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Eur Food Res Technol (2008) 228:8391 DOI 10.

1007/s00217-008-0909-8

ORIGINAL PAPER

Polycyclic aromatic hydrocarbons (PAH) in various types of tea


Katja Ziegenhals Wolfgang Jira Karl Speer

Received: 12 March 2008 / Revised: 4 June 2008 / Accepted: 8 June 2008 / Published online: 28 June 2008 Springer-Verlag 2008

Abstract For the analysis of the 16 PAH (EFSA-PAH), which are classied as priority for different food groups by the Scientic Committee on Food (SCF) and the Joint FAO/WHO Experts Committee on Food Additives (JECFA) in tea, a sensitive analytical Fast-GC/HRMS method was used. The sample preparation included accelerated solvent extraction (ASE) and the highly automated clean up steps, gel permeation chromatography and solid phase extraction. The analytical parameters, limit of detection (0.010.02 lg/kg) and limit of quantication (0.03 0.06 lg/kg), were determined. The repeatability (RSD, n = 3) of different PAH in fruit tea ranged from 0.1 to 11%. It was observed that the total contents of the 16 PAH in tea samples ranged from 14 to 2,662 lg/kg. The analysed tea samples showed an increasing presence of PAH in the following order: herbal and fruit tea (n = 7) \ black tea (n = 11) \ green tea (n = 11) \ white tea (n = 3) \ mate-tea (n = 8). The correlation coefcient (R) between BaP and the sum of the 16 EFSA-PAH was established considering the contamination amount in all the 40 tea samples analysed. Keywords Tea EFSA Fast-GC GC/HRMS PAH

K. Ziegenhals W. Jira (&) Max Rubner-Institut (MRI), Federal Research Institute of Nutrition and Food, Analysis Division, E.-C.-Baumann-Str. 20, 95326 Kulmbach, Germany e-mail: wolfgang.jira@mri.bund.de K. Speer Institute of Food Chemistry, Technical University of Dresden, Dresden, Germany

Abbreviations 5MC 5-Methylchrysene ASE Accelerated solvent extraction BaA Benzo[a]anthracene BaP Benzo[a]pyrene BbF Benzo[b]uoranthene BcL Benzo[c]uorene BgP Benzo[g,h,i]perylene BjF Benzo[j]uoranthene BkF Benzo[k]uoranthene CPP Cyclopenta[c,d]pyrene CHR Chrysene D/A Dichloromethane/acetone DeP Dibenzo[a,e]pyrene DhA Dibenzo[a,h]anthracene DhP Dibenzo[a,h]pyrene DiP Dibenzo[a,i]pyrene DlP Dibenzo[a,l]pyrene EFSA European Food Safety Authority EPA Environmental Protection Agency GPC Gel permeation chromatography HRMS High resolution mass spectrometer IcP Indeno[1,2,3-cd]pyrene IQR Interquartile range JECFA Joint FAO/WHO Experts Committee on Food Additives KOH Potassium hydroxide LLE Liquid liquid extraction LOD Limit of detection LOQ Limit of quantication Na2SO4 Sodium sulphate PAH Polycyclic aromatic hydrocarbons SCF Scientic Committee on Food SIM Selected ion monitoring SPE Solid phase extraction

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Introduction Polycyclic aromatic hydrocarbons (PAH) consist of two or more condensed aromatic carbon rings. They are ubiquitous compounds generated by pyrolysis of organic materials. Some of them show carcinogenic properties. The best known carcinogenic PAH compound is BaP which has been used as a leading substance until now. Gaseous and particlebound PAH can be transported over a long distance. After deposition they are able to accumulate in vegetation [1]. This could cause human exposure to PAH through food consumption. Tea leaves which possess a high surface area can be contaminated with PAH. During the preparation of tea also technical processes such as roasting and drying PAH are formed and can accumulate on the tea leaves. For producing green tea the young tea leaves are withered, steamed or pan red, dried, graded and packaged. Black tea is made by fermenting the slightly wilted leaves before smoked ring, amed ring or steaming. Producing white tea consists of air-drying unopened leaf buds, short heating and again air-drying. Drying tea leaves using combustion gases from burning wood, oil or coal may also be responsible for the accumulation of PAH [2]. The accumulation as such depends on the surface of the leaves, the thickness of the cuticula and plant composition [3, 4]. The layer of wax which covers the surface of fresh tea leaves may absorb PAH from the air. The stomata of these tea leaves offer a pathway for PAH to enter the leaves [5]. Furthermore, tea plants are able to uptake PAH from contaminated soil or water by their roots, but the subsequent transport to the leaves by xylem is very low [5]. Some papers deal with the content of the 16 priority PAH recommended by the US Environmental Protection Agency (16 EPA-PAH: naphthalene, acenaphthylene, acenaphthene, uorene, phenanthrene, anthracene, uoranthene, pyrene, BaA, CHR, BbF, BkF, BaP, IcP, DhA, BgP) in tea. The contamination in tea ranged between 3 and 20 lg BaP/kg (3 Russian mixtures, 5 brands of Chinese tea, 1 Russian. Smoke tea, 1 Chinese smoke tea) [6], 4971,162 lg PAH/kg (two green teas, two brick teas) [7], 137,536 lg PAH/kg (11 different brands) [80], 48 1,703 lg PAH/kg (34 different brands) [9], 9,650 1,200 lg PAH/kg (black tea samples of the same producing area) [2] and 3238,800 lg PAH/kg (8 brands of Chinese tea) [10]. The PAH contents in tea infusion were reported too [1115], highlighting that a maximum of 11% of the PAH present in the tea leaves are transferred into the infusion [3]. Since 1 April 2005 the Commission Regulation (EC) No 208/2005 of 4 February 2005 provides maximum levels for BaP in different food groups [16]. Now, these maximum levels are part of Commission Regulation

