Journal of Chromatography A, 1102 (2006) 268272

Development and validation of a solid phase micro-extractiongas chromatographymass spectrometry method for the determination of furan in baby-food
Federica Bianchi , Maria Careri, Alessandro Mangia, Marilena Musci
Dipartimento di Chimica Generale ed Inorganica, Chimica Analitica, Chimica Fisica, Universit` degli Studi di Parma, a Parco Area delle Scienze 17/A, 43100 Parma, Italy Received 21 July 2005; received in revised form 17 October 2005; accepted 27 October 2005 Available online 17 November 2005

Abstract An efcient and simple method for the determination of furan in baby-food (vegetables and fruits) by solid phase micro-extractiongas chromatographymass spectrometry (SPMEGCMS) was developed and validated. Experimental design was used to investigate the effects of temperature and time of extraction. The calculated regression model was used to nd the experimental conditions providing the optimal SPME extraction yield. Validation was carried out in terms of limit of detection (LOD), limit of quantitation (LOQ), linearity, precision and trueness. LOD and LOQ values in the low ng kg1 were achieved, whereas linearity was established over two order of magnitude. Good precision was obtained both in terms of intra-day repeatability and between-day precision on two concentration levels (RSD% lower than 3.6%). Recovery values of 91.5 6.2% and of 96.1 1.3% (n = 3) were calculated at 75 ng kg1 and 75 g kg1 level. Finally, the applicability of the method to the determination of furan in a number of commercial and home-made baby-food samples was demonstrated. 2006 Elsevier B.V. All rights reserved.
Keywords: Furan; Solid phase micro-extraction; Baby-food; Food safety; GCMS; Method validation

1. Introduction Food safety is regarded as a widespread priority task. In addition to the actions undertaken by food-related industries, the EU legislative committees are reviewing current legislation, both introducing new recommendations aimed to the establishment of general principles issuing rules to increase quality standards in food and xing hazard limits for the EU citizen health [1]. Recently, great attention has been paid to the presence of both natural and xenobiotic contaminants in raw and processed foods. Among naturally-occurring substances, great concern is addressed to the analysis of furan, this compound being classied by the International Agency for Research on Cancer (IARC) in the group 2B as possibly carcinogenic to humans [2]. The Food and Drug Administration (FDA) is requesting the submission of data and information regarding the occurrence of furan in food, as well as sources of exposure, mechanisms of forma-

Corresponding author. Tel.: +39 0521 905433; fax: +39 0521 905556. E-mail address: federica.bianchi@unipr.it (F. Bianchi).

tion and toxicology [3]. In fact, although furan is a well known heat treatment related by-product and has been detected in many thermally treated foods [4], only few methods principally based on static headspace techniques have been developed and applied for furan determination in food [510]. SPME is an alternative solvent-free sampling technique widely used for the analysis of volatile compounds, but nowadays only a SPMEGCMS method with the use of a cryofocussing unit was recently developed for furan determination in heated foodstuffs [11]. The aim of this work was the development and validation of a more simple method for the selective and sensitive analysis of furan in baby-food, based on SPMEGCMS. An experimental design was used to study the effects of two parameters on the SPME extraction, i.e. temperature of extraction and time of extraction. Data obtained allowed to calculate the main and interaction effects of the factors under investigation, thus indicating the optimised extraction conditions. The SPMEGCMS method was then validated under optimised conditions. According to the EURACHEM guidelines, in-house validation was carried out in terms of detection

0021-9673/$ see front matter 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.10.056

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limit (LOD), quantitation limit (LOQ), linearity, precision and trueness. Excellent results in the low ng kg1 level were obtained for LOD and LOQ, thus proving the potential of the method for the determination of furan at ultratrace levels in baby-food. The method developed was then applied to determine furan in commercial and home-made baby-food samples. 2. Experimental 2.1. Chemicals Furan (+99%, purity), furan-d4 (99.3%, purity) and methanol (99.8% purity) were purchased from SigmaAldrich (Milan, Italy). Standard and working solutions, prepared from the 2500 mg l1 methanolic stock solution by dilution in Ultra Resi-Analysed purge and trap water (J.T. Baker, Deventer, The Netherlands), were stored in completely lled vials closed with hermetic seals at 4 C until analysis. 2.2. Experimental design Fresh carrots (4 g) were boiled, minced and homogenized with 4 ml of Ultra Resi-Analysed purge and trap water. Experiments were carried out on samples spiked with furan at the nal concentration of 1 g kg1 . The vials were immediately closed just after the spiking procedure. A 22 two-level full factorial design (FFD) was performed [12] to investigate the effects of temperature of extraction (T) and time of extraction (t): low and high levels were T = 3080 C, t = 1040 min. This experimental plan allows us to evaluate the effects of the main factors and their interactions. The order of the experiments was randomised in order to avoid possible memory effect due to the analytical procedure. A F-test comparing the experimental and calculated responses at the centre of the experimental domain was performed to evaluate the existence of relevant quadratic effects and a star design was added to the factorial design experiments if a relevant quadratic effect was observed [13,14]. The nal regression model was then calculated using the Central Composite Design (CCD) experiments, obtained both from the FFD and the star design and used to nd the optimal extraction conditions. All statistical analyses were carried out by using the statistical package SPSS 10.0 for Windows (SPSS, Bologna, Italy). 2.3. SPME analysis SPME experiments were carried out using a 75 m carboxenpolydimethylsiloxane (CAR-PDMS) ber (Supelco, Bellefonte, PA, USA) by using manual device. The ber was exposed to the headspace of the 10 ml sample vial operating under the optimized extraction conditions, i.e. extraction temperature: 30 C and extraction time: 10 min. A constant magnetic stirring was always applied.

