Food Chemistry 141 (2013) 436–443

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Food Chemistry

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Analytical Methods

Simultaneous derivatisation and preconcentration of parabens in foodand other matrices by isobutyl chloroformate and dispersive liquid–liquid microextraction followed by gas chromatographic analysis

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.012

⇑ Corresponding author.E-mail address: mohanitrc@gmail.com (M.K.R. Mudiam).

1 Equal first author(s).

Rajeev Jain a,b,1, Mohana Krishna Reddy Mudiam a,⇑,1, Abhishek Chauhan a, Ratnasekhar Ch a, R.C. Murthy a,Haider A. Khan b

a Analytical Chemistry Section, CSIR-Indian Institute of Toxicology Research, M G Marg, Lucknow 226 001, UP, Indiab Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi 110 006, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 6 July 2012Received in revised form 20 February 2013Accepted 2 March 2013Available online 14 March 2013

Keywords:ParabensDLLMEIsobutyl chloroformate derivatisationGC–FID

A simple, rapid and economical method has been proposed for the quantitative determination of para-bens (methyl, ethyl, propyl and butyl paraben) in different samples (food, cosmetics and water) basedon isobutyl chloroformate (IBCF) derivatisation and preconcentration using dispersive liquid–liquid mic-roextraction in single step. Under optimum conditions, solid samples were extracted with ethanol (dis-perser solvent) and 200 lL of this extract along with 50 lL of chloroform (extraction solvent) and 10 lL ofIBCF was rapidly injected into 2 mL of ultra-pure water containing 150 lL of pyridine to induce formationof a cloudy state. After centrifugation, 1 lL of the sedimented phase was analysed using gas chromato-graph-flame ionisation detector (GC–FID) and the peaks were confirmed using gas chromatograph-posi-tive chemical ionisation-mass spectrometer (GC–PCI–MS). Method was found to be linear over the rangeof 0.1–10 lg mL�1 with square of correlation coefficient (R2) in the range of 0.9913–0.9992. Limit ofdetection (LOD) and limit of quantification (LOQ) were found to be 0.029–0.102 lg mL�1 and 0.095–0.336 lg mL�1 with a signal to noise ratio of 3:1 and 10:1, respectively.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Parabens are known to be p-hydroxybenzoates formed by theesterification of p-hydroxybenzoic acid and are used as preserva-tives for various food, cosmetics and pharmaceutical products.The parabens normally used are methyl paraben (MP), ethyl para-ben (EP), propyl paraben (PP) and butyl paraben (BP). The wide useof parabens as preservatives is due to their low toxicity, neutral pH,no perceptible odour and thermal stability, which also make themideal as antimicrobial agents (Soni, Carabin, & Burdock, 2005). Usu-ally, a combination of parabens is used rather than the single ex-ploit of paraben to achieve high antimicrobial activity. However,in recent times, estrogenic activities of parabens were documentedsince the first finding of presence of parabens in human breast tis-sue (Dabre et al., 2004). The Council of the European Community(Directive 76/768/EC) has limited a maximum concentration ofindividual paraben to 0.4% (w/w) and a total parabens should beless than 0.8% (w/w) in consumer products (Routledge, Parker,Odum, Ashby, & Sumpter, 1998).

Analysis of parabens by GC requires derivatisation due to their po-lar nature. The GC derivatisation reagents such as N,O-bis (trimethyl-silyl) trifluoroacetamide (BSTFA), N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) and pentafluoropropionic acid anhy-dride (PFPA) requires a reaction temperature of 60 �C for 10 min un-der moisture-free conditions, overnight reaction at roomtemperature and reaction time of 20 min, respectively (Fan, Kubwab-o, Rasmussen, & Otazo, 2010; Lee, Peart, & Svoboda, 2005; Perez, Alb-ero, Miguel, & Brunete, 2012; Pietrogrande & Basaglia, 2007). AlkylChloroformates (ACFs) are derivatising reagents in GC, possesses sev-eral advantages over silylating reagents such as, reaction within oneminute at room temperature directly in the aqueous medium. (Citova,Sladkovsky, & Solich, 2006; Kaspar, Dettmer, Gronwald, & Oefner,2008; Luo et al., 2010; Mudiam et al., 2011; Yonamine, Tawil, Moreau,& Silva, 2003; Zahradnickova, Hartvich, Simek, & Husek, 2008).

