RAPID MSE AND HPLC METHOD OF BENZO(a)PYRENE IN FISH by pu92g68F

VIEWS: 8 PAGES: 17

									    1411 11 1                                      143




                                           1




     1                      111
1

1 1                                   12
111                                                         12
                                           18681   181     131


31823                                 68 31


                                  1




       1461
    elsaeidm@ksu.edu.sa                                  11421


                          -1-
Res. Bult., No. (143), Food Sci. & Agric. Res. Center, King Saud Univ., pp. (1-17) 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,
0.1370.016, 0.1800.015 and 2.6810.272 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.

_____________________________________________________________


                                            -2-
Department of Plant Protection, College of Food and Agricultural Sciences, King
Saud University, Riyadh-11451, Saudi Arabia     Email: elsaeidm@ksu.edu.sa

                            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 28.4g 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


                                     -3-
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)].
        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

                                -4-
       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
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


                                 -5-
(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
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 was120ºC 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
C18-2,5m. 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., 1µm film thickness; J&W
Scientific, Folsom, CA) One micro liter of the fish sample extract was
injected split less injector temperature of 250 ºC, on the GC/MS for
analysis. The temperature program for the GC was as follows:
isothermal for 1 min at 70 ºC, increased at a rate of 10 ºC/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)

                                -6-
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
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 0.2280.017, 0.3340.011, 0.1220.019 and 0.1210.010 ppm
respectively.
       The current data shows a low concentration of BaP in canned
Sardine 0.1410.015 and Tuna 0.1330.018 ppm; Meanwhile the data
in the same table showed higher concentration of BaP residue in
smoked Mackerel 2.1790.230 and Herring 3.1830.314 ppm. The


                                 -7-
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 0.1810.015ppm.
Table (1): Average Benzo(a)Pyrene residues (ppm) and relative
              standard deviation (RSD) in fish samples extracted by
              MSE,and determined by HPLC.

                                        BaP Residues (ppm)
            Fish Samples
                                              RSD

                Fresh

 Mullet                                     0.2280.017

 Cat fish                                   0.3340.011

 Tillapia                                   0.1220.019

 Shrimp                                     0.1210.010

               Canned

 Sardine                                    0.1410.015

 Tuna                                       0.1330.018

 Salmon                                          ND

              Smoked

 Mackerel                                   2.1790.230

 Herring                                    3.1830.314

            Salted cured

 Mullet                                     0.1810.015


                               -8-
ND= Not Detected.




                         BaP RESIDUES IN FISH

              3

             2.5

              2                                                FRESH
BaP(mg/kg)




                                                               CANNED
             1.5
                                                               SMOKED
              1                                                SALTED

             0.5

              0
                               BaP(mg/kg)
                            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)



                                        -9-
    Fresh         Spiked       Salted         Drip         Total

 0.2280.017 1.2280.017 0.8390.022      0.3880.019 1.2270.041
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 98.72.6,
92.53.3, 97.52.9, 98.42.6 respectively. The recovery percentage
 RSD of canned fish were 98.62.7, 96.73.6,and 97.43.7 for
Sardine, Tuna and Salmon respectively. The recovery percentage 
RSD of smoked Mackerel and Herring fish were 96.82.4 and
97.31.6 respectively. The recovery percentage  RSD of salted cured
Mullet fish 99.21.7 .

Table (3): BaP Recovery and relative standard deviation (RSD)
           percentages of spiked fish samples extracted by MSE and
           determined by HPLC.
            Fish Samples                Recovery %  RSD
                Fresh
    Mullet                                   98.72.6
    Cat fish                                 92.53.3
    Tilapia                                  97.52.9
    Shrimp                                   98.42.6
               Canned
    Sardine                                  98.62.7
    Tuna                                     96.73.6
    Salmon                                   97.43.7
               Smoked


                                -10-
   Mackerel                             96.82.4
   Herring                              97.31.6
          Salted cured
   Mullet                               99.21.7




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



                             -11-
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.




