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Bulletin UASVM Animal Science and Biotechnologies, 65(1-2)/2008

pISSN 1843-5262; eISSN 1843-536x


Weber C.I., Gh. Murean, B. Georgescu
University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and
Biotechnologies, 3-5 Manastur Street, 400372 Cluj-Napoca, Romania
email: webercristiani@yahoo.com
Key words: organochlorine pesticides, cow milk, gas chromatography (GC), electron capture
detector (ECD), mass spectrometry (MS).
Abstract. The pesticides have been widely used throughout the world since the middle of the 20th
century. Organochlorine pesticides do not readily degrade in the environment and are lipophilic with a tendency
to bioaccumulate, so they can be found at high concentrations in fatty foods, including the cow milk. The
accumulation of organochlorine compounds in fatty foods is still a matter of major concern although the use of
most organochlorine compounds (OCs) has been banned or restricted in the most of the countries, due to the
uncertainty about the adverse effects that those residues may have after a lengthy exposure at low doses. The
sequence steps in pesticide residues analysis are sampling, sample preparation, extraction, clean-up and
identification and quantitative determination of pesticide residues. Since the early 1970s, most routine pesticideresidue determination has been conducted by gas chromatography (GC) coupled with electron capture detector
(ECD) or/and mass spectrometry (MS).


Organochlorine pesticides were used to help increase the yield and improve the quality
of the crops (Nawaz, 2003). They were mainly used in agriculture, animal production (Beyer
et al., 2007) and for public-health purposes (Nawaz, 2003). These substances have been
widely used throughout the world since the middle of the 20th century (Beyer et al., 2007).
The dairy cows are mainly exposed to these contaminating residues through their food
(Mallatou, 1997 et al., Martinez et al., 1997, Nag et al., 2008). After the residues have been
metabolized, they are stored in the fat reserves from which they enter in circulation and are
eliminated through milk (Cerkvenik et al., 2000, Nag et al., 2008). The OCPs (organochlorine
pesticides) accumulate in different environmental compartments and in the food chain thus
causing elevated contamination in the human body (Hura et al., 1999, Patel et al., 2005, Nag
et al., 2008). The main pathway of the human exposure to organochlorine compounds is food,
especially the dairy products (Hura et al., 1999, Mamontova et al., 2004). The accumulation
of organochlorine compounds in fatty foods is still a matter of major concern although the use
of most organochlorine compounds had been banned or restricted in industrialized countries
since the 1970s, due to the uncertainty about the adverse effects that those residues may have
after a lengthy exposure at low doses (Kodba et al., 2007, Soler et al., 2007). Organochlorine
pesticides (aldrin, chlordane, DDT (dichlorodiphenyltrichloroethane), dieldrin, endrin, HCB
(hexachlorobenzene), heptachlor, mirex, toxaphene) are currently under regulation of the
Stockholm Convention (2001) on Persistent Organic Pollutants (Muir et al., 2006).
At international level, the Codex Alimentarius Commission of the United Nation's Food
and Agriculture Organization and the World Health Organization (FAO/WHO) has
established maximum residue levels (MRLs) for pesticides in a variety of foods that have

official status across the world. (Nawaz, 2003). Because milk is placed at the highest level of
the trophic chain, it is used as an indicator of the bioaccumulation of the environmentally
persistent micropollutants such as organochlorine pesticides (Ahmed, 2001).
In Europe, for determination of OCPs and PCBs (polychlorinated biphenyls) in fatty
foods, an analytical standard EN 1528 is used as the reference tool (Kodba et al., 2007).