Different sample preparation procedures were applied to theextraction and preconcentration of parabens in different matricessuch as sewage water and cosmetic samples (Canosa, Rodriguez,Rubi, Bollain, & Cela, 2006; Ferreira, Moder, & Laespada, 2011;Lee et al., 2005; Saraji & Mirmahdieh, 2009; Villaverde-de-Saaet al., 2010). The techniques used are solid-phase extraction, singledrop microextraction, non-porous membrane assisted liquid–li-quid extraction, solid-phase microextraction and stir bar sorptive

R. Jain et al. / Food Chemistry 141 (2013) 436–443 437

extraction. The stir bar sorptive extraction involves a coating ofpolymeric material like polydimethylsiloxane (PDMS) on a mag-netic bar as similar to GC stationary phase. The coated stir barhas been immersed in the liquid samples for extraction of chemicalanalytes. All these techniques suffer with a drawback of longerextraction time (20 to 90 min), and solid-phase extraction requiresa large amount of toxic solvents, which are being environmentallymalignant. It must also be noted that none of the methods reportedtheir use for the analysis of a wide variety of matrices (water, cos-metic and food samples). Recently, Rezaee and co-workers intro-duced a new microextraction technique, DLLME for theextraction and/or preconcentration of analytes before analysis (Re-zaee et al., 2006). As per our knowledge, no literature was availablefor the simultaneous derivatisation, extraction and preconcentra-tion of parabens using ACF and DLLME. Coupling of ACF derivatisa-tion with DLLME will not only enable the simultaneousderivatisation and preconcentration of parabens but also improvesthe sensitivity of the analysis.

In the present manuscript, a simple and rapid analytical methodhas been developed for the simultaneous derivatisation, extractionand preconcentration of parabens in different matrices (cosmetics,water and food) using ACFs as derivatising reagents and DLLME asextraction and preconcentration technique in a single step.

2. Experimental

2.1. Chemicals and reagents

All chemicals and standards used for this study were of analyt-ical grade unless otherwise stated. The standards of parabens (MP,EP, PP and BP) were supplied by Sigma (St. Louis, MO, USA). Ethylchloroformate (ECF), isobutyl chloroformate (IBCF), Chloroform,trichloroethylene, dichloromethane, chlorobenzene, methanol,ethanol, acetone, acetonitrile and sodium chloride (NaCl) were ob-tained from Merck (Darmstadt, Germany). Water used for thisstudy was of ultrapure grade produced from the Milli-Q waterpurification system (Millipore, Bedford, MA, USA). The stock solu-tion of mixture of parabens was prepared in a concentration of1 mg mL�1 each in acetonitrile. Working standard solution wasprepared daily by diluting the stock solution accordingly.

2.2. Collection of real samples

Various food items (pickles, vinegar, sauces, fruit juices), cos-metic items (face wash, fairness cream, shaving cream, moisturiz-ers, hair gel) and carbonated drinking water of different brandswas purchased from the local market for the analysis of parabens.Tap and drinking water was collected from laboratory premises inorder to verify the presence of parabens. River water, influent andeffluent sewage water samples were collected from Gomti River(Lucknow, India) and sewage-treatment plants located in ChinhatIndustrial Area (Lucknow, India), respectively. All water sampleswere stored at 4 �C whereas food and cosmetic samples werestored at room temperature before analysis.

2.3. Sample preparation

2.3.1. Food samplesPickle samples were cut into small pieces and homogenised. An

amount of one gram of sample was extracted with 5 mL of ethanolby sequential vortexing (for 3 min), sonication (for 5 min) and cen-trifugation (at 5000 rpm for 5 min). The supernatant ethanol wasfiltered and diluted up to 20 times before analysis. Vinegar andfruit juices were filtered and 0.5 mL of filtrate was diluted with

water (three times) and this filtrate was directly subjected forDLLME without any prior extraction.

2.3.2. Cosmetic samplesAn amount of 1 g of cosmetic sample was weighed in a 15 mL

centrifuge tube and extracted with 5 mL of ethanol by vortex for3 min followed by sonication for 5 min. The resultant mixturewas centrifuged for 5 min at 5000 rpm to settle down the solidmaterial, and the supernatant (ethanol) was collected in a separatetube and filtered. The filtrate was diluted (50 times) with ethanoland subjected to simultaneous derivatisation and DLLME.