(EC) No 1881/2006 of 19 December 2006 [17]. In addition, the Commission recommends that the member states analyse the contents of 15 PAH compounds classied as priority (15 SCF-PAH) to check the suitability of BaP as a marker [18]. These 15 SCF-PAH are BaA, CHR, CPP, 5MC, BbF, BkF, BjF, BaP, DhA, IcP, BgP, DlP, DeP, DiP and DhP. In particular, DlP has been in the spotlight of scientic interest recently because toxicological investigations indicated that DlP probably has a much stronger carcinogenic potential than BaP [19, 20]. Additionally, the EFSA recommends analysing BcL assessed to be relevant by JECFA [21]. Up to now, methods for analysing the 15 SCF-PAH were developed at the Max Rubner-Institut (MRI) in Kulmbach, Germany, and checked successfully in interlaboratory comparison studies for primary smoke avourings and edible oils [2224]. Within the collaborative study for edible oils BcL was additionally analysed. For the analysis of the 16 EFSA-PAH compounds in different food groups a Fast-GC/HRMS-method was developed [25]. Considering the number of papers dealing with PAH presence in tea, the high occurrence of PAH in this matrix has been widely proved. However, no data on the presence of the 16 EFSA-PAH have been reported yet. Most of the methods used to determine PAH in tea samples involve different types of extraction and clean up procedures. ASE [26], saponication [27] and soxhlet extraction [7, 28] are possible extraction methods. SPE [7] and GPC [26] can be used as clean up steps. For the detection and quantication of PAH in tea HPLC [2, 13, 2931] and GC with ame ionization detector [28] were used in further investigations. There are only a few papers that focus on PAH analyses in tea using GC low resolution mass spectrometry [15, 27]. Most studies rely on 5 50 g sample weight [7, 8, 10, 2628, 30] with a limit of quantication (LOQ) of 0.15 lg/kg [10, 27, 29]. With the described method and the use of GC high resolution mass spectrometry, a sample weight of 1.52 g is sufcient in order to obtain a LOQ in the range of 0.03 0.06 lg/kg. The main objective of the present study was to determine the contents of these PAH compounds in different types of tea. After a comparison of different extraction methodologies, the ASE was optimized for the extraction of PAH in the tea matrix. For the analysis of 40 tea samples in this study ASE, GPC and SPE were used. The extraction method developed delivered satisfactory results and is less time-consuming than the saponication and soxhlet extraction. Due to its speed, automation and sensitivity this sample preparation is the preferred method.

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Materials and methods Materials and reagents 40 samples of tea bags and loose tea were purchased from different manufacturers. These 40 samples included 11 green tea, 11 black tea, 3 white tea, 8 mate tea and 7 herbal and fruit tea samples. All solvents, except methanol and dichloromethane were obtained in picograde quality from Promochem (Wesel, Germany). Methanol was obtained in uvasol quality from Promochem (Wesel, Germany) and dichlon romethane in pestanal quality from Riedel-de Hae (Seelze, Germany). Celite 545 (p.A., 0.020.1 mm), KOH (per analysis) and anhydrous sodium sulphate (per analysis) were acquired from Merck (Darmstadt, Germany). The drying material (poly(acrylic acid), partial sodium salt-graft-poly(ethylene oxide)) was purchased from Sigma Aldrich (Munich, Germany), Bio Beads SX3 (200400 mesh) from Bio-Rad Laboratories (Munich, Germany) and silica gel from Merck (Darmstadt, Germany). Glass microbre lters (18 mm i.d.) were obtained from Dionex (Idstein, Germany). The PTFE-Filters (1 lm pore size, 25 mm i.d.) and the SPE-Cartridges (12 mm i.d.) were purchased from Alltech (Unterhaching, Germany). Standard solutions The isotope labelled PAH standard solutions (for quantication: benzo[a]anthracene-13C6, chrysene-13C6, 5-methylchrysene-d3, benzo[b]uoranthene-13C6, benzo[k] 13 uoranthene- C6, benzo[a]pyrene-13C4, benzo[g,h,i]perylene-13C12, dibenzo[a,h]anthracene-d14, indeno(1,2,3-cd) pyrene-d12, dibenzo[a,e]pyrene-13C6, dibenzo[a,i]pyrene13 C12 and for recovery: benzo[a]anthracene-d12, benzo[a]pyrene-d12, benzo[g,h,i]perylene-d12 in isooctane) were purchased from Promochem (Wesel, Germany) and the uorinated PAH standards (for quantication: 5-uorobenzo[c]uorene, 13-uorodibenzo[a,l]pyrene) from Biochemical Institute for Environmental Carcinogens Prof. Dr. Gernot Grimmer-Foundation (Grosshansdorf, Germany). The PAH contents in the standard solution ranged from 60 to 170 ng/mL. All standards were dissolved in isooctane. The standard mixture was prepared by merging the stock solutions of the single PAH compounds followed by a subsequent dilution with isooctane. The internal standard for quantication (50 lL of standard solution) was added before the extraction procedure and the recovery standard (50 lL of standard solution) before injection.

