AnalyticalMethods

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aFaculty of Life Science and Technology

Technology, Kunming 650500, P. R. Chi

13888316388bChemical Engineering Institute, Kunming

Kunming 650500, P. R. China

Cite this: Anal. Methods, 2013, 5, 3920

Received 20th December 2012Accepted 7th April 2013

DOI: 10.1039/c3ay26587k

www.rsc.org/methods

3920 | Anal. Methods, 2013, 5, 3920

Ultrasound-assisted cloud point extraction for thedetermination of sulfonamides in honey samples byfluorescence spectrophotometry

Fang Yu,a Chang Liu,a Yuhong Guob and Yaling Yang*a

An ultrasound-assisted cloud point extraction (UA-CPE) method using nonionic surfactant nonylphenol

ethoxylate (NP-7) as the extractant was developed for determination of two sulfonamides (SAs,

sulfadiazine sodium and sulfamerazine) in honey samples. The procedure was based on the

derivatization SAs in honey with fluorescamine yielding fluorescent complexes, which were extracted

into the surfactant-rich phase and analyzed by molecular fluorescence spectrophotometry. The polar of

SAs of derivatives was reduced and strong fluorescence was produced at lex ¼ 410 nm. Variable

parameters affecting the derivatization and UA-CPE procedure were evaluated and optimized. The

calibration graph was linear from 10 to 10 000 ng mL�1 with coefficient of determination more than

0.9991. The limits of detection (LOD) were 0.5 ng mL�1 for sulfadiazine sodium and 5.0 ng mL�1 for

sulfamerazine with the relative standard deviations of the method ranging from 2.2% to 4.1%.

Recoveries of the compounds on spiked honey samples at fortified level of 1 mg mL�1 were 92.2% for

sulfadiazine sodium and 84.1% for sulfamerazine, respectively. Therefore, the developed method is

practical and reliable, with potential application to determine the content of SAs in honey samples.

1 Introduction

Sulfonamides (SAs) are one of the most used broad-spectrumsynthetic antibiotics, widely used in the prevention and treat-ment of bacterial bee diseases such as American foulbroud(Bacillus larvae) and European foulbroud (Streptococcuspluton).1 They are usually employed for the treatment ofbacterial infections in veterinary practices, mainly due to theirlow price. There has been growing concern about the use ofsulfonamide antibiotics because they may lead to residualcontamination by these compounds in foods of animalorigin.2,3 The European Union has established in CommissionRegulation (EC) no. 281/96 (ref. 4) a maximum residue limit of100 mg kg�1 for the total amount of sulfonamides in muscle,liver and milk and so on.

Several analytical methods have been developed for thedetermination of residual sulfonamides in foodstuffs (honeyincluded) including enzyme immunoassays,5–7 ultra-violetspectrometry,8,9 liquid chromatography (LC),10–12 high perfor-mance liquid chromatography (HPLC),13–15 gas chromatog-raphy (GC),16 thin layer chromatography,17 ow injectionanalysis18 and liquid chromatography-mass spectrometry

, Kunming University of Science and

na. E-mail: yilyil8@163.com; Tel: +86

University of Science and Technology,

–3926

(LC-MS),19 gas chromatography-mass spectrometry (GC-MS).20

Spectrouorimetric techniques are a simple and convenientmethod, and derivatization is required to determine SAs.

Most sample preparation procedures reported for SAsinvolve the use of repetitive extractions followed by clean upand concentration steps. There is an absence of suitablesolvents to quantitatively extract such a wide range of polaranalytes, so liquid–liquid extraction,21 solid phase extraction,22

liquid–liquid extraction followed by solid-phase extraction,23

enzymatic-microwave assisted extraction,24 and ionic liquid-based microwave-assisted dispersive liquid–liquid micro-extraction25 have been developed for SAs in samples. Thesemethods have specic advantages and disadvantages, forexample, solid phase extraction and liquid–liquid extractionare limited because of the large consumption of toxic volatileorganic solvents.