2.3.3. Non-alcoholic beverages and water samplesCarbonated drinking water was degassed for 10 min in water

bath of ultra sonicator. An aliquot of 0.5 mL of sample was dilutedwith Milli-Q water and subjected to simultaneous derivatisationand DLLME. Real water samples (tap water, drinking water, riverwater and sewage water) were filtered, diluted appropriately andthen subjected to simultaneous derivatisation and DLLME.

2.4. Simultaneous derivatisation and DLLME procedure

For simultaneous derivatisation and DLLME, 2 mL of water sam-ple was placed in a 15 mL glass tube with a conical bottom. Anamount of 150 lL of pyridine was added to this solution as a cata-lyst for ACF derivatisation reaction. Two hundred microlitres ofethanol (disperser solvent), 50 lL of chloroform (extraction sol-vent) and 10 lL of IBCF (derivatising reagent) were rapidly injectedinto the aqueous sample. The resultant mixture was vortexed for30 s and centrifuged at 4000 rpm for 3 min in order to sedimentthe extraction solvent at the bottom of the conical tube. Simulta-neous derivatisation and extraction of parabens with IBCF andchloroform respectively and their dispersion into the aqueous sam-ple in a single step resulted into a formation of a cloudy solution.An aliquot of 1 lL of this sedimented phase was injected into agas chromatograph–flame ionisation detector (GC–FID) systemfor analysis. The ethanol extracts (200 lL) of solid samples weresubjected to simultaneous derivatisation and DLLME procedureas described above. The ethanol extracts itself acts as a dispersersolvent for DLLME of solid samples.

2.5. Chromatographic analysis

The separation and identification of IBCF derivatives of para-bens were achieved on a Perkin Elmer Clarus 500 gas chromato-graph equipped with an Elite-5 capillary column (5% diphenyl-95% dimethyl polysiloxane; 30 m � 0.25 mm i.d. � 0.25 lm filmthickness). Initial oven temperature was kept at 100 �C for 2 min,increased up to 250 �C at a rate of 10 �C min�1 and kept for5 min. Nitrogen (99.99%) was used as a carrier gas at a flow rateof 1 mL min�1. The injection was performed at 250 �C in splitlessmode. The flame ionisation detector was maintained at 300 �Cand flame was ignited with air and hydrogen with a flow rate of350 and 30 mL min�1 respectively.

Confirmation of derivatisation reaction of parabens with IBCFwas achieved on Gas Chromatography–Positive Chemical Ionisa-tion–Mass Spectrometry (GC–PCI–MS). A Trace GC Ultra coupledto TSQ Quantum XLS Mass Spectrometer (Thermo Fisher, USA)equipped with TG-5MS capillary column (5% phenyl–95% meth-ylpolysiloxane; 30 m length � 0.25 mm I.D. � 0.25 lm film thick-ness) was used for this purpose. The same gas chromatographicconditions described for GC–FID analysis were also adopted forGC–PCI–MS analysis. Helium (99.99%) was used as carrier gas.Transfer line and source temperature was kept to be 290 and220 �C. The MS was operated in full scan mode in the range of

Fig. 1. PCI mass spectras of IBCF derivatives of parabens: (a) methyl paraben (b) ethyl paraben (c) propyl paraben and (d) butyl paraben.

438 R. Jain et al. / Food Chemistry 141 (2013) 436–443

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50–300 amu. Methane at a flow rate of 2 mL min�1 was used forGC–PCI–MS.