The ber was conditioned in the injection port of the gas chromatograph at 300 C under helium ow for 1.5 h prior to use. Desorption was carried out at the temperature of 230 C for 3 min. A ber blank was run between each sample to reduce memory effects. 2.4. GCMS analysis A HP 6890 Series Plus gas chromatograph (Agilent Technologies, Milan, Italy) equipped with MSD 5973 mass spectrometer (Agilent Technologies) was used for GCMS analysis. Helium was used as the carrier gas at a ow rate of 1 ml min1 ; the gas chromatograph was operated in splitless mode with the PTV injector (Agilent Technologies) maintained at the temperature of 230 C and equipped with a PTV multi-bafed liner (i.d. 1.5 mm, Agilent Technologies). Chromatographic separation was performed on a 60 m 0.25 mm, df 0.5 m HP-INNOWAX capillary column (Agilent Technologies). The following GC oven temperature program was applied: 40 C for 8 min, 12 C min1 to 200 C, 200 C for 2 min. Transfer line and source were maintained at the temperature of 220 and 230 C, respectively. The mass spectrometer was operated in selected-ion monitoring mode (SIM) by recording the current of the following ions: m/z 68 and 39 for furan and m/z 72 and 42 for furan-d4 . The corresponding ion ratios were used to conrm the identication of the analyte. A dwell time of 100 ms was used for all the ions. Preliminarily, full scan EI data were acquired to determine appropriate masses for SIM under the following conditions: ionisation energy: 70 eV, mass range: 35150 amu, scan time: 3 scan/s. All the analyses were performed setting the electron multiplier voltage at 1200 V. Signal acquisition and elaboration were performed using the HP Chemstation (Agilent Technologies). 2.5. Validation procedure Validation of the SPMEGCMS method was performed on a blank boiled carrot sample (no signal was detected at the retention time of the analyte) according to EURACHEM guidelines [15]. Detection and quantitation limits were calculated following the approach already used in previous works [14,16] by constructing a calibration curve in the 50500 ng kg1 range. Linearity was established over two order of magnitude in the 1100 g kg1 range. Furan-d4 was used as internal standard at the nal concentration of 30 g kg1 . Six concentration levels were analyzed performing three measurements at each concentration level. Statistical analysis (Bartlett, lack-of-t and Mandel test) were performed to check the goodness of t and linearity [16,17]. The signicance of the intercept (signicance level 5%) was established running a t-test. Intra-day repeatability and between-day precision, estimated over 3 days [12] were calculated in terms of RSD% on two concentration levels, performing three replicates at each level. Trueness was evaluated in terms of recovery by spiking blank cooked carrots with 75 ng kg1 and 75 g kg1 of furan.

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Recovery (R%) [15] was calculated as follows: R% = cobs 100 cspike

Divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) 2 cm50/30 m; Carboxen-polydimethylsiloxane (CAR-PDMS) 75 m. With the aim to load the highest amount of analyte, aqueous solutions containing furan at the concentration of 1 g l1 were analyzed for 15 min at the temperature of 40 C, following a previously described approach [11,14]. For each ber, three replicated measurements were performed. Data obtained showed a different behavior among the bers. As already obtained by Goldman et al., the CAR-PDMS resulted the ber with the better performance. 3.2. Experimental design A 22 two-level factorial design was used to evaluate the signicance of the main and interaction effects of the factors investigated. The experimental domain was dened taking into account instrumental and operative limits, namely: temperature values lower than 30 C could not be maintained over long time, owing to the thermal variability of the laboratory, whereas temperature values higher than 80 C could produce analyte desorption from the ber, whereas extraction times greater than 40 min would determine long analysis times. Eight replicated measurements at the centre of the experimental domain were performed in order to evaluate both the experimental error and the existence of relevant quadratic effects. The presence of curvature was tested by the F-test, as described in Section 2. A star design was added to the factorial design experiments since relevant quadratic effects were shown obtaining a Fcalc = 125 versus a Ftab(1,7, = 0.05) = 5.6. Using the CCD experiments the following nal regression model was then calculated: y = 146000(12000) 93000T (11000) +46000T 2 (16000) r2 = 0.91

