Res. Bult., No. (143), Food Sci. & Agric. Res. Center, King Saud Univ., pp.

(5-20) 2006

MICROWAVE SOLVENT EXTRACTION (MSE) AND HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) AS A RAPID DETERMINATION METHODS OF BENZO(A)PYRENE IN FISH
Mohamed H. EL-Saeid

ABSTRACT: Benzo(a)pyrene (BaP) is one of Polycyclic Aromatic Hydrocarbons (PAHs)

which cause health problems such as red blood cell damage (leading to anemia), DNA damage, genotoxicity, lung cancer, developmental and reproductive effects and the best known of the carcinogenic PAHs. Maximum Contaminant Level (MCL) has been set by USEPA at 0.2 ppb in drinking water and 1 ppm in fish. Several types of fish samples such as fresh, canned, salted (salted cured) and smoked fish samples were collected from Houston local market and analyzed by HPLC Diode Array detector, using the mobile phase 100 % acetonitrile and 2.00 ml/min. as a flow rate. The detection and run time of BaP was 3 min. with only 0.01 ppm as a Minimum Detection Limit (MDL). Microwave Solvent Extraction (MSE) was used with 25ml of acetone : petroleum ether (1:1 v:v) for 15 min. The aim of this paper was firstly, to determine the effectiveness of the use of Microwave Solvent Extraction (MSE) and HPLC and GC/MS techniques in the analysis of the levels of BaP residues in fresh, canned, salted (salted cured) and smoked fish samples. Secondly, to study the effect of fish salting on the BaP concentration levels and contributes to the promotion of consumer safety by excluding BaP residues contamination from fish markets.The average of the Benzo(a)pyrene in fresh, canned, salted and smoked fish samples were 0.210 0.014, 0.1370.016, 0.1800.015 and 2.6810.272 mg/kg respectively. On the other hand, BaP was not detected in Salmon canned fish samples. Results shows that BaP content in Mullet fish was decreased by salting for 5 dayes, 68.32% of BaP present in Spiked Salted fish and 31.59 % present in Spiked Salted fish drip. MSE and HPLC modified methods were highly sensitive, accurate, and rapid analytical techniques capable of detecting the minimum concentrations (0.01 ppm) of the BaP residues.

Keywords: Fish, PAHs, BaP, MSE and HPLC.

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Department of Plant Protection, College of Food and Agricultural Sciences, King Saud University, Riyadh-11451, Saudi Arabia Email: elsaeidm@ksu.edu.sa

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INTRODUCTION Humans may be exposed to PAHs from foods, air, water, and tobacco smoke. There are two major sources of occurrence the Benzo(a)pyrene (BaP) in foods. The most important source is the deposition and uptake of BaP and other PAHs from polluted air in food chain. The other significant source is the formation and deposition of PAHs during heat processing such as roasting, smoking and grilling. The presence of BaP cause many healthy problems such as red blood cell damage, leading to anemia, DNA damage, genotoxicity, lung cancer, developmental and reproductive effects and the best known of the carcinogenic PAHs [USEPA (1998), Pokorski, et al (2000), Anastasio, et al (2004), USEPA (2002), Butler, et al (1993), Telli-Karakoc, et al (2002), Salama, et al (2001) and Yang, et al (2000)]. PAHs was determined in some seafood from Egypt, such as tilapia fish (Oreochromis aureus), crabs (Portuns pelagicus), bivalves (Venerupis decussata), clams (Strombus tricornis) and gastropods (Munes spp.). Where these sea foods are locally favorite consumed foods in the area around the lake (Ismailia governorate). Results showed that crabs contained significantly higher concentrations of both total and carcinogenic PAHs ranging from 1318.6 to 3767.4 and 1230.3 to 3442.2 g kg-1, respectively. Meanwhile, clams contained significantly lower levels with a mean value of 28.4g kg-1 for total and 24.4 g kg-1 for carcinogenic PAHs. The most frequently detected PAHs in the tested samples were indeno(1,2,3-cd)pyrene followed by BaP, dibenzo(a,h)anthracene, and benzo(b) fluoranthene which are characterized as carcinogenic compounds [Mostafa (2002)]. Six PAHs were measured in 54 fish samples using GC/MS and were greater than the acceptable tolerance limit (1 g/kg). They were found in 68.5, 40.7, 51.9 and 83.3% of the fish samples, respectively [Al-Saleh, and Al-Doush, (2002)]. Also, twenty seafood samples, which are common edible species and important items in Korea, were analyzed for PAHs. The levels of 16 PAHs in seafood were 161-2243 pg/g wet wt. The levels of potentially carcinogenic PAHs of 6 species were 9-123 pg/g wt wt [Moon, et al (2002)]. -6-

