Determinacion en Fish

7/28/2019 Determinacion en Fish

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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), DNAdamage, genotoxicity, lung cancer, developmental and reproductive effects and the bestknown 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 asfresh, 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 infresh, canned, salted (salted cured) and smoked fish samples. Secondly, to study the effect offish 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 0.2100.014,

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 fishwas 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 highlysensitive, 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.

_____________________________________________________________Department of Plant Protection, College of Food and Agricultural Sciences, King

Saud University, Riyadh-11451, Saudi Arabia Email: [email protected]


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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)].


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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


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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 MicrowaveSolvent 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 235CDiode 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


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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 fishsamples 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 ppmrespectively.

The current data shows a low concentration of BaP in canned

Sardine 0.1410.015 and Tuna 0.1330.018 ppm; Meanwhile the datain 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.


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Table (1): Average Benzo(a)Pyrene residues (ppm) and relative

standard deviation (RSD) in fish samples extracted by

MSE,and determined by HPLC.

Fish SamplesBaP Residues (ppm)

RSD

Fresh

Mullet 0.2280.017

Cat fish 0.3340.011

Tillapia 0.1220.019

Shrimp 0.1210.010

Canned

Sardine 0.1410.015

Tuna 0.1330.018

Salmon ND

Smoked

Mackerel 2.1790.230

Herring 3.1830.314

Salted cured

Mullet 0.1810.015

ND= Not Detected.


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BaP RESIDUES IN FISH

0

0.5

1

1.5

2

2.5

3

BaP(mg/kg)

FISH SAMPLES

BaP(mg/kg) FRESH

CANNED

SMOKEDSALTED

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 anddetermined by HPLC.

Fish Samples Recovery % RSDFresh

Mullet 98.72.6

Cat fish 92.53.3

Tilapia 97.52.9

Shrimp 98.42.6

Canned

Sardine 98.62.7

Tuna 96.73.6

Salmon 97.43.7Smoked

Mackerel 96.82.4

Herring 97.31.6

Salted cured

Mullet 99.21.7


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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.


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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 theenvironment, 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 BaPdetected 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


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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 BaPresidues 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 HPLCis 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%.

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March 4-6, 2004, Schreiner University, Kerrville ,Texas


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Determinacion en Fish