 1107

has also been used to detect the SAs [20–

23] but complex derivatization processes

have been needed due to the non-volatile

nature of SAs.

CE has become a powerful separa-

tion technique with its wide applications

due to its high resolution, high efficiency,

rapid analysis and small consumption of

both sample and solvent in comparison

with HPLC. Some methods have been

reported using CE with different detec-

tors for the analysis of SAs from phar-

maceuticals and meat tissues [24–29].

Most of the works are focused on the

separation studies rather than quantita-

tive analysis.

SAs are usually extracted from edible

tissues with organic solvents (acetone,

ethyl acetate, chloroform, acetonitrile

and methanol), alone or combined.

Supercritical fluid extraction (SFE) [30],

matrix solid phase dispersion (MSPD)

[31–33] and accelerated solvent extrac-

tion (ASE) [34] have also been used for

extraction of SAs from pork, calf and

chicken samples. Since there is a need to

remove protein, fat and other potential

interferences from sample matrix, sam-

ple pre-treatment is required. Liquid-

liquid extraction (LLE) combined with

solid-phase extraction (SPE) has been

demonstrated as quite effective proce-

dure. But these methods rather use the

combination of different SPE cartridges

or need tedious clean-up steps [24, 25,

35, 36].

Our basic goal was to achieve sim-

plicity with better selectivity and sensi-

tivity for the isolation of SAs from meat.

LLE was done for the extraction of

sulfamethazine (SMZ), sulfamerazine

(SMR), sulfadimethoxine (SDM), and

sulfamethoxazole (SMX) from meat

samples. SPE was used for the purifica-

tion of desired analytes from sample

matrix. This simplified extraction pro-

tocol is quite sensitive and selective for

SAs. The method was found to be useful

alternative to the commonly applied

techniques for sample preparation. Four

SAs in the sample was analyzed by CZE

within 15 min. The established SPE-CE

method gave a shorter overall analysis

time, provided good resolution for four

SAs and produced excellent linearity

over a wide concentration range.

Experimental

Chemicals

Acetonitrile and methanol were HPLC

grade, purchased from J & K Chemical

(Beijing, China). Ethyl acetate, n-hexane,

sodium sulfate, sodium hydroxide,

potassium dihydrogen phosphate and

1-propanol all were analytical grade

reagents and purchased from Beijing

Chemicals (Beijing, China). Sulfamera-

zine (SMR), sulfamethazine (SMZ),

sulfamethoxazole (SMX) and sulfadi-

methoxine (SDM), with purity 99% were

purchased from Sigma-Aldrich (St

Louis, MO, USA). Cleanert PEP-SPE

cartridges (3 mL, 60 mg) were supplied

by Agela Technologies (Beijing, China).

Food mixer was obtained from Supor

Group (Zhejiang, China). The water

was purified and deionized by a Milli-Q

system (Millipore, Bedford, MA, USA).

The solvents and samples for CE were

filtered through 0.45 lm filters and

degassed in an ultrasonic bath before

injection.

Standard Solution

Individual standard solutions of

100 mg L-1 were prepared in metha-

nol:water (50:50 v/v) and stored in dark

at 4 �C. Working solutions at various

concentrations were freshly prepared by

diluting with water.

Buffer solution was prepared by dis-

solving the appropriate quantity of

KH2PO4 in deionized water and pH was

adjusted by 0.05 mol L-1 H3PO4 or

NaOH solutions.

Apparatus

Experiments were carried out with

Beckman Coulter P/ACE MDQ capil-

lary electrophoresis system equipped

with a photodiode array detector (PDA).

Uncoated fused-silica capillary was used

(60.2 cm 9 75 lm id, 50 cm to detec-

tor). Samples were injected by applying a

pressure of 0.5 psi for 15 s. The applied

voltage is 20 kV and capillary tempera-

ture was maintained at 25 �C. At the

beginning of each run, the capillary was

conditioned by flushing with 1 mol L-1

NaOH for 10 min, water for 5 min and

finally with electrolyte for 20 min. For

pH measurements, a Sartorius PB—10

pH meter was employed.