where cobs is the mean concentration of the fortied sample and cspike is the spiked concentration. All the measurements were replicated three times. 2.6. Samples The following commercial baby-food samples were purchased in local stores: vegetables (nine samples of carrots, French beans, mixed vegetables and zucchini deriving from different producers) and fruits (nine samples of banana, applebanana and pear deriving from different producers). The vegetables samples were prepared by heating the closed jars in hot water, whereas the fruit samples were analysed without previous heating, thus reproducing the typical behaviour of the consumers. Home-made baby-food samples were also analysed: fresh fruit samples (apple, pear, banana and kiwi) and fresh vegetable samples (potatoes, zucchini and green-peas) were purchased in local stores. The fruit samples were minced and analysed, whereas the vegetables were boiled, homogeneised and analysed. 3. Results and discussion The aim of this work was to develop a simple and sensitive method based on SPMEGCMS analysis for furan detection and determination in baby-food. All the analyses were performed by using a 60 m 0.25 mm, df 0.50 m INNOWAX column in order to obtain the elution of the analyte at a retention time compatible with quantitative purposes. The column length as well as the lm thickness allowed to elute furan with an adequate retention time, at 7.5 min, thus obtaining a very good furan chromatographic signal without the use of a cryofocussing module as reported in a previous work [11]. The proper selection both of the chromatographic conditions (column, temperature program) and the acquisition mode (SIM) allowed to avoid matrix interference. Regarding the preparation of the solutions, obviously, being furan very volatile and characterised by a low molecular weight, all the solutions were stored in completely lled vials. In fact, when partially lled vials were used, a general decrease in the chromatographic response was evidenced during the time as a consequence of the furan partition in the headspace. 3.1. Fiber selection In order to obtain the highest furan yield and the absence of memory effects, ber selection was performed by testing three different coatings: Polydimethylsiloxane-divinylbenzene (PDMS-DVB) 65 m;

The model was used to nd the experimental conditions providing the optimal SPME extraction yield, i.e. temperature of extraction = 30 C. As for the time of extraction, since it was not a signicant effect, a value of 10 min was used in order to reduce analyses time. As a general result, being furan very volatile, it has to be observed that a low temperature value is sufcient to improve its partition in the gas phase: on the contrary, higher temperatures could produce its desorption from the ber. The use of a 10 min extraction reduced the analysis time, thus allowing to perform about 20 analyses in a working day (8 h). In a further step, the method was validated operating under these conditions. 3.3. Method validation The method was validated in terms of detection limit, quantitation limit, linearity, precision and trueness by using the experimental setting providing the optimised conditions.

F. Bianchi et al. / J. Chromatogr. A 1102 (2006) 268272 Table 1 Furan content in baby-food samples Baby-food samples Processed baby-food Applebananab Bananab Mixed vegetablesb Carrotsb French-beansb Zucchinib Apple Pear Banana Kiwi Potatoes Zucchini Green-peas Furan ( g/kg)a 2.78 4.3 99.1 15.38 50.09 140.9 c c c c 0.336 0.014 0.11 0.025 1.03 0.25 0.12 0.2 9.7 0.56 2.94 2.4

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Home-made baby-food (fruits)

Home-made baby-food (vegetables)

a b c

Mean of nine independent samples. Different producers. <LOD.