Thus, effective, simple, and fast extraction procedures arestill in demand. Cloud point extraction (CPE) has beenrecognized as an alternative to the conventional extractionprocedures because of its performance, low cost, and lowertoxicity, based on surfactant mediated phase separation. Ithas been successfully used for the preconcentration ofdifferent species. Classical CPE is Triton X-100 or Triton X-114(a nonionic surfactant)-CPE, which has been successfullyused for the selective extraction of various compounds.The surfactant–aqueous solvent becomes cloudy whenthe temperature is above cloud-point temperature, usually15 to 20 �C higher than cloud-point temperature.26 The

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surfactant-phase (SRP) and the aqueous-phase (AQ) areformed, and SRP has a smaller volume in contrast to the largevolume AQ. The surfactant solvent has a small volume, so itshould be diluted with a minimum volume of interrelatedorganic solvent. The diluted SRP is then used for analysis byspectrouorimetry.

Derivatization and CPE are the two major procedures in thisstudy. Aer the SAs derivatization with uorescamine, stronguorescence can be produced27,28 and the SAs are more conve-niently detected. It is easier to concentrate the compounds inthe surfactant phase because of the polarity of derivatives isreduced. NP-7 is nonylphenol ethoxylate, its cloud point is about20 �C. To the best of our knowledge, NP-7 has never been appliedin CPE for the determination of SAs. Compared with Triton X-114, a better recovery and higher enrichment factor wereobtained by the CPE system with NP-7 under the same experi-mental conditions. Ligang Chen et al.29 reported the determi-nation of sulfonamides based on Triton X-114 as the extractionmedium, but the disadvantage of this method is the hightemperatures required. Also, NP-7 has no uorescence intensityat wavelength 410 nm eliminating the effects of the matrix. Thismethod has been successfully applied to determine SAs residuesin honey with satisfactory results.

2 Experimental2.1 Chemicals

The SAs (sulfadiazine sodiumand sulfamerazine)were purchasedfrom Aladdin Chemistry Co., Ltd. (Shanghai, China) and bothare of analytical grade. Fluorescamine of analytical grade waspurchased from Aladdin Chemistry Co., Ltd. (Shanghai, China).Non-ionic surfactant NP-7 analytical grade was purchased fromAladdin Chemistry Co., Ltd. (Shanghai, China).

The stock standard solution of sulfadiazine sodium wasprepared in water at a concentration of 1 g L�1. The workingstandard solution (100 mg L�1) was freshly prepared by dilutingthe stock solution with water. The solution of sulfamerazinewas prepared in water at a concentration of 100 mg L�1. Theuorescamine solution was prepared in methanol at a concen-tration of 0.02% (0.2 g L�1). The NP-7 solution was prepared inwater at a concentration of 10% (v/v, 0.1 mL mL�1). Buffersolution formulated from phosphoric acid, acetic acid and boricacid (pH ¼ 5, 0.4 mol L�1) was used.

All the stock solutions and working solutions werestored at 0–4 �C in a refrigerator and found to be stable fortwo months.

2.2 Apparatus

Fluorescence measurements were performed on ShimadzuRF-5301 PC spectrouorimeter (Kyoto, Japan) equipped with ahydrogen discharge lamp and 1.0 cm quartz cell. The slit widthof both excitation and emission monochromators was set at5 nm and scan rate was 250 nm min�1. A XMTB digitaltemperature controller (Tianjin, China) was used to implementCPE. An ultrasonic cleaners (Shanghai, China) was used toassist the CPE procedure.

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2.3 Sample preparation

Honey samples were obtained from local supermarkets aroundthe city of Kunming. 20 mL of SAs working solution (100 mg L�1)and 10mL of water were added into 4 mL of honey. The solutionwas shaken by vortex oscillator, and ltered with a lter paper.The ltrate was then removed into a new centrifuge tube, anddiluted to 10 mL with doubly distilled water. The diluted solu-tion was mixed by vortex followed by centrifuging at 10 000 rpmfor 10 min. The supernatant was collected into another tube,and used in following the derivatization and CPE procedure.

2.4 Derivatization/CPE procedure

60 mL uorescamine solution (0.2 g L�1) and 1 mL of buffersolution (pH ¼ 5) were added to a 10 mL screw-cap glasscentrifuge tube, and the solution was diluted in 5 mL withdoubly distilled water. The 5 mL solution was placed for 40 minfor derivatization, forming the derivative solution. 1 mL(sulfadiazine sodium) or 1.8 mL NP-7 solution (10%, v/v) wasadded to the derivative solution and the mixtures diluted withdoubly distilled water to 10 mL. Then, ultrasound-assistedextraction for 30 min for sulfadiazine sodium or 20 min forsulfamerazine at 49 kHz, and aerwards were placed in athermostatic water bath at 35 �C for 30 min, to obtain completeseparation of the surfactant-rich phase. The water phase wasremoved to a new tube carefully, and extracted twice with NP-7,to achieve a nal volume of 3.0 mL by adding methanol. Thediluted NP-7 phase was measured at lex/lem ¼ 410 nm/495 nm.