Analytical procedures The analytical procedure described by [26] was modied. The sample preparation of this analytical method included ASE, GPC and SPE. For the detection of PAH compounds a GC/HRMS method was used. Different types of extraction procedures were compared: accelerated solvent extraction (ASE), saponication with liquid-liquid-extraction (LLE) and ultrasonic-extraction with dichloromethane/ acetone (50:50, v/v). Extraction Saponication 1.52 g tea and 30 mL 2 M methanolic potassium hydroxide (KOH) were heated under reux for 1 h. After cooling, the total volume was ltered through glass wool into a separating funnel. An extraction step with 30 mL cylohexane followed three times. The combined cyclohexane extracts were washed three times with 30 mL portions of bidistilled water. After washing, the combined cyclohexane phases were ltered through Na2SO4/Celite (70:30, w/w), and the solvent was removed with a rotary evaporator. Ultrasonic extraction This extraction procedure was described by Lin and Zhu [2] (LLE-D/A). The sample amount was also 1.52 g of tea. Tea samples were extracted by ultrasonication with a solution of dichloromethane/acetone (1:1, v/v). The extraction procedure was repeated three times. The combined extracts were ltered through anhydrous Na2SO4/ Celite (70:30, w/w). The solvent was removed with a rotary evaporator. Accelerated solvent extraction (ASE) The homogenised sample (1.52 g tea) was levigated with the same amount of the drying material poly(acrylic acid), partial sodium salt-graft-poly(ethylene oxide). The resulting material was poured into 33-mL cells which are locked with glass microber lters at the outlet end of the extraction cells. The extraction was performed with an ASE 200 from Dionex (Sunnyvale, USA) and carried out with n-hexane at 100 C and 100 bar for a constant time of 10 min. The ush volume was 60%, and the purge time was 120 s. Two extraction cycles were accomplished (ASE-1). The solvent mixture used in the extraction procedure was modied twice, and the extraction cycles were increased as follows: The second ASE-method used four

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extraction cycles with n-hexane (ASE-2). The third ASEmethod used n-hexane/acetone (50:50, v/v) instead of nhexane (ASE-3). Gel permeation chromatography (GPC) The evaporated ASE extract was dissolved in 4.5 mL cyclohexane/ethylacetate (1:1, v/v) and ltered through a polytetrauoroethylene (PTFE) lter with a pore size of 1 mm. The GPC column (25 mm i.d.) was lled with 60 g Bio Beads S-X3. Samples were eluted at a ow rate of 5 mL/min applying cyclohexane/ethylacetate (1:1, v/v). The waste time was 036 min and the collect time 36 65 min. The GPC solvent was removed with a rotary evaporator and the eluate was dried in a nitrogen stream. Solid phase extraction (SPE) The SPE was performed automatically with a modied ASPEC Xli (automatic sample preparation with extraction columns) [32] from Gilson (Bad Camberg, Germany). A total of 1 g silica dried for 12 h at 550 C and subsequent deactivating with 15% bidestilled water was lled into commercial 8-mL SPE columns (12 mm i.d.). After conditioning the columns with 3 mL cyclohexane the samples were applied and eluted with 10 mL cyclohexane. Fast-GC/HRMS analysis

column (60 m 9 0.25 mm 9 0.25 lm) purchased from Varian (Darmstadt, Germany). Helium was used as carrier gas at a constant pressure of 27 psi. The injection temperature was 300 C and the injection volume was 1 lL (splitless). The following temperature program was used: isothermal at 50 C for 1 min, at 25 C/min to 280 C, at 1 C/min to 330 C, isothermal at 330 C for 30 min. The quantication of PAH by GC/HRMS was performed by using a VG Autospec (Waters, Manchester, UK) working in the EI positive ion mode using an electron energy of 35 eV. The transfer line temperature and the ion source temperature were maintained at 280 and 250 C, respectively. The resolution of the MS was tuned to 8,000 (10% valley denition). The PAH were analysed in a 4function SIM experiment. Validation tests For validation, fruit tea samples were spiked with several dilutions of a standard mixture of native PAH compounds. Five PAH concentration levels 1, 5, 10, 15 and 20 lg/kg were studied. This standard mixture of native PAH compounds and the internal standard for quantication were added before the extraction. For the sample preparation the modied ASE method, GPC and SPE were used, for detection of PAH compounds the Fast-GC/HRMS method was applied. Results