3. Results and discussion

3.1. Optimisation of derivatisation conditions

Reaction of ACFs with polar analytes requires less than 1 min atroom temperature and the reaction can be performed in aqueousmedium which eliminates the use of anhydrous conditions nor-mally used for silylation using BSTFA. Initially, two mostly usedACFs viz. ECF and IBCF were evaluated for better derivatisationyield. With preliminary results, IBCF was found to be superior asit was producing a greater detector response in comparison toECF. However, ECF produced more volatile derivatives than IBCFderivatives under the same oven temperature programming andeluted prior to IBCF derivatives (retention times: MP 11.82 and13.51 min, EP 12.66 and 14.25 min, PP 13.66 and 15.23, BP 14.69and 16.21 for ECF and IBCF derivatives, respectively). Therefore,considering the detector response and very short difference(<2 min) in the retention times, IBCF was selected as derivatisingreagent for further experiments. To optimise the volume of IBCFand pyridine for the derivatisation of parabens, a set of experi-ments were conducted by varying the volume of IBCF in the rangeof 10–200 lL. The derivatisation efficiency of IBCF was found to behighest at a volume of 10 lL and significantly decreased at 50 lLthen remained constant (data now shown). Therefore, 10 lL ofIBCF was selected for further experiments. Pyridine was used tocatalyse the reaction and also as an acid scavenger for the comple-tion of derivatisation. Different volumes of pyridine were variedover the range of 50–200 lL at a constant volume of IBCF at10 lL. The maximum derivatisation efficiency was obtained froma pyridine volume of 150 lL (data not shown). Therefore, 10 lL

Fig. 2. Optimisation of DLLME parameters: (a) selection of disperser solvent, (b) effect oextraction solvent.

of IBCF and 150 lL of pyridine were selected for furtherexperiments.

3.2. GC–MS characterisation of derivatives

Gas chromatograph–Electron Ionisation–Mass spectrometry(GC–EI–MS) and GC–PCI–MS were used to confirm the derivatizedproducts of parabens with IBCF. Reaction of parabens with IBCF re-sults an increment of molecular weight of 100 amu as the activehydrogen of the phenolic hydroxyl from paraben molecules get re-placed by the isobutyl formate group (C5H9O2; m/z 101) from IBCF.However, a clear molecular ion was not obtained in the EI massspectra of paraben-IBCF derivatives, as this isobutyl formate moi-ety gets readily detached from the molecule upon electron impact,therefore, positive chemical ionisation (PCI) was performed in or-der to get molecular ion of the derivatives. The mass spectra’s ofparabens obtained after GC–PCI–MS analysis were shown inFig. 1a–d which shows a clear protonated molecular ion [M+H]+,at m/z 253, 267, 281 and 295. Loss of isobutyl formate (C5H9O2;m/z 101) moiety results in the formation of real molecular weightof parabens (MP = m/z 152; EP = m/z 166; PP = m/z 180; BP = m/z194). Further, the loss of –CH3O (m/z 31) from MP, –CH3CH2O(m/z 45) from EP, –CH3CH2CH2O (m/z 59) from PP and –CH3CH2-

CH2CH2O (m/z 73) from BP, respectively, results in the formationof a common ion at m/z 121. Additionally, loss of –CH3CH2 (m/z29) from EP; –CH3CH2CH2 (m/z 43) from PP and –CH3CH2CH2CH2

(m/z 59) from BP, respectively, results in the formation of hydroxylbenzoic acid moiety having m/z of 138.

3.3. Effect of disperser solvent and its volume on DLLME

In DLLME, disperser solvent plays a crucial role, as it allows thedispersion of extraction solvent into the aqueous sample where it is

f volume of ethanol, (c) selection of extraction solvent and (d) effect of volume of

Table 1Determination and recovery of parabens in different cosmetic products.

Analyte Concentration (lg g�1) Added (lg g�1) Total (lg g�1) Found (lg g�1) % Mean recovery (%RSD)

Fruit moisturiserMP 57 100 157 137.8 87.8 (2.15)

57 200 257 249.8 97.2 (1.25)EP 224.1 200 424.1 391 92.2 (2.42)

224.1 500 724.1 693 95.7 (1.69)PP 638.3 500 1138.3 1095 96.2 (0.71)

638.3 1000 1638.3 1607 98.1 (1.14)BP 30.7 100 130.7 116.8 89.4 (2.74)

30.7 200 230.7 218.5 94.7 (1.81)

Shaving creamMP 55.3 100 155.3 134.2 86.4 (1.46)

55.3 200 255.3 239 93.6(2.45)PP 1258.2 500 1758.2 1725 98.1 (1.14)

1258.2 1000 2258.2 2290 101.4 (0.82)

Hair conditionerMP 82.35 100 182.35 160.1 87.8 (2.61)

82.35 200 282.35 262.3 92.9 (1.84)

Table 3Recoveries of parabens in different food samples.