3 Results and discussion

Fluorescence emission spectrum is shown in Fig. 1. Fig. 1(A) isthe uorescence emission spectrum of sulfadiazine sodium,Fig. 1(B) is the spectrum of sulfamerazine.

3.1 Optimization of derivatization

The inuence of different experimental parameters includingthe concentration of uorescamine, pH, derivatizationtemperature and time were investigated.

3.1.1 Effect of derivatization reagent concentration. Theamount of uorescamine is important for the derivatizationbecause sufficient reagent is needed in the reaction and thisaffects uorescence intensity. The concentration of uoresc-amine investigated ranged from 0.2 mg L�1 to 20 mg L�1.Fig. 2(A) shows the optimum concentration of uorescamine forboth sulfadiazine sodium and sulfamerazine is 1.2 mg L�1.

3.1.2 Effect of derivatization pH. The pH is a critical factorfor uorescence detection and is very important for the deriv-atization of SAs and uorescamine. It is known that uores-cence intensity is sensitive to pH. The effect of pH in the rangeof 2.0–10.0 was investigated. Fig. 2(B) shows uorescenceintensity of the derivative is highest at pH 5.0, therefore pH 5.0was chosen for the derivatization.

3.1.3 Effect of derivatization temperature and time. Reac-tion temperature and time can affect derivatization progressbecause the reaction can complete with appropriate tem-perature and enough time. Fig. 2(C) shows the optimum

Anal. Methods, 2013, 5, 3920–3926 | 3921

Fig. 1 Fluorescence spectrum of sulfamerazine–fluorescamine–NP-7. (A) is the fluorescence emission spectrum of sulfadiazine sodium, and (B) is the spectrum ofsulfamerazine. Experimental conditions: 1.0 mg L�1 of SAs; 1.2 mg L�1

fluorescamine; 1.0 mL of buffer solution (pH ¼ 5.0); 1.0% (v/v) NP-7 for sulfadiazine sodium and2.0% (v/v) NP-7 for sulfamerazine.

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temperature for the reaction was 20 �C (room temperature).The reaction equilibration time was 40 min, indicated inFig. 2(D).

Fig. 2 (A) Effect of derivatization reagent concentration. Experimental conditions:temperature) for 30min. (B) Effect of pH. Experimental conditions: 0.2mg L�1 of SAs;Effect of derivatization temperature. Experimental conditions: 0.2 mg L�1 of SAs; 1.2 m(D) Effect of derivatization time. Experimental conditions: 0.2 mg L�1 of SAs; 1.2 mg

3922 | Anal. Methods, 2013, 5, 3920–3926

3.2 Optimization of CPE conditions

NP-7 was chosen as the extraction medium because it iscommercially available and has a low cloud-point temperature

0.2 mg L�1 of SAs; 1.0 mL of buffer solution (pH ¼ 5.0); derivation at 20 �C (room1.2mg L�1 of fluorescamine; derivation at 20 �C (room temperature) for 30min. (C)g L�1 of fluorescamine; 1.0 mL of buffer solution (pH¼ 5.0); derivation for 30min.

L�1 of fluorescamine; 1.0 mL of buffer solution (pH ¼ 5.0); derivation at 20 �C.

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Fig. 3 (A) Effect of NP-7 concentration on the recovery. Extraction conditions: pH ¼ 5.0; equilibrium temperature: 35 �C; equilibrium time: 30 min; ultrasonicationtime: 30 min. (B) Effect of pH on the recovery. Extraction conditions: concentration of NP-7 1.8% (v/v); equilibrium temperature: 35 �C; equilibrium time: 30 min;ultrasonication time: 30 min. (C) Effect of equilibration temperature on the recovery. Extraction conditions: concentration of: NP-7 1.8% (v/v); pH ¼ 5.0; equilibriumtime: 30 min; ultrasonication time: 30 min. (D) Effect of equilibration time on the recovery. Extraction conditions: concentration of NP-7 1.8% (v/v); pH ¼ 5.0; equi-librium temperature: 35 �C; ultrasonication time: 30 min.