Fast-GC/HRMS was performed using a Trace-GC chromatograph (ThermoFisher, Germany) equipped with a split/splitless injection port. Separation was performed on a TR-50MS column (10 m 9 0.1 mm 9 0.1 lm) (ThermoFisher, Germany). The injection temperature was 320 C; injection volume was 1 lL (splitless). Helium with a constant ow of 0.6 mL/min was used as carrier gas. The following temperature program was used: isothermal at 140 C for 1 min, at 10 C/min to 240 C, at 5 C/min to 270 C, at 30 C/min to 280 C, at 4 C/min to 290 C, at 30 C/min to 315 C and at 3 C/min to 330 C. Identication of PAH by GC/HRMS was performed using a sector mass spectrometer DFS (ThermoFisher, Germany) working in the EI positive ion mode and applying an electron energy of 45 eV. The temperatures of the source and the transfer line were heated up to 280 and 300 C. The resolution of the MS was tuned to 8,000 (10% valley denition). GC/HRMS analysis

Extraction methods Two different extraction methods (ASE and LLE) were compared to extraction by saponication. In addition, the ASE was modied twice. The results of the extraction efciency are shown in Fig. 1. The results by using saponication were set to 100% of extraction efciency. The rst two ASE-methods were not able to reach comparable extraction efciencies for the PAH. In some cases the content accounted for only half of the content determined with saponication and LLE. Next to the ASE using a solvent mixture of n-hexane/acetone the ultrasonic extraction using D/A resulted in similar PAH contents in comparison to the saponication (deviation: ASE-3 max. 8%, LLE-D/A max. 21%). The ASE-3 method, which used a solvent mixture of nhexane/acetone, was preferred for the following investigations. Validation tests

The GC/HRMS analysis of PAH was performed on a HP 5890 II gas chromatograph with a split/splitless injection port. The GC was equipped with a VF-17 ms capillary

The average recovery for all PAH ranged between 75 and 117%. All PAH compounds showed good linearity within

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Eur Food Res Technol (2008) 228:8391 Fig. 1 Results of the efciency of the extraction methodologies (ASE-1, ASE-2, ASE-3, LLED/A) in comparison with saponication followed by LLE using cyclohexane (100%)

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the tested concentration range with determination coefcients (R2) in the range of 0.99640.9999. The relative standard deviation (RSD) was between 0.1 and 11% (n = 3 replicates). The LOD, which is dened as the lowest measured content, from which it is possible to deduce the presence of an analyte with reasonable statistical certainty (signal-tonoise ratio = 3:1), was 0.02 lg/kg for the dibenzopyrenes and 0.01 lg/kg for the other PAH compounds. The LOQ, which is dened as the lowest content of the analyte that can be measured with reasonable statistical certainty (signal-to-noise ratio = 9:1), was 0.06 lg/kg for the dibenzopyrenes and 0.03 lg/kg for the other PAH. To check the suitability of this method for tea samples with BaP contents out of the range tested ([20 lg/kg), three mate-tea samples with high PAH contents were analysed in two ways. The rst method was the standard procedure of sample preparation. Therefore, the isotope labelled standards in the range of 38.5 ng were added before ASE extraction. For the second method, the ASEextract was separated into three parts and higher amounts of isotope labelled standards in the range of 3085 ng were added to each part. Both results were compared (Table 1). The deviations in both results ranged from 1 to 28%. Consequently, this method is also suitable for analysing tea samples whose BaP contents are out of the tested range. Comparison of Fast-GC/HRMS and GC/HRMS Two separation methodologies were compared in this study. The rst method was a Fast-GC-method performed within 25 min. The second one was a GC-method which used a 60 m capillary column. The runtime of the second method was 90 min. Both columns have a 50% phenylpolysiloxan phase. Both methods ran on different mass spectrometers with different sensitivities. The LODs and the LOQs of the GC/HRMS method were different from