Matrix MP EP PP BP

Added Found %R Added Found %R Added Found %R Added Found %R

Picklea 50 43.14 86.28 (1.31)c 50 40.78 81.56 (1.82)c 50 42.43 84.86 (2.14)c 50 44.69 89.38 (1.96)c

250 228 94.20 (1.25)c 250 231.6 92.64 (2.22)c 250 231.2 92.48 (1.94)c 250 233.65 93.46 (1.46)c

Tomato saucea 50 41.71 83.42 (2.15)c 50 41.15 82.30 (2.18)c 50 43.67 87.35 (3.14)c 50 43.36 86.73 (2.58)c

250 229 91.61 (1.02)c 250 225.7 90.31 (1.36)c 250 232 92.82 (2.45)c 250 233.4 93.39 (2.37)c

Vinegarb 0.5 0.490 98.00 (1.08)c 0.5 0.473 94.68 (2.29)c 0.5 0.473 94.68 (2.64)c 0.5 0.482 96.44 (2.79)c

2.5 2.42 97.15 (1.98)c 2.5 2.38 95.29 (1.74)c 2.5 2.35 94.16 (2.39)c 2.5 2.40 96.17 (1.61)c

Fruit Juiceb 0.5 0.481 96.23 (1.26)c 0.5 0.487 97.49 (1.37)c 0.5 0.475 95.19 (2.09)c 0.5 0.474 94.86 (2.24)c

2.5 2.41 96.49 (1.56)c 2.5 2.42 96.97 (1.47)c 2.5 2.41 96.79 (1.15)c 2.5 2.38 95.53 (1.19)c

Carbonated waterb 0.5 0.458 91.63 (2.67)c 0.5 0.483 96.72 (1.61)c 0.5 0.487 97.59 (1.29)c 0.5 0.486 97.34 (1.07)c

2.5 2.33 93.48 (2.19)c 2.5 2.36 94.79 (2.64)c 2.5 2.40 96.37 (2.16)c 2.5 2.45 98.13 (1.67)c

%R = %Recovery.a Values are in lg g�1.b Values are in lg mL�1.c % RSD.

Table 2Recovery of parabens in different water samples.

Matrix MP EP PP BP

0.5 lg mL�1

(%RSD)2.5 lg mL�1

(%RSD)0.5 lg mL�1

(%RSD)2.5 lg mL�1

(%RSD)0.5 lg mL�1

(%RSD)2.5 lg mL�1

(%RSD)0.5 lg mL�1

(%RSD)2.5 lg mL�1

(%RSD)

WW(IL)

95.12 (2.10) 99.70 (0.83) 90.12 (2.15) 95.14 (1.96) 91.52 (2.19) 91.36 (1.36) 90.01 (2.63) 92.72 (2.17)

WW(OL)

93.81 (1.68) 99.04 (0.94) 94.69 (2.89) 97.37 (2.16) 94.25 (2.46) 91.88 (1.96) 96.79 (1.37) 97.35 (1.56)

TW 92.91 (2.0) 94.93 (1.84) 98.36 (1.56) 101.22 (2.34) 90.29 (1.96) 94.61 (2.36) 93.76 (2.46) 95.61 (1.69)RW 97.05 (1.15) 99.50 (1.28) 95.63 (2.14) 97.89 (1.68) 97.91 (1.17) 98.69 (1.61) 91.56 (2.82) 95.35 (2.08)DW 93.94 (2.64) 95.01 (2.18) 96.34 (2.36) 93.65 (1.89) 89.69 (2.59) 91.97 (2.65) 94.19 (1.64) 96.61 (1.96)

Abbreviations: WW (IL): waster water (Inlet); WW (OL): waste water (Outlet); TW: tap water; RW: river water; DW: Drinking water.