Fig. 4 Effect of ultrasonication on the recovery. Extraction conditions: pH ¼ 5.0;equilibrium temperature: 35 �C; equilibrium time: 30 min; concentration of NP-7:1.8% (v/v).

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and high density, which facilitates the concentration procedure.The factors that affect the CPE such as NP-7 concentration,extraction pH, ultrasonication time, extraction temperature andtime were investigated.

3.2.1 Effect of NP-7 concentration. The concentration ofsurfactant should be large enough to enable high recovery. Theeffect of NP-7 concentration from 0.8% to 3.0% (v/v) on SAs'recoveries was investigated (Fig. 3(A)). As shown in Fig. 3(A) at alower concentration of non-ionic surfactant liquid, the extrac-tion recoveries of SAs were low, and increased with the increaseof surfactant concentration from 0.8% to 1.8% for sulfadiazinesodium and sulfamerazine, respectively. For a NP-7 concentra-tion of 1.8% (v/v) for SAs, the recoveries remained constantabove this level. In principle, low surfactant concentration issuitable to obtain a high concentration factor. So 1.8% (v/v) forSAs was chosen as the optimum surfactant concentration andused in the following experiments.

3.2.2 Effect of extraction pH. The recovery and uorescenceintensity may be inuenced by pH. The pH was adjusted with0.4 mol L�1 buffer solution and 0.1 mol L�1 NaOH. The recov-eries of SAs were studied over the pH range 2.0–9.0 (Fig. 3(B)).The experimental results indicated that the recoveries were

Anal. Methods, 2013, 5, 3920–3926 | 3923

Fig. 5 Fluorescence intensity of SAs of different concentrations. Experimental conditions: 1.2 mg L�1 of fluorescamine; 1.0 mL of buffer solution (pH¼ 5.0); derivation at20 �C for 40 min; 1.8% (v/v) NP-7; 30 or 20 min of ultrasonication time; extraction at 35 �C for 30 min. The concentrations of sulfadiazine sodiumwere 10 ngmL�1, 20 ngmL�1, 50 ngmL�1, 100ngmL�1, 1mgmL�1, 3mgmL�1, 5mgmL�1, 7 mgmL�1. Concentrations of sulfamerazinewere 10ngmL�1, 50 ngmL�1, 100ngmL�1, 1 mgmL�1, 3 mgmL�1, 5 mg mL�1, 6 mg mL�1, 8 mg mL�1.

Table 1 Determination of SAs in honey samplesa

SAsAdded(mg kg�1)

Found(mg kg�1)

Recovery(%)

RSD(%, n ¼ 6)

SD 0 ND1 0.922 92.2 2.75 4.48 89.6 3.17 6.22 88.9 3.4

SM1 0 ND1 0.841 84.1 2.35 4.10 82.0 3.77 5.84 83.4 3.2

a ND: not detected.

Fig. 6 Mechanism of sulfamerazine reacting with fluorescamine.

Fig. 7 Effect of different methods on the recoveries of SAs. Experimentalconditions: 1.0 mg L�1 of SAs; 1.2 mg L�1 of fluorescamine; 1.0 mL of buffersolution (pH ¼ 5.0); derivation at 20 �C for 40 min; 1.8% (v/v) NP-7; 30 or 20 minof ultrasonication time; extraction at 35 �C for 30 min.

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satisfactory at pH 5.0. pH 5.0 was chosen as the optimum pH forfurther study.

3.2.3 Effect of equilibration temperature and time. It isknown that in CPE two phases are formed when the aqueoussolution of a non-ionic surfactant is heated above the cloud-point temperature. The best temperature and enough time areneeded to obtain the satisfactory recoveries. Also, the ratio ofwater in the surfactant phase can be reduced at highertemperature, but too high a temperature is not good for heat-sensitive materials. Thus, a temperature range of 20–70 �C wasstudied, shown in Fig. 3(C). At 35 �C the maximum extractionrecoveries were observed. Therefore 35 �C was used in the CPEprocedure. The equilibration time was also investigated in therage of 10–60 min (Fig. 3(D)). It was found that the surfactant

3924 | Anal. Methods, 2013, 5, 3920–3926

phase cannot form completely before 20 min has passed. Themaximum extraction recoveries were presented at 30 min, andbeyond 30 min, the recoveries were almost constant. Therefore,30 min was chosen as the optimum equilibration time.