The mean of BaP content of some smoked fish was 9.0 ug/kg for Tilapia, 26.2 g/kg for Claris, and 33.6 g/kg for Alestes. Analyses of the fresh unprocessed forms of these foods had lower levels in fish as compared with the smoked fish [Ogbadu, and Ogbadu, (1989) ]. Also, BaP content in 19 fish species from 3 Baltic bays ranged from 0 to 1.14 g/kg wet wet [Veldre, et al (1985)]. The fish from sea and freshwater reservoirs from Estonian region was not high and ranged from 0.09-1.14 g/kg and 0.18-5.96 g/kg, respectively [Veldre, et al (1980) ]. The BaP in 30 samples of cod, pink salmon, mackerel, and horse mackerel smoked by conventional methods was 4.15-60 g/kg. Only 0.7-1.7 g/ kg of electro statically smoked sprats. [ Petrun, and Rubenchik, (1966)]. The effect of salting on BaP residues in Atlantic herring was reported and detectable after salting [Dossu-Yovo, et al (2001)]. Microwave Solvent Extraction (MSE) achieves rapid extractions, potential advantages than conventional extraction, superior recovery, reduced solvent expenditure by using solvents at high temperature and pressure than other solvent extraction techniques, no cleanup, Safe and high ability as food extraction technique [CEM application note # E003, Hasty, and Revesz (1995), EL-Saeid, (1999), Noble, (1993), Colis, and Neas, (1988) and USEPA (1986)] The purpose of this study is to determine the effectiveness of the use of MSE and HPLC modified techniques in the analysis of the levels of BaP residues in fresh, canned, salted (salted cured) and smoked fish collected from Houston local markets to contribute to the promotion of consumer safety by excluding BaP contamination from markets, also to study the effect of salting on BaP residues in Mullet fish. MATERIALS AND METHODS 1. Sample collection and preparation Fish samples used in this study were collected from Houston local markets. Five of each fresh fish samples (Mullet, Catfish, Tilapia and Shrimp); canned fish samples (Sardine, Tuna and Salmon); and -7-

smoked fish samples (Mackerel and Herring). Salted cured fish samples (Mullet) was prepared (adding 250g salt/kg in skin and gills for 5days.) 2. Analytical Standards of BaP. Benzo(a)Pyrene (Figure 1) standard with purity of 99.9 % was obtained from Chem Service Inc. (West Chester, PA).

Fig (1): Structure of Benzo(a)Pyrene present in the environment and detected in fish. 3. Recovery assays Untreated fish samples were spiked with BaP at levels from 0.001-1.0 mg/kg. The spiked samples were homogenized and allowed to equilibrate for 1h analysis. Three replicates were analyzed to calculate the recovery and relative standard deviation (RSD) % by MSE & HPLC methods. 4. Benzo(a)pyrene Extraction CEM application note No. E003 was modified and used during conducting of the present study (EL-Saeid, 1999). A Microwave Solvent Extraction (MSE) system model MES-1000 (CEM Corporation, Matthews, NC, USA) with Lined Extraction Vessels (LEV) was used. This system consists of a 950 watt microwave instrument which has been specifically designed for use with organic solvents. Extraction vessels are double-walled vessels specifically

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adapted for use with organic solvents. Extraction conditions was conducted with 25g fish sample, extraction solvent was 25 ml of Acetone : Petroleum Ether (1:1), pressure was 125 psi, microwave power was 90%, temperature was120C and time of extraction 15 min. 5. Benzo(a)pyrene Analysis 5.1. Analysis by HPLC. HPLC determination method were conducted by a Perkin Elmer 410 LC pump/ ISS 200 LC sample processor using a 235C Diode Array detector, LC pump 410, Column 250x2.6-mm LUNA C18-2,5m. The determination conditions were 100 % acetonitrile, 2.00 ml/min. as a flow rate, pressure 1250-1600 psi, wavelength 255 and 365 nm. and run time 5 min. 5.2. Analysis by GC/MS. The quantification of BaP in fish samples extract was performed on a HP 5890 series II plus GC coupled to an HP 5972 Mass Selective Detector. The GC columns were a DB-5 fused silica capillary column (30m x 0.32mm i.d., 1m film thickness; J&W Scientific, Folsom, CA) One micro liter of the fish sample extract was injected split less injector temperature of 250 C, on the GC/MS for analysis. The temperature program for the GC was as follows: isothermal for 1 min at 70 C, increased at a rate of 10 C/min to 220 and isothermal for 10 min. 6. Minimum Detection Limit (MDL) To determine the MDL of BaP we used the dilution levels ranged from 0.001-1.0 ppm. RESULTS The Benzo(a)Pyrene (BaP) residues are found frequently in the environment, water and fish . Microwave Solvent Extraction (MSE) with HPLC technique was modified and developed to obtain the optimum conditions to extract and determined the BaP in a short time, with high recovery %. MDL, extraction-determination times, averages -9-