the Fast-GC/HRMS as different mass spectrometers were used. The LODs of GC/HRMS method for BcL, BaA, CHR, CPP, 5MC, BbF, BkF, BjF, BaP, BgP were 0.01 lg/ kg and the LOQs 0.03 lg/kg. For IcP, DhA, DlP, DeP, DiP, DhP the limit of detection was set to 0.03 lg/kg and the limit of quantication to 0.09 lg/kg. With the help of this comparison it was shown that the short method is able to determine the same content for the PAH except for BcL (Table 2). Furthermore in some tea samples different contents for CPP, 5MC and IcP were observed using FastGC/HRMS and GC/HRMS. With both methods a separation of triphenylene (TP) and CHR was not possible. Therefore, sum contents of both PAH were listed.
Table 1 Comparison of the PAH results using different standard concentrations to check the suitability of the methods for the analysis of tea with BaP contents out of the tested linearity range (lg/kg) Standard method (standard conc. 60170 ng/mL) Tea A BcL BaA CPP 5MC BbF BkF BjF BaP IcP DhA BgP DlP DeP DiP DhP 47.0 83.6 93.8 5.8 63.5 26.0 35.9 75.8 64.9 3.8 61.8 2.6 6.7 5.1 0.5 Tea B 17.0 59.8 116.8 10.0 4.8 67.8 26.7 36.5 79.3 92.2 5.3 106.2 1.6 8.9 7.4 0.4 Tea C 194.5 374.6 746.4 168.8 19.2 258.1 87.5 123.5 236.5 184.5 13.3 221.2 5.6 16.2 12.1 0.8 Separated extracts (standard conc. 6001,700 ng/mL) Tea A 50.5 84.2 129.6 85.5 8.4 63.0 26.7 36.7 78.0 64.8 4.8 72.6 2.2 6.4 6.8 0.6 Tea B 17.8 57.1 119.5 8.4 6.7 64.3 26.7 35.2 76.0 87.9 4.8 104.5 1.8 7.8 6.5 0.6 Tea C 215.2 367.4 674.5 157.9 23.4 255.7 87.9 126.8 248.1 184.2 13.6 220.7 7.5 15.4 11.9 1.0

CHR 125.8

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Table 2 Comparison of the 16 EFSA-PAH contents determined with Fast-GC/HRMS and GC/HRMS Tea A GC BcL BaA 1.6 15.8 Tea B Fast-GC GC 4.3 14.1 19.0 3.2 1.4 14.5 7.0 8.2 11.4 9.7 1.4 11.3 0.3 1.3 0.5 0.2 4.2 Tea C Tea D Tea E Fast-GC GC 0.4 1.6 4.9 0.2 0.3 1.7 1.1 1.2 0.8 1.0 0.1 0.6 0.5 6.4 9.6 3.9 2.6 3.1 1.5 1.4 1.5 1.6 0.7 1.1 0.1 Tea F Fast-GC GC 2.9 6.2 8.8 0.2 2.0 3.3 1.5 1.7 1.9 2.2 0.4 1.6 0.4 1.9 3.2 Tea G Fast-GC GC 3.2 3.0 1.9 9.6 Tea H Fast-GC 1.2 3.6 8.3 1.0 0.3 2.6 1.6 1.7 1.8 1.2 0.1 1.1 0.2 0.1

Fast-GC GC 8.7

Fast-GC GC 0.4 1.5 5.3 0.3 0.7 1.4 1.0 1.0 0.7 0.6 0.1 0.4

Fast-GC GC 4.4 9.7 0.7 3.8 9.0 1.1 0.4 3.0 1.7 2.0 2.0 1.3 0.2 1.4 \0.09 \0.09

15.6 16.2 45.9 44.9 95.2 81.9 18.0 17.0 3.6 2.5 42.7 38.9 17.9 17.1 22.5 23.7 35.2 37.8 27.9 36.8 4.0 1.6 2.8 2.2 0.8 3.1 1.1 3.5 2.1 0.5 40.4 38.9

21.7 20.5 41.4 34.4 2.5 2.0 10.8 4.2 1.7 9.4

CHR 21.2 + TP CPP 1.4 5MC BbF BkF BjF BaP IcP DhA BgP DlP DeP DiP DhP 0.9 14.0 7.6 8.7 11.5 9.5 1.8 10.6 0.4 0.8 \0.03 \0.03

20.2 17.3 0.9 1.1 6.0 2.7 3.3 2.4 2.9 0.6 2.4 0.3 0.6 0.5 0.3 0.7 0.7 5.6 2.5 3.2 2.2 3.2 0.4 2.6 0.2 0.7 0.5 0.2

16.1 14.8 2.7 1.3 8.2 4.1 5.3 6.9 4.7 0.8 5.1 0.4 1.1 0.6 0.4 2.4 0.9 8.0 4.2 5.3 6.7 6.5 0.8 5.4 0.3 1.3 0.7 0.2

20.9 18.7 12.4 11.4 16.0 15.1 14.2 13.0 2.3 0.9 1.0 1.6 0.2 1.7 0.5 1.7 1.0 0.1 15.0 14.2