3.2.4 Effect of ultrasonication. Ultrasound can assist theaqueous phase and non-ionic surfactant phase to mixsymmetrically and to contribute to extraction of SAs. It takeslonger to get total phase separation and obtains recoveries arelower without ultrasound. So ultrasound-assistance is neces-sary. Ultrasonication time in the range of 5–50 min was evalu-ated. Fig. 4 shows that the highest recovery is presented at30 min for sulfadiazine sodium and 20 min for sulfamerazine,and then decreases for longer times. Therefore, 30 min forsulfadiazine sodium and 20 min for sulfamerazine were chosenas the preferred ultrasonication times in further experiments.

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3.3 Analytical applications

Under optimal experimental conditions, there were good linearrelationships between the responses and the analyte concen-trations of SAs in the range 10–10 000 ng mL�1 (Fig. 5) in SAs-uorescamine-NP-7 system with a correlation coefficient (r) of0.9994 for sulfadiazine sodium and 0.9991 for sulfamerazine,the limits of detection (LOD) for the determination of sulfadi-azine sodium and sulfamerazine were 0.5 and 5.0 ng mL�1.Fig. 5(A) is the linear graph of sulfadiazine sodium and Fig. 5(B)is for sulfamerazine. Recoveries and RSD (n ¼ 6) varied from80.2% to 93.0%, and from 2.2% to 4.1% by detecting SAs atdifferent concentrations in honey. The recoveries of theproposed method were 89.7% for sulfadiazine sodium and83.5% for sulfamerazine, when the concentrations of SAsare 0.2 mg mL�1.

To validate the accuracy and reproducibility of the proposedmethod, the method was applied for the determination of SAsin honey samples and spiked three different levels of sulfadia-zine sodium and sulfamerazine (1.0, 5.0 and 7.0 mg mL�1). Theresults are shown in Table 1. From Table 1, it can be seen thatthe recoveries for the analytes were 80.2–93.0% with RSD (n¼6)less than 5.0. The results demonstrated that the developedmethod is effective for the analysis of SAs in real samples.

3.4 Interaction mechanism

The interaction mechanism is illustrated by sulfadiazinesodium. Sulfadiazine sodium can lose a Na+ and gain ahydrogen ion, the structure is described in Fig. 6. The reactionbetween sulfadiazine sodium and uorescamine, and also thepotential derivative is shown in Fig. 6. The derivatizationreduces the polar of sulfadiazine sodium and produces astructure, which in turn produces strong uorescence at lex ¼410 nm. NP-7 is a non-ionic surfactant and is weakly polar,which produces a non-polar cavity. Non-ionic surfactant NP-7can therefore extract the derivative from aqueous phase.

3.5 Comparison of NP-7-CPE and Triton X-114-CPE

The use of nonionic surfactant Triton X-114 of classical CPE toextract SAs performs a low recovery under the same conditionswith NP-7-CPE (Fig. 7). Method 1 is a system of SAs–uoresc-amine–NP-7, and method 2 is a system of SAs–uorescamine–Triton X-114. From the Fig. 7 it can be seen that the recoveries ofmethod 1 for SAs are higher than the recoveries of method 2.The temperature of SAs–uorescamine–NP-7 CPE system is35 �C, lower than the other surfactant, which is milder for targetcompounds, and especially appropriate for heat-sensitivematerials. Fluorescence detection is rapid and reliable.

4 Conclusions

The inuence of various factors on the extraction of sulfon-amides has been studied. To the best of our knowledge this isthe rst report on using NP-7-CPE for the determination of SAsin food samples. NP-7-CPE was shown to be a viable pretreat-ment and clean-up technique, and compared with traditionalCPE extraction, the technique showed good reproducibility,

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strong specicity, and high sensitivity. A new molecular uo-rescence spectroscopy detection technique for sulfonamidesis developed, which is simple, rapid and effective. It wassuccessfully applied to determine the content of sulfonamideresidues in honey samples with satisfactory results. Ultrasound-assistance shortened derivatization time and improvedrecoveries.

Acknowledgements

This work was supported by Analysis Test Research Center ofKunming University of Science and Technology, Yunnan Prov-ince, China.

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