of residues, effect of salting cured and recovery % of BaP results will discuss. Minimum Detection Limits (MDL) and Extraction/Determination time of BaP. The MDL of BaP was 0.01 ppm by using the HPLC/ UV at 265 nm. The extraction by MSE was done in 15 min. for 11 fish samples. On the other hand the determination time by using the HPLC was 3.0 min. BaP residues in fish samples extracted by MSE and determined by HPLC. Table (1) and Figure (2) present the concentration of BaP residues (ppm) in 4 major local fish samples extracted by MSE and determined by HPLC. The results revealed that the BaP detected in all tested fish samples. The highest levels of BaP residues was observed in smoked fish, but on other hand the lowest level was observed in canned fish followed fresh (except mullet and cat fish) and salted cured fish samples. This variation in BaP residues content between different fish samples may be due to on or more than one reason such as smoking, water contamination, atmosphere contamination and cross contamination. The residue of tested BaP in studied 4 groups of fish samples was found to be higher than the official acceptable tolerance limit of PAHs in fish [1]. BaP residue results data tabulated in table (1) shown the Mullet, Cat fish, Tillapia and Shrimp fresh fish samples were 0.2280.017, 0.3340.011, 0.1220.019 and 0.1210.010 ppm respectively. The current data shows a low concentration of BaP in canned Sardine 0.1410.015 and Tuna 0.1330.018 ppm; Meanwhile the data in the same table showed higher concentration of BaP residue in smoked Mackerel 2.1790.230 and Herring 3.1830.314 ppm. The BaP concentration levels in smoked fish was higher because of the smoking processing produce the BaP than the normal. The BaP in fresh slated cured Mullet was 0.1810.015ppm. -10-

Table (1): Average Benzo(a)Pyrene residues (ppm) and relative standard deviation (RSD) in fish samples extracted by MSE,and determined by HPLC. Fish Samples Fresh Mullet Cat fish Tillapia Shrimp Canned Sardine Tuna Salmon Smoked Mackerel Herring Salted cured Mullet
ND= Not Detected.

BaP Residues (ppm) RSD

0.2280.017 0.3340.011 0.1220.019 0.1210.010

0.1410.015 0.1330.018 ND

2.1790.230 3.1830.314

0.1810.015

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BaP RESIDUES IN FISH

3 2.5

BaP(mg/kg)

2 1.5 1 0.5 0 BaP(mg/kg)

FRESH CANNED SMOKED SALTED

FISH SAMPLES

Fig (2): B(a)P residues present in fish samples.

Effect of salting on BaP content in Mullet fish. The data from the current study also showed the effect of salting on BaP content in Mullet fish it was decreased by in mullet fish by salting for 5 dayes. Table (2) shows 68.32% of BaP present in Spiked Salted fish and 31.59 % present in Spiked Salted fish drip. Table (2): Effect of 5 days salting on BaP concentration in Mullet fish Spiked by 1 ppm.
BaP Concentration (ppm) Fresh Spiked Salted Drip Total

0.2280.017 1.2280.017 0.8390.022

0.3880.019 1.2270.041

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BaP Recovery percentages of spiked fish samples extracted by MSE and determined by HPLC. The results of BaP recovery percentages of spiked fish samples extracted by MSE and determined by HPLC/UV were tabulated in (Table-3 and Fig-3). These data shows high recovery percentages of BaP by using MSE and HPLC modified method in fresh, canned, smoked and Salted cured fresh samples. The recovery percentage RSD of Mullet, Cat fish, Tilapia and Shrimp fresh fish 98.72.6, 92.53.3, 97.52.9, 98.42.6 respectively. The recovery percentage RSD of canned fish were 98.62.7, 96.73.6,and 97.43.7 for Sardine, Tuna and Salmon respectively. The recovery percentage RSD of smoked Mackerel and Herring fish were 96.82.4 and 97.31.6 respectively. The recovery percentage RSD of salted cured Mullet fish 99.21.7 . Table (3): BaP Recovery and relative standard deviation (RSD) percentages of spiked fish samples extracted by MSE and determined by HPLC. Fish Samples Fresh Mullet Cat fish Tilapia Shrimp Canned Sardine Tuna Salmon Smoked Mackerel Herring Salted cured Mullet -1396.82.4 97.31.6 99.21.7 98.62.7 96.73.6 97.43.7 Recovery % RSD 98.72.6 92.53.3 97.52.9 98.42.6