440 R. Jain et al. / Food Chemistry 141 (2013) 436–443

immiscible. The main criterion for the selection of the disperser sol-vent is the miscibility with both the extraction solvent and theaqueous sample in order to induce the phenomena of dispersion.Four widely used disperser solvents viz. ethanol, methanol, acetoneand acetonitrile were initially screened in order to select the bestdisperser solvent. The experiment showed the highest detector re-sponse when ethanol was used as a disperser solvent followed bymethanol, acetone and acetonitrile (Fig. 2a). Furthermore, volumeof ethanol was optimised by varying the volume between 100and 1000 lL at a constant volume of 100 lL of trichloroethylene(extraction solvent). Extraction efficiency of DLLME was signifi-cantly increased by the increase in the volume of ethanol up to200 lL and tends to decrease after 200 lL (Fig. 2b). This is because

of increasing solubility of extraction solvent in aqueous phase whenthe larger amount of disperser solvents was used (above 200 lL).Therefore, 200 lL of ethanol was selected as optimum volume ofdisperser solvent for further experiments.

3.4. Effect of extraction solvent and its volume

After finding out the suitable disperser solvent and its optimumvolume, a set of experiments were conducted to screen differentextraction solvents having density higher than the water (chloro-form, dichloromethane, trichloroethylene and chlorobenzene). Inall these experiments, the volume of ethanol was kept at its opti-mum level i.e. 200 lL. From Fig. 2c, it can be clearly seen that high-

Fig. 3. (A) GC–FID chromatogram of parabens in different matrices; (a) standard spiked in water at 2.5 lg mL�1, (b) MP, EP, PP and BP in fruit moisturiser, (c) MP and PP inshaving cream, (d) MP in hair conditioner, (e) spiked in fruit moisturiser at 200, 500, 1000 and 1000 lg g�1 of MP, EP, PP and BP, respectively and (f) blank sample followingsame DLLME procedure. Peak identification: 1-MP, 2-EP, 3-PP, 4-BP. (B) GC–FID chromatogram of parabens in food and water samples; (a) standard spiked in water at2.5 lg mL�1, (b) PP paraben in inlet waste water sample; (c) PP in tomato sauce sample and (c) MP in fruit juice. Peak identification: 1-MP, 2- EP, 3-PP, 4-BP.

R. Jain et al. / Food Chemistry 141 (2013) 436–443 441

442 R. Jain et al. / Food Chemistry 141 (2013) 436–443

est extraction efficiency of DLLME was obtained when chloroformwas used as extraction solvent. Further, the volume of chloroformwas optimised by taking different volumes of chloroform ranges50–250 lL at an interval of 50 lL with ethanol (200 lL) as dis-perser solvent and IBCF (10 lL) as derivatising reagent. The peakresponse of parabens was decreased gradually from 50–250 lL,this may be due to dilution of the sedimented phase at larger vol-umes (Fig. 2d). Therefore, 50 lL of the chloroform was selected forfurther experiments.

3.5. Effect of ionic strength and pH

To investigate the effect of salt on the extraction efficiency ofDLLME, NaCl was added in the range of 0–10% (w/v). Results re-vealed that, salt addition had no significant effect on the extractionefficiency of DLLME as the peak response was found to remain con-stant as the ionic strength increases. Therefore, no salt was addedfor further experiments.

In order to find out the effect of pH on DLLME, a set of experi-ments were conducted in which the pH of the aqueous sample be-fore DLLME was adjusted to 2, 4, 7, 10 and 12 (adjusted by 5 M HClfor acidic and 5 M NaOH for basic). The experiment showed that,pH at 7 was found to be optimum for DLLME extraction efficiency.At pH 7, the extraction efficiency of DLLME for parabens was foundto be maximum and response was gradually decreased at eitherside of pH 7. This may be due to the decrease in the derivatisationyield at pH above and below 7 and may also be due to the hydro-lysis of the derivatives formed, thus reducing the extraction effi-ciency (Blanco, Casais, Mejuto, & Cela, 2009; Soni et al., 2005).The maximum extraction efficiencies of parabens at neutral pHmay also due to the fact that, after derivatisation, the parabens re-mains neutral as their hydroxyl moiety gets replaced during o-alk-oxycarbonylation. This is also in agreement to the previous studiesin which alkyl phenols were extracted at neutral pH (Luo et al.,2010).