\0.09 0.06 \0.09 0.08 \0.03 0.08 \0.03 0.06

\0.03 0.07 \0.03 0.1 \0.03 0.1

\0.09 \0.06

\0.03 \0.02

PAH in tea In this investigation, 40 tea samples were analysed and the contents of the 16 EFSA-PAH determined (Table 3). The method for the tea analysis included ASE-3, GPC, SPE and Fast-GC/HRMS. The highest content of BaP was detected in mate tea (237 lg/kg). This value was in the same order of magnitude as the BaP contents in 10 different mate teas (190714 lg/kg) determined by Ruschenburg et al. [27]. The analysed tea samples (see Fig. 2) show the increasing presence of the total PAH content (median values) in the following order: herbal and fruit tea (22 lg/kg) \ black tea (33 lg/kg) \ green tea (72 lg/kg) \ white tea (156 lg/ kg) \ mate-tea (873 lg/kg). The contents of BaP reported by Eskinja et al. (up to 1.2 lg/kg) [29] in white lime tea, chamomile tea, and mallow root tea were in agreement with the data found in herbal and fruit tea samples (Table 4). Within the following denition: interquartile range, abbreviated IQR = Q3 (75%) Q1 (25%). outliers: below Q1 1.5 9 IQR or above Q3 + 1.5 9 IQR, extreme value: below Q1 3 9 IQR or above Q3 + 3 9 IQR, no outliers and no extreme values were detected in any of the tea samples. The sum of all the PAH was correlated with the BaP contents. Considering the samples analysed in this study, the following formula can be calculated: y = 10.6x 3.8 (y = sum of PAH; x = BaP concentration), with a correlation coefcient of R = 0.99 (n = 40). Additionally, correlations between the other 15 EU priority PAH and the PAH sum content were calculated.

With the exception of CPP (R = 0.83) and DhP (R = 0.88) all correlation coefcients were higher than 0.9. Especially BaA and the three benzouoranthenes seem to be good markers in order to calculate the total content of the 15 + 1 EU priority PAH in tea. These correlations are important, because the current EU legislation has the goal to obtain data on the occurrence and the specic concentration pattern of PAH in various matrices in order to assess whether BaP or other PAH may serve as markers for all priority PAH [33].

Discussion To summarize, next to saponication, ASE using a solvent mixture of n-hexane/acetone (50:50, v/v) provides for the best conditions for the extraction of PAH from tea matrices. This step of sample preparation, the ASE, can be automated, is less time-consuming and needs less solvent than the other extraction methodologies described. Using the ASE-method, GPC, SPE and Fast-GC/HRMS, 40 different tea samples were analysed in order to determine the content of all 16 EFSA-PAH. The repeatability (RSD, n = 3) of this method ranged between 0.1 and 11%. Both, the limit of detection (0.010.02 lg/kg) and the limit of quantication (0.030.06 lg/kg) were determined. The content of the 16 EFSA-PAH in the 40 brands of tea which were analysed during this study ranged from 14 to 2,662 lg/kg. The highest content of BaP was

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Eur Food Res Technol (2008) 228:8391 Table 3 PAH contents in various types of tea (lg/kg) (n = 40) BcL Green tea Green tea Green tea Green tea Green tea Green tea Green tea Green tea Green tea Green tea Green tea Mate-tea Mate-tea Mate-tea Mate-tea Mate-tea Mate-tea Mate-tea Mate-tea Black tea Black tea Black tea Black tea Black tea Black tea Black tea Black tea Black tea Black tea Black tea Herbal and fruit tea Herbal and fruit tea Herbal and fruit tea Herbal and fruit tea Herbal and fruit tea Herbal and fruit tea Herbal and fruit tea White tea White tea White tea 4.4 3.2 1.2 3.0 12.0 7.2 17.4 16.2 0.9 17.1 4.3 BaA 9.7 3.0 3.6 3.6 5.9 4.3 40.4 29.0 1.8 22.4 11.7 CHR CPP + TP 14.8 17.3 8.3 11.2 28.9 28.7 61.5 47.7 6.7 42.4 16.7 2.4 0.7 1.0 0.3 0.9 0.4 13.4 6.0 0.2 3.0 5.7 5MC BbF 0.9 0.7 0.2 0.8 2.1 1.4 3.3 2.9 0.4 3.9 1.0 7.7 5.6 4.6 2.5 2.9 8.5 0.1 0.6 2.0 0.3 0.3 0.8 0.6 1.5 0.3 0.7 0.4 0.4 0.7 0.4 0.5 0.6 0.7 0.8 1.4 1.7 2.6 8.0 5.6 2.6 5.3 9.0 6.8 BkF BjF 4.2 2.5 1.6 2.4 3.9 3.1 5.3 3.2 1.7 3.2 5.2 3.9 18.5 13.2 1.3 11.0 8.4 BaP 6.7 2.2 1.8 2.7 3.9 2.6 32.6 17.5 1.6 16.2 12.5 IcP 6.5 3.2 1.2 4.0 4.9 3.5 20.3 12.9 1.4 14.2 11.8 DhA BgP 0.8 0.4 0.1 0.3 0.4 0.4 2.3 1.7 0.1 1.9 1.0 5.4 2.6 1.1 4.4 4.1 3.0 21.6 11.2 14.2 10.2 DlP 0.3 0.2 0.0 0.2 0.2 0.1 0.6 0.4 0.7 0.3 4.1 5.6 2.6 1.6 1.1 0.8 6.5 2.4 0.0 0.1 0.1 0.1 0.2 0.1 0.2 0.2 DeP 1.3 0.7 0.2 0.5 0.8 0.6 1.9 2.0 2.8 0.6 9.6 16.1 6.7 8.9 3.5 2.3 10.9 8.0 0.1 0.5 0.4 0.2 0.1 0.5 0.6 0.8 0.2 0.7 0.1 0.1 0.5 0.2 0.2 0.2 0.3 0.5 1.3 1.7 0.9 DiP 0.7 0.5 0.1 0.2 0.2 0.1 1.5 1.2 1.5 0.6 8.2 12.1 5.1 7.4 2.1 1.4 8.0 8.2 0.1 0.4 0.1 0.1 0.1 0.3 0.3 0.4 0.1 0.5 0.1 0.1 0.3 0.1 0.2 0.1 0.1 0.2 0.5 1.0 0.6 DhP 0.2 0.2 \0.06 \0.06 \0.06 0.1 0.3 0.1 0.2 0.1 0.9 0.8 0.5 0.4 0.5 0.1 0.8 0.6 \0.06 0.1 0.1 \0.02 0.1 0.1 0.1 0.1 \0.06 0.1 \0.02 \0.06 \0.06 \0.06 \0.06 \0.02 \0.06 \0.06 0.2 0.1 0.1