LOD and LOQ values, expressed as concentration, were calculated by constructing an appropriate calibration curve in the 50500 ng kg1 range. Excellent results were obtained for LOD (25.7 ng kg1 ) and LOQ (41.7 ng kg1 ) in boiled carrots, thus proving the potential of the method for the determination of furan at ultratrace levels. Good linearity (y = 5.29x 0.05, r2 = 0.998, n = 18) was demonstrated over two order of magnitude in the 1100 g kg1 range by applying Mandels tting test (Fcalc = 8.40, Ftab(1,16,CI = 99%) = 8.53). Method precision was evaluated testing two concentration levels, i.e. 50 ng kg1 and 100 g kg1 . Good results were obtained both in terms of intra-day repeatability and betweenday precision: RSD% values lower than 1.2% at the highest concentration and lower than 3.5% at the lowest one were calculated for intra-day repeatability, whereas between-day precision was evaluated verifying homoscedasticity and performing ANOVA on the data acquired over 3 days. ANOVA showed that mean values were not signicantly different among the 3 days obtaining p values of 0.069 and 0.615 for the highest and lowest level, respectively at 95% condence level. RSD% lower than 3.6% at both concentration levels were calculated. Extraction recoveries of 91.5 6.2% and of 96.1 1.3% (n = 3) were calculated at 75 ng kg1 and 75 g kg1 , respectively by addition of furan to blank boiled carrots samples. These results show the good efciency of the developed method in terms of extraction recovery as well as of precision, without using the standard addition method recommended by FDA [5]. 3.4. Baby-food samples Finally, applicability of the validated method for the determination of furan in baby-food samples was demonstrated (Fig. 1). Different commercial and home-made vegetable and fruit samples were submitted to analysis: data obtained are reported in Table 1.

Fig. 1. GCSIMMS chromatograms of: (A) a commercial baby-food sample (zucchini) and (B) an home made baby-food sample (zucchini).

As shown in the table furan was detected and quantitated at different concentration levels depending both on the matrix and on the cooking technique. In particular, all the processed babyfood showed higher furan levels in comparison with the homemade products. Even though the number of samples considered does not make possible a denitive conclusion, this behaviour could be ascribed to the different cooking technologies. In fact, all the commercial processed foods are submitted to the heating treatments in closed pots, whereas the home-made baby-food are cooked in open containers. Under these circumstances, the analyte, being extremely volatile can be more easily released from the matrix. As for the matrices analysed, it was observed that all the fruit samples revealed a furan content lower than the vegetable samples. In the case of the processed baby-food samples, this difference could be explained taking into account that the fruit samples are generally pasteurised, whereas the vegetables are sterilised: the different heating treatment could be responsible for furan production. Nowadays, no Acceptable Daily Intake values have been established: however, on the basis of our results, considering a consumption of 350 g/die of canned baby-food and assuming a body weight of 7.5 kg of a 6 month baby, this would result in a maximum exposure of 7 g/kg b.w./day. Future investigations will be aimed to the analysis of other matrices, like meat in order to verify if different technological treatments are able to inuence furan production. 4. Conclusions A simple method for ultratrace determination of furan in baby-food (vegetables and fruits) was developed and validated.

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F. Bianchi et al. / J. Chromatogr. A 1102 (2006) 268272 [6] http://www.fda.gov/ohrms/dockets/ac/cfsan04.html, accessed 27 September 2005. [7] http://www.cfsan.fda.gov/dms/furandat.html, accessed 27 September 2005. [8] A. Becalski, S. Seaman, J. AOAC 88 (2005) 102. [9] T. Kuballa, S. Stier, N. Stricow, Deutsche Lebensmittel-Rundschau 101 (2005) 229. [10] A. Becalski, D. Forsyth, V. Casey, B.P.Y. Lau, K. Pepper, S. Seaman, Food Add. Contam. 22 (2005) 535. [11] T. Goldmann, A. Perisset, F. Scanlan, R.H. Stadler, Analyst 130 (2005) 878. [12] G.E.P. Box, W.G. Hunter, J.S. Hunter, Statistic for Experimental, Wiley, New York, 1978. [13] A.I. Khuri, J.A. Cornell, Response Surfaces, Marcel Dekker, New York, 1987. [14] F. Bianchi, M. Careri, C. Corradini, A. Mangia, M. Musci, CAC 1 (2005) 129. [15] The Fitness for Purpose of Analytical Methods: A Laboratory Guide to Method Validation and Related Topics, EURACHEM Guide, First English Edition 1.0-1998, LGC (Teddington) Ltd., http://www. eurachem.ul.pt/. [16] B. Antolini, F. Bianchi, M. Bottazzi, M. Careri, M. Musci, Chromatographia 60 (2004) 323. [17] N. Draper, H. Smith, Applied Regression Analysis, Wiley, New York, 1981.

The analytical approach was based on the use of SPME coupled with GCMS analysis for food safety evaluation purposes. The selection of the SPME ber and the use of an experimental design allowed to calculate the optimal sampling conditions. These conditions were found in correspondence with a temperature of extraction of 30 C. A time of 10 min was chosen in order to obtain shorter analyses time. Under these experimental setting values, the method proved accurate and sensitive allowing the determination of the investigated analyte at low ng kg1 levels. The method developed was successfully applied to analyse a number of commercial and home-made baby-food samples, thus revealing differences related to the production technique. References
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