3.6. Effect of centrifugation time and speed

Centrifugation time and speed were studied within the range of3–15 min and 3000–8000 rpm, respectively. The results shownthat this parameter has no significant effect on extraction effi-ciency of DLLME procedure, as after mixing of extraction solvent,disperser solvent and aqueous sample equilibrium is achieved veryquickly, and centrifugation assists only in settling down the extrac-tion solvent but not affect the extraction efficiency of DLLME.Therefore, centrifugation time of 3 min and speed of 4000 rpmwere selected for further experiments.

3.7. Analytical performance

The developed method was validated for its linearity and preci-sion. A five point calibration curve was plotted using x-axis as con-centration and y-axis as detector response for MP, EP, PP and BPspiked in water in the concentration range of 0.1–10, 0.35–10,0.25–10 and 0.12–10 lg mL�1, respectively. The square of correla-tion coefficient (R2) was found to be 0.9952, 0.9937, 0.9992 and0.9913 for MP, EP, PP and BP respectively. Intra-day (repeatability)and inter-day precision (reproducibility) were calculated by ana-lysing five replicates at low and high concentration levels of cali-bration graph on a same day and at five different days and wereexpressed as percent relative standard deviation (%RSD). Intra-day precision for MP, EP, PP and BP were found to be 1.12%,1.50%, 1.59% and 2.87%, respectively whereas inter-day precisionfor MP, EP, PP and BP were found to be 5.52%, 6.30%, 6.78% and6.86%, respectively. LODs and LOQs were calculated according tothe procedure recommended by Eurachem guide (EURACHEM

Guide., 1998). The LODs for MP, EP, PP and BP were found to bein the range of 0.029, 0.102, 0.071 and 0.033 lg mL�1 whereasLOQs were 0.095, 0.336, 0.234 and 0.108 lg mL�1, respectively.

Recovery of parabens in different matrices (foods, cosmeticsand waters) were studied by standard addition of known amountof parabens into the matrices. In cosmetic samples, standard para-bens were added in the range of 100–1000 lg g�1 and the recover-ies were found to be in the range of 86.4–101.4% (Table 1). Therecoveries in liquid samples (water, vinegar, fruit juice and carbon-ated water) were tested at two different concentrations (0.5 and2.5 lg mL�1) and the percent recovery was found to be in the rangeof 89.69–101.22 (Table 2 and Table 3). Food samples (tomato sauceand pickle) were fortified at 50 and 250 lg g�1 and the percentrecovery was found to be in the range of 81.56–94.20 (Table. 3).The reason of choosing different concentrations for different matri-ces for recovery studies is to bring the peak response in the work-ing calibration range.

3.8. Application to real samples

The developed method has been successfully applied for thedetermination of parabens in different foods, cosmetics and watersamples as described. Parabens were detected in all the cosmeticsamples including fruit moisturisers, hair conditioners and shavingcreams (Table 1). In water samples, only PP was detected in inletwaste water sample at a concentration of 2.55 lg mL�1

(±0.007 lg mL�1) and all other water samples were found to befree of parabens. PP was detected at 26.1 lg g�1 (±0.18 lg g�1) intomato sauce whereas MP was found in fruit juice at 0.13 lg mL�1

(±0.002 lg mL�1). The overlay GC–FID chromatogram of standardparabens, real and spiked cosmetic samples and blank were shownin Fig. 3A. GC–FID chromatogram of food and water samples isshown in Fig. 3B.

4. Conclusion

A rapid and simple method has been developed based on simul-taneous derivatisation and preconcentration using IBCF and DLLMEfor parabens. The IBCF derivatisation of parabens overcomes thedisadvantages of widely used silylation as it does not require heat-ing, and the derivatisation is completed within 1 min directly inthe aqueous medium. Further the derivatisation yields a large peakresponse in comparison to underivatised parabens. The methodwas found to be rapid, simple, sensitive and economical in compar-ison to the earlier reported methods. The method has been suc-cessfully applied to different samples of food and other matrices.Furthermore, the developed method will find wide applicationsfor the routine analysis of parabens in various samples by foodand environmental laboratories.

Acknowledgments

The authors are indebted to Dr. K.C. Gupta, Director, CSIR-IITRfor his support in providing the necessary infrastructural facilities.Author R.J. and A.C. are grateful to UGC and RS is thankful to CSIRfor providing fellowship to carry out this research work. Fundingfrom CSIR-OLP-0004 is gratefully acknowledged.

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