89

Total PAH 71.6 46.2 24.6 42.1 82.6 66.1 286.1 194.2 19.6 180.8 105.2 1,068.8 2,662.1 701.9 640.1 365.8 325.8 1,997.9 1,043.9 19.4 61.8 33.3 19.6 14.0 69.2 57.2 80.8 27.8 58.4 20.0 13.8 35.9 16.1 19.5 21.7 26.6 36.4 107.9 156.1 349.7

33.4 17.1 20.8 11.5 2.2 19.7 12.6 1.2 9.8 7.8

1.7 \0.02 \0.02 \0.02 \0.02

54.6 117.7 210.0 131.0 46.9 16.9 16.2 20.0 83.6 125.7 59.7 116.7 44.9 38.4 81.9 87.3 93.8 10.0 17.0 14.6

97.0 38.4 63.5 26.0 67.7 26.7 38.9 17.1 34.1 13.9 90.6 34.6 2.3 6.8 3.3 2.6 1.7 6.7 5.9 8.1 1.5 6.2 1.9 1.3 4.5 1.8 1.7 1.7 2.4 3.6 14.5 18.7 1.4 3.6 1.5 1.7 1.1 3.9 3.1 4.7 1.0 3.6 1.1 0.6 1.9 0.8 0.9 1.0 1.1 1.7 7.0 9.4

52.7 115.2 100.2 35.9 36.5 23.7 19.5 48.7 1.5 4.4 1.7 1.9 1.2 4.5 3.4 5.4 1.0 4.1 1.2 0.8 2.5 1.1 1.1 1.2 1.5 2.5 8.2 11.4 22.1 75.8 79.3 37.8 24.8 96.7 1.3 4.3 1.9 1.3 0.8 6.9 8.4 8.2 14.1 5.3 1.8 0.8 3.1 1.4 1.4 1.3 1.3 2.4 11.4 15.1 19.0 64.9 92.2 36.8 29.2 80.2 1.4 6.3 2.2 1.7 1.0 4.4 5.7 8.1 1.4 6.1 1.2 0.9 2.7 1.2 1.1 1.3 1.4 2.1 9.7 13.0 15.9

7.9 113.6 3.8 3.1 2.3 6.2 0.1 0.4 0.4 0.1 0.1 0.4 0.5 0.9 0.1 0.5 0.1 0.1 0.3 0.1 0.2 0.2 0.1 0.3 1.4 1.7 1.9 61.7 38.9 34.4 95.3 94.1 0.9 4.2 1.6 1.3 4.0 7.8 6.5 6.1

194.4 374.6 746.3 168.8 19.0 258.1 87.5 123.5 236.5 184.4 13.3 221.1 5.3 106.2

124.8 324.6 597.6 0.7 5.0 2.9 0.5 0.4 5.2 3.1 6.9 1.5 4.8 2.4 1.6 2.7 1.5 1.4 2.9 4.6 4.8 4.3 8.7 24.4 2.5 8.7 6.2 2.0 1.6 13.1 4.6 10.2 1.3 7.0 1.8 1.2 3.5 1.9 2.1 2.6 2.7 3.1 14.1 20.5 80.1 6.5 15.4 8.8 5.7 4.9 17.3 11.9 18.1 3.4 12.1 6.8 4.5 10.4 4.0 7.3 6.7 8.5 11.6 19.0 34.4 95.1