Sample Preparation

The meat and liver samples were ground

and homogenized by using a food mixer.

Solvent extraction was used for the

extraction of SAs from the samples.

Acetonitrile was found to be the suitable

solvent for extraction. 5 g (meat or liver)

homogenized sample was placed in a

50 mL centrifuge tube and vortexed with

15 mL acetonitrile, centrifuged for

10 min at 5,000 rpm and filtered through

0.45 lm membrane filter. 5 mL of

1-propanol was added and clear super-

natant was evaporated to dryness under

reduced pressure at 40 �C, reconstitutedwith 10 mL water and passed through

Cleanert PEP-SPE polymer cartridge.

The cartridge was conditioned with

3 mL methanol and water prior to the

loading of sample. After sample loading,

the cartridge was rinsed by water to

wash out the interferences and then dried

for about 10 min. Finally, analytes were

eluted with 4 mL methanol. Eluted

methanol was evaporated to dryness

under a gentle stream of nitrogen and

reconstituted with 1 mL methanol/water

(50/50, v/v) mixture.

Results & Discussion

Optimization of CE

Electropherograms were taken at

254 nm wavelength. pH of the buffer

solution plays a major role in the sepa-

ration of SAs. Phosphate buffer at dif-

ferent pH (5.5–8.5) was investigated

in order to ascertain any influence on

SAs separation and quantification. Best

resolution was obtained at a pH of 6.3.

Ionic strength of the buffer is also a

critical parameter for the separation in

CE. Different concentrations of buffer

(30, 35, 40, 45, 50 and 60 mmol L-1)

were tested. At low concentration, the

1108 Chromatographia 2009, 69, May (No. 9/10) Full Short Communication

migration time was shorter which led to

poor resolutions. Increase in concentra-

tion resulted in increasing of the migra-

tion time. 45 mmol L-1 was selected as

the optimized concentration. The effects

of capillary temperature, applied voltage

and injection volume were also opti-

mized. It was verified that the tempera-

ture (ranging 15–30 �C) and applied

voltage (ranging 15–30 kV) had much

influence on the migration of analytes

and separation efficiencies. Temperature

of 25 �C and 20 kV applied voltage were

chosen as the best compromise between

running time, efficiency of separation

and sensitivity. Moreover, it was also

observed that 15 s injections were most

efficient (5 s injections correspond to

approximately 20 nL injection volume of

sample) and gave a satisfactory separa-

tion in a reasonable analysis time

(15 min). A typical electropherogram of

SMZ, SMR, SDM and SMX at the

optimized conditions is depicted in

Fig. 1.

Isolation of SAs from Meat

Beside the instrument parameters and

buffer composition etc., clean-up step is

the most important factor that influ-

ences the sensitivity. As SAs bind to the

proteins of meat, therefore the effect of

proteins in the extraction and sample

cleaning can not be neglected [37].

Different solvent systems such as, ethyl

acetate, acetonitrile, mixture of aceto-

nitrile and acetone, mixture of ethyl

acetate and acetone were employed for

the extraction of SAs [38, 39]. Acetoni-

trile and ethyl acetate were found better

than mixture solvent because of their

abilities to precipitate proteins present

in the sample matrix. After extraction,

proteins in sample matrix were dena-

tured with two different approaches.

1-propanol was used in one approach

while 1 mol L-1 HCl neutralized with

1 mol L-1 NaOH was used in another

approach. Both of the approaches were

found reasonably well for the denatur-

ing of proteins but 1-propanol was

preferred due to the recovery efficiency.

Due to this fact, the mixture of 1-pro-

panol with ethyl acetate and acetonitrile

was also used for the extraction of SAs

but not much significance was observed.

So 1-propanol was utilized for the

denaturing of proteins in the sample

matrix. Cleanert PEP-SPE polymer

cartridge was employed for the further

cleaning of interferences.

Validity of Analysis

The analytical performance parameters

of the method including selectivity, pre-

cision, limits of detection and recovery

were validated. The analysis has been

carried out on the basis of the replicate

analysis of sample according to the

recommendations of EU [40].