The sequence steps in pesticide residues analysis are: sampling, sample preparation,
extraction, clean-up and identification and quantitative determination of organochlorine
pesticide residues (Mukherjee et al., 1996, Ahmed, 2001).
1) Sampling. Acquisition of a suitably representative samples is fundamental to any
measurement program and should not be overlooked (Muir et al., 2006). Samples must be
analyzed without any delay, as some pesticide residues may degrade rapidly (Nawaz, 2003).
For cows milk sampling are used different container types, such as glass bottles (Mamontova
et al., 2004, Nag et al., 2008) or high-density polyethylene containers (Ghidini et al., 2002).
After sample collection, this is immediately cooled to about +4 0C, the determination being
made in the same day (Mallatou et al., 1996, Ghidini et al., 2002). For long time storage, the
samples are frozen at minimum -18 0C (Pinelli et al., 2005). Some researchers have used even
lower temperatures: -20 0C (Losada et al., 1996, Nawaz, 2003), -25 0C (Mallatou et al., 1997),
-30 0C (Mamontova et al., 2004) and -70 0C (Muir et al., 2006).
2) Sample preparation. Due to the low residue levels and the complex nature of the
matrices in which the target compounds are present, it is needed an efficient sample
preparation (Beyer et al., 2007). Homogenization is a very important process in sample
preparation, so that blending is used for liquid samples (cow milk). The solid samples (whole
fruit or vegetable) are weighed, chopped or homogenized in a mortar, blender, shaker or
stirrer, or sonicator with a solvent (or a sorbent) to disintegrate the matrix. (Ahmed, 2001).
3) Extraction. The character of the matrix, especially the fat content, dictates the method
to be employed for extraction, cleanup and analysis of sample (Lambropoulou et al., 2007,
Ridgway et al., 2007). Products with fat above 2% are considered fatty (Ahmed, 2001). The
methodology varies from simply keeping the sample in a solvent overnight to a more
exhaustive technique such as Soxhlet extraction (Mukherjee et al., 1996). Solvent extraction
aided by shaking is based on the partitioning of analytes between two immiscible liquids in
the case of liquid-liquid extraction or between liquid and solid phases, in the case of solidphase extraction (Beyer et al., 2007).
Liquid-liquid extraction (LLE) applies to liquid matrices (Ahmed, 2001) and is
commonly used in the analysis of fatty samples (Nawaz, 2003). Of the liquid solvents
employed, n-hexane, benzene and ethyl acetate have been examples of non-polar solvents,
whereas water-miscible solvents such as dichloromethane, methanol, MeCN (acetonitrile)
and/or acetone and water have been employed at the polar end for extraction of residues from
high-moisture commodities (Ahmed, 2001, Nawaz, 2003).
Solid-phase extraction (SPE) was developed as an alternative to LLE for the separation,
purification, concentration and/or solvent exchange of solutes for solutions. SPE can be used
directly as an extraction technique for liquid matrices, or as a clean-up method for solvent
extracts (Ahmed, 2001).
In addition to these classical extraction techniques, other more recent approaches, for
example QuEChERS (quick, easy, cheap, effective, rugged and safe), supercritical fluid
extraction (SFE), pressurized-liquid extraction (PLE), microwave-assisted extraction (MAE),

matrix solid-phase dispersion (MSPD), solid-phase microextraction (SPME), and stir-barsorptive extraction (SBSE), have resulted in new possibilities in sample treatment and
advantages such as a substantial reduction of the extraction time (Lambropoulou et al., 2007).
The efficiency of extraction process depends on the physicochemical properties of the
solvents and analytes, including pH, polarity, temperature, sample/solvent ratio, presence of
water, and degree of analyte/matrix binding (Ridgway et al., 2007).
For multiresidue extraction methods (MRMs), it is not possible to establish the optimum
extraction conditions for all residues with differing physical and chemical properties so the
choice of solvent polarity is usually a compromise (Nawaz, 2003). An inherent problem with
a multiresidue approach is that matrix coextractives increase as the method includes a wider
polarity range of analytes, and no current method is suited for extracting all pesticides from
all matrices (Ahmed, 2001).
In general, the analysis of organochlorine pesticide residues in milk follows the
conventional approach of the multimatrix MRMs in which the total residues are extracted
together with the total fatty material (Ahmed, 2001). The most used solvents for extraction in
organochlorine pesticides analysis in cow milk are: hexane (Losada et al. 1996), petroleum
ether (Cerkvenik et al., 2000), petroleum ether-acetonitrile (Ghidini et al., 2002) and nhexane-acetone (Mamontova et al., 2004). Other solvents used for milk extraction are
chloroform-methanol, diethyl ether-petroleum ether, acetone-hexane, hexane-acetonitrileethanol and with ethyl acetate-methanol-acetone (Mukherjee et al, 1996, Ahmed, 2001).
Armendariz et al. (2004) has used centrifugation for fat extraction from cow milk, mixing
with sodium sulfate and transfer to a glass column and elution with hexane.
4) Clean-up. Whichever technique is used for extraction, sample extracts not only
contain the target analytes but may also contain coextractives, such as pigments, lipids
(Ahmed, 2001), proteins (Nawaz, 2003), carbohydrates (Beyer et al., 2007). These
coextractives must have to be removed prior to instrumental analysis to avoid possible
contamination of instruments and to eliminate compounds that interfere during the
determination step (Nawaz, 2003).
Clean-up techniques currently include liquid-liquid partitioning (LLP) (Nawaz et al.,
2003), open column chromatography, adsorption chromatography (AC), gel permeation
chromatography (GPC) (Patel et al., 2005), thin-layer chromatography, steam distillation and
low temperature precipitation (Mukherjee et al, 1996, Ahmed, 2001). The most commonly
used method of clean-up is adsorption chromatography (Nawaz, 2003), applying the SPE
technique (Beyer et al., 2007). A number of materials are available for adsorption
chromatography, including alumina, florisil, cellulose, diatomaceous earth (celite), carbon
(charcoal, graphite), silver nitrate, and silica. Alumina and silica are effective for the clean-up
of fatty samples for organochlorine pesticide residue analysis. Silver nitrate is used for the
removal of sulfur-containing interferences. Carbon has a high affinity for plant pigments and
is particularly suitable for the clean-up of green leafy vegetable extracts with a high
chlorophyll content. These sorbents are used in cartridges, disks, membrane filters, and
impregnated fiber tips (Nawaz, 2003).
Gel permeation chromatography (GPC) is relatively effective at removing fats and oils
and is applicable to a wide range of OCPs, but a further SPE clean-up step is often required to
remove any remaining lipid and other matrix components (Patel et al., 2005). It can be used
for separating large molecules (lipids, pigments and polymeric coextractives), because its
principle of operation is based on size exclusion (Nawaz, 2003, Beyer et al., 2007). Florisil is
the most used sorbent, and is particularly suited for fatty foods (Ahmed, 2001).