31.7 29.1 191.8 88.4 138.3 186.3 140.8 23.1 0.2 1.1 0.2 0.2 0.2 0.9 1.3 0.7 0.4 0.6 0.1 0.2 0.1 0.2 0.1 0.5 0.2 0.5 3.2 4.2 6.1

87.6 132.9 244.1 100.4

0.6 \0.06

1.5 \0.06 1.1 \0.06 1.0 \0.06 2.7 1.3 1.2 1.2 1.3 2.3 11.3 14.2 13.8 0.2 0.1 0.1 0.2 0.2 0.1 0.3 0.5 0.4

44.6 22.2

detected in mate tea (237 lg/kg). During the processing of mate leaves, which are widely consumed in South America, the leaves are dried by direct smoldering and roasting. This procedure is responsible for the higher presence of PAH in mate tea than in other teas [8]. To

intensify the avour of green tea, a similar technique to that of preparing mate tea can be used. In the nal analysis, the determined PAH contents show that the PAH contamination depends on the drying process of tea leaves and special procedures during the manufacturing

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Eur Food Res Technol (2008) 228:8391

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Fig. 2 Box Plots of the BaP contents in different types of tea (n = 40)

of different types of tea. Thanks to the good correlation coefcient (R = 0.99) between the sum of all the PAH and the BaP content, it is possible to claim that BaP is a suitable marker for estimating the total contamination of the EFSA-PAH in tea samples.

Table 4 Correlation coefcients (R) for all PAH in tea (signicant for P \ 0.05, n = 40) BcL BcL BaA CHR +TP CPP 5MC BbF BkF BjF BaP IcP DhA BgP DlP DeP DiP DhP R PAH 1.00 0.98 0.98 0.84 0.90 0.98 0.95 0.95 0.96 0.93 0.87 0.91 0.92 0.91 0.89 0.83 0.98 BaA 0.98 1.00 1.00 0.76 0.95 0.99 0.99 0.99 0.97 0.94 0.93 0.89 0.95 0.91 0.88 0.83 0.99 CHR + TP 0.98 1.00 1.00 0.76 0.94 0.99 0.98 0.98 0.97 0.94 0.92 0.90 0.94 0.91 0.88 0.82 0.99 CPP 0.84 0.76 0.76 1.00 0.62 0.81 0.75 0.73 0.84 0.84 0.61 0.89 0.80 0.86 0.88 0.84 0.83 5MC 0.90 0.95 0.94 0.62 1.00 0.92 0.96 0.98 0.92 0.89 0.99 0.78 0.95 0.85 0.81 0.81 0.93 BbF 0.98 0.99 0.99 0.81 0.92 1.00 0.99 0.98 0.99 0.98 0.91 0.94 0.95 0.95 0.92 0.87 1.00 BkF 0.95 0.99 0.98 0.75 0.96 0.99 1.00 1.00 0.98 0.96 0.96 0.90 0.97 0.93 0.90 0.87 0.98 BjF 0.95 0.99 0.98 0.73 0.98 0.98 1.00 1.00 0.97 0.96 0.98 0.88 0.97 0.92 0.89 0.87 0.98 BaP 0.96 0.97 0.97 0.84 0.92 0.99 0.98 0.97 1.00 0.99 0.92 0.96 0.97 0.97 0.96 0.91 0.99 IcP 0.93 0.94 0.94 0.84 0.89 0.98 0.96 0.96 0.99 1.00 0.90 0.97 0.96 0.99 0.98 0.92 0.98 DhA 0.87 0.93 0.92 0.61 0.99 0.91 0.96 0.98 0.92 0.90 1.00 0.79 0.96 0.86 0.83 0.85 0.92 BgP 0.91 0.89 0.90 0.89 0.78 0.94 0.90 0.88 0.96 0.97 0.79 1.00 0.89 0.98 0.98 0.88 0.94 DlP 0.92 0.95 0.94 0.80 0.95 0.95 0.97 0.97 0.97 0.96 0.96 0.89 1.00 0.94 0.92 0.93 0.96 DeP 0.91 0.91 0.91 0.86 0.85 0.95 0.93 0.92 0.97 0.99 0.86 0.98 0.94 1.00 0.99 0.93 0.96 DiP 0.89 0.88 0.88 0.88 0.81 0.92 0.90 0.89 0.96 0.98 0.83 0.98 0.92 0.99 1.00 0.93 0.93 DhP 0.83 0.83 0.82 0.84 0.81 0.87 0.87 0.87 0.91 0.92 0.85 0.88 0.93 0.93 0.93 1.00 0.88 R PAH 0.98 0.99 0.99 0.83 0.93 1.00 0.98 0.98 0.99 0.98 0.92 0.94 0.96 0.96 0.93 0.88 1.00

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