Fig (3): HPLC of B(a)P (A) standards (B&C) residues present in smoked fish samples.

BaP Confirmation by GC/MS and UV spectrometer methods. Fig (4 and 5) shows the confirmation data of the BaP extracted by MSE from fish samples and confirmed by GC/MS and UV spectrometer. -14-

Figure (4): BaP by GC/MS at DB-5, 30m column and Ion 252.00 and 250.00

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Figure (5) BaP UV Sectrum at 250-500nm.

DISCUSSIONS The Benzo(a)Pyrene (BaP) residues are found frequently in the environment, water and fish . Microwave Solvent Extraction with HPLC technique was modified and developed to obtain the optimum conditions to extract and determine the BaP in a short time, with high recovery %. The results of the MDL, extraction and determination times, averages of residues, effect of salting cured and recovery % of BaP showed effective positive results. The MDL of BaP was 0.01 ppm by using the HPLC/ UV at 265 nm. The extraction by MSE was done in 15 min. for 11 fish samples. On the other hand the determination time by using the HPLC was 3.0 min. The concentration of BaP residues (ppm) in 4 major locally fish samples extracted by MSE and determined by HPLC revealed that the BaP detected in all tested fish samples, the highest levels of BaP residues being observed much more in smoked fish while the lowest level was observed in canned fish followed fresh (except mullet and cat fish) and salted cured fish samples. This variation in BaP residues content between different fish samples may be due to various factors such as -16-

smoking, water contamination, atmospheric and cross contaminations. On health risk grounds, the residues of BaP in the 4 groups of fish samples were abnormally higher than the official acceptable tolerance limit of PAHs in fish [1]. Hence, it is found that the mullet, catfish, tilapia and shrimps samples were 0.2280.017, 0.3340.011, 0.1220.019 and 0.1210.010 ppm respectively. In the same note, the current data showed also a low concentration of BaP in canned sardine (0.1410.015) and tuna (0.1330.018 ppm). On the contrary, higher concentration of BaP residues were found in smoked mackerel (2.1790.230) and herring (3.1830.314 ppm). In the course of the investigation, it was observed that salting of the fish has a reduction effect on BaP content in mullet fish within 5 days (Table 2).

CONCLUSION Trace level of BaP in the investigated blank and spiked fish samples can be extracted only 15min. by using modified Microwave Solvent Extraction (MSE) method. Determination method by HPLC is considerably fast with low detection limit (0.01ppm). The MSE run time for 11 fish samples was completely achieved in 15 min. The effect of salting on the BaP concentration in salted Mullet fish was decreased 31.59%. REFERENCES Al-Saleh, Iman, and Al-Doush, Inaam (2002). Gas chromatographymass spectrometric determination of polycyclic aromatic hydrocarbons in five species of fish from three sites in the Arabian Gulf. International Journal of Environmental Health Research, 12(2),193-200. Anastasio, A; Mercogliano, R; Vollano, L; Pepe, T. and Cortesi, M.L (2004) Levels of Benzo(a)pyrene in Mozzarella di Bufala

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Veldre, I.; Itra, A.; Paalme, L.; Kangur, M. (1980) Accumulation, distribution, and excretion of benzo[a]pyrene in fish. Voprosy Onkologii 26(10), 80-82. Yang, S.C.; Jeng, S.N.; Kang, Z.C. and Lee, H. (2000) Identification of benzo[a]pyrene 7,8-Diol 9,10-Epoxide N2Deoxyguanosine in human lung adenocarcinoma cells exposed to cooking oil fumes from frying fish under domestic condition. Chem. Res. Toxicol. 13, 1046-1050. EL-Saeid, M.H, 107s annual meeting of Texas Academy of Science, March 4-6, 2004, Schreiner University, Kerrville ,Texas

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elsaeidm@ksu.edu.sa