                                -12-
Figure (4): BaP by GC/MS at DB-5, 30m column and Ion 252.00 and
            250.00




          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

                                 -13-
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
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 0.2280.017, 0.3340.011,
0.1220.019 and 0.1210.010 ppm respectively.
        In the same note, the current data showed also a low
concentration of BaP in canned sardine (0.1410.015) and tuna
(0.1330.018 ppm). On the contrary, higher concentration of BaP
residues were found in smoked mackerel (2.1790.230) and herring
(3.1830.314 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 chromatography-
     mass spectrometric determination of polycyclic aromatic
     hydrocarbons in five species of fish from three sites in the
     Arabian Gulf. International Journal of Environmental Health


                                 -14-
     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
     Campana Cheese Smoked According to Different Products. J.
     Agric Food Chem. 52, 4452-4455.

Butler, J.P.; Post, G.B.; Lioy, P.J.; Waldman, J.M.; and
     Greenberg, A. (1993) Assesment of carcinogenic risk form
     personal exposure to BaP in the total human environmental
     exposure study (THESIS). J. Air Waste Manag. Assoc. 43, 970-
     977.

Colis, M.J. and Neas, E.D. (1988) Introduction to microwave sample
      preparation: theory and practice, chap 2. In Kingston H.M;
      Jassie, L.B. eds. Washington, DC: American Chemical Society.

CEM application note # E003. PAHs from Sludge HPLC method.
   CEM Corporation, Friendswood, TX 77549.

Dossu-Yovo, P.; Gel’dysh, T.G.and Palagina, I.A.(2001) Changes
     in toxic elements, benzo(a)pyrene, pesticides and radionuclides
     in Atlantic herring during salting. Piishchevaya Teknologiya, (5-
     6), 87.

EL-Saeid, M.H. (1999) New Techniques for residue analysis of
    pesticides in foods (Ph.D. Dissertation) Al-Azhar Univ., Cairo,
    Egypt.

Hasty, E. and Revesz R. (1995) Total petroleum hydrocarbon
     determination by Microwave Solvent Extraction. American
     Laboratory (2), 66-74.

Moon, Hyo-Bang; Choi, Hee-Gu; Kim, Sang-Soo; Lee, Pil-Yong.
    (2002). Accumulation and characterization of PAHs in sea
    foodfrom the costalareasof Korea. Journal of Fisheries Science
    and Technology ), 5(2),127-135.


                                -15-
Mostafa, Gamal A. (2002). Monitoring of polycyclic aromatic
    hydrocarbons in seafoods from Lake Timsah, Egypt.
    International Journal of Environmental Health Research, 12(1),
    83-91.

Noble, D. (1993) Here today, gone tomorrow, halogenated solvent in
     analytical chemistry. Anal. Chem. 65 (15) 694A.

Ogbadu, G. H.; Ogbadu, L. J. (1989), Levels of benzo(a)pyrene in
    some smoked ready-to-eat Nigerian foods. Lebensmittel-
    Wissenschaft und Technologie. 22(5),313-14.

Petrun, A. S.; Rubenchik, B. L. (1966) Possibility of carcinogenic
     3,4-benzopyrene in electrostatically smoked fish. Vrachebnoe
     Delo, (2), 93-5

Pokorski, L. P. (2000) Introdution of Toxicology, Chemical
    Carcinogenesis II. University of Michigan-Dearborn.
    Department of Natural Science. February 22, 2000.

Salama, S.A.; Sierra-Torres, C.H.; Oh, H.Y.; Hamada, F.A. and
     Au, W.W. (2001) Variant metabolizing gene alleles determine
     the genotoxicity of benzo[a]pyrene. Environmental and
     Molecular Mutagenesis 37 (1), 17-26.

Telli-Karakoc, F.; Ruddock, P.J.; Brid, D.J.; Hewer, A.; Van
      Schanke, A.; Phillpis, D. H. and Peters, L.D. (2002)
      Correlative changes in metabolism and DNA damage in turbot
      exposed to benzo[a]pyrene. Marine Environmental Research 45,
      (3-5), 511-515.

USEPA (2002) Integrated Risk Information System. Washington, DC,
    http:/www.epa.gov/iriswebp/iris/index.

USEPA (1998) United States EPA’s contaminated sediment
    management strategy. EPA #823/R-98-001.



                               -16-
USEPA (1986) Test methods for evaluating solid waste. SW-846, 3rd
    ed. Method 3540, 3450-3.

Veldre, I.; Itra, A.; Trapido, M.; Paalme, L. (1985) Accumulation
     of benzo(a)pyrene in fish. Eesti NSV Teaduste Akadeemia
     Toimetised, Bioloogia 34(4), 262-6.

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 N2-
    Deoxyguanosine 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




                               -17-

								
To top