Drug free meat samples and spiked

samples were tested. Absence of interfer-

ences from sample matrix in the migra-

tion range of SAs in electropherograms

confirmed the selectivity of method.

Linearity of method was evaluated by

plotting calibration curves in the range

of 0.5–50 lg mL-1 with triplicate anal-

ysis. It was found to be linear over this

range with acceptable correlation coeffi-

cient (r2 = 0.99). Limits of detection

(LOD) were calculated by using S/N

ratio of 3. LOD of each SAs ranged from

3.7 to 6.0 lg kg-1. The LOD values were

comparable to the reported methods

[12, 13]. Precision of method is sufficient

determined by comparing the migration

time and peak area of SAs standards.

Table 1 represents the within-day and

between-day repeatability in terms of

percentage RSD. RSD values of within-

day precision for migration times were

1.3–2.4% and 1.1–3.7% for peak areas.

While between-day RSD values for

migration time were 3.7–5.8% and 4.0–

6.4% for peak areas, showing that the

method is reproducible.

Fig. 1. Electropherogram of four sulfonamides at 25 lg mL-1. Separation conditions:45 mmol L-1 phosphate buffer (pH = 6.3), 20 kV, 25 �C, fused-silica capillary,60.2 cm 9 75 lm id, 50 cm to detector. 1 = SMZ, 2 = SMR, 3 = SDM, 4 = SMX

Table 1. Evaluation of the method

Analyte Within-daya

RSD%

Between-daya

RSD%

Slopeb Intercept r2 LODlg kg-1

LOQlg kg-1

tm Area tm Area

Sulfamethazine 1.3 1.1 4.1 4.0 3537.8 12.1 0.99 5.8 10.7Sulfamerazine 1.7 1.1 5.4 6.4 3251.7 -11.2 0.99 4.5 10.5Sulfadimethoxine 2.1 3.7 3.7 4.8 4111.7 -14.2 0.99 6.0 13Sulfamethoxazole 2.4 2.6 5.8 5.8 4206.6 -4.7 0.99 3.7 24

a n = 5b Calibration data (six points, three replicate of each point)

Full Short Communication Chromatographia 2009, 69, May (No. 9/10) 1109

Recovery

To check the applicability and trueness

of method, extraction recovery was

determined. For recovery studies of

SAs in meat samples, three spiked

samples at concentration of 50, 100

and 500 lg kg-1 were used. The results

are shown in Table 2. Overall recover-

ies were 94.5% (SMZ), 85.0% (SMR),

83.9% (SDM) and 83.3% (SMX)

with RSD less than 10%. The data

confirmed the trueness of proposed

method having adequate sensitivity and

accuracy.

Robustness

Robustness of method was checked by

the eventual variations introduces in

different parameters. Parameters inves-

tigated were buffer composition (pH =

6.3 ± 0.1, ± 0.2), temperature (25 ±

1.0 �C), injection volume (15 ± 1 s),

rinse time, applied voltage (20 ± 1 kV).

No significant variation was observed.

The method was proved to be robust and

could be able to be applied in the routine

analysis.

Analysis of Real Sample

Applicability of the method on real

sample was validated by the analysis of

chicken and beef tissue and liver samples

obtained from local market. Electro-

pherograms of tissue samples spiked at

10 lg kg-1 and 50 lg kg-1 of each of

SAs obtained after the application of

present method are shown in Fig. 2 a

and b. No interferences from the matrix

were observed.

Conclusion

Application of a simple and sensitive

method for the extraction and analysis

of mostly used SAs from chicken and

beef tissue samples were evaluated. CE

separation and determination conditions

of SAs were optimized with phosphate

buffer (pH = 6.3, 45 mmol L-1). Matrix

proteins were denatured by 1-propanol.

Cleanert PEP-SPE polymer cartridge

was used for the cleaning of potential

interferences. The proposed simplified

clean-up procedure enables the quantifi-

cation of SAs residues at concentration

lower than the MRL established by EU.

Obtained recoveries were over 94%. The

presented method is feasible to study the

occurrence of selected compounds in

edible meat.

Acknowledgments

This work was supported by Natural

Science Foundation of China (No.

20375004).

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