Column chromatography using alumina, silica gel and florisil, or a combination of these
adsorbents has been successfully applied to the analysis of contaminants in several food
matrices. This method can be employed for purifying extracts of many OCPs and PCBs,
resulting in products that are ready for further analysis by means of gas or liquid
chromatography with a proper detector.
Saponification and sulphuric acid treatment are other techniques used to remove
interfering compounds that were reported by many authors (Beyer et al., 2007).
Armendariz et al. (2004) has used concentrated sulphuric acid treatment for extract
purification (clean-up). Adsorption chromatography was used by Ghidini et al. (2002) for
clean-up in his studies on pesticide residues determination from cow milk. Mamontova et al.
(2004) has used for extract clean-up liquid chromatography on two columns by gel
chromatography column, the first column filled with bio-bead S-X3 and the second with silica
gel/aluminium oxide. In her studies, Cerkvenik et al., (2000) has used partial deactivated
aluminium oxide (Al2O3).
5) Identification and quantitative determination. The monitoring of the pesticide
residues in foods is often at the microgram per kilogram level or lower and has to be
supported by strict analytical quality-control standards, so that the analysis produces precise
and accurate residue data (Kodba et al., 2007). Before samples are analyzed, the analyst has to
demonstrate that the intended analytical method can achieve adequate specificity, sensitivity,
linearity, accuracy and precision at the relevant analyte concentration and in appropriate
matrices (Nawaz, 2003).
As the majority of pesticides are relatively volatile, gas chromatography (GC) has
proved to be an excellent technique for pesticide determination and is by far the most widely
used. Typically, GC is useful for analyzing nonpolar and semi-polar, volatile and semivolatile chemicals (Lehotay et al., 2002, Lambropoulou et al., 2007). The identification and
separation capabilities of GC can be increased when combined with the confirmation
capabilities of the mass-spectrometry (MS) (Patel et al., 2005, Soler et al., 2007).
The use of GC-MS to determine the residual pesticides in agricultural products helped
to obtain the veracity and accuracy in many groups of pollutants (Liu et al., 2006). Most GC
methods employ element-selective detectors such as electron capture detector (ECD),
nitrogen-phosphorus detector (NPD), flame photometric detector (FPD), electrolytic
conductivity detector and microwave induced plasma-atomic emission detection (Ahmed,
For organochlorine pesticide residues determination from cow milk and milk products,
researchers are using gas-chromatograph (GC) coupled with electron capture detector (ECD)
(Losada et al., 1996, Mallatou et al., 1997, Martinez et al., 1997, Hura et al., 1999, Cerkvenik
et al., 2000, Ghidini et al., 2002, Armendariz et al., 2004, Mamontova et al., 2004, Pinelli et
al., 2005, Darnerud et al., 2006, Darko et al., 2007, Nag et al., 2008).
A wide range of GC column types are used for residue analysis and the choice depends
on the physicochemical properties of the analytes (Ahmed, 2001, Nawaz, 2003,
Lambropoulou et al., 2007). Packed glass columns were used in pesticide residues analysis in
the past (Losada et al., 1996, Mallatou et al., 1997, Cerkvenik et al., 2000). Fused silica
capillary columns are now most widely used during analysis of pesticides, allowing the
separation of a large number of pesticides with similar physico-chemical characteristics.
Typical capillary columns are 2530 m long with an internal diameter range of 0,10,5 mm
and a stationary phase of 0,110 m thickness (Cerkvenik et al., 2000, Ahmed, 2001, Ghidini
et al., 2002, Nawaz, 2003, Mamontova et al., 2004, Darko et al., 2007, Nag et al., 2008). For
the separation of nonpolar pesticide residues such as OCP and pyrethroids are used nonpolar

stationary phases (Nawaz et al, 2003), like DB-1 (or OV-1) and DB-5 (or BPX-5) (van der
Hoff, 1999, Ahmed, 2001). Lambropoulou et al. (2007) has indicated another frequently used
non-polar stationary phases for capillary GC, for pesticide residues determination in food, for
example polydimethylsiloxane or phenylmethylpolysiloxane (DB-5, HP-5, ZB-5, RTX-5, HP5, SPB-5). Among the most used carrier gas for pesticide residues determination from food
matrices are helium (Mukherjee et al., 1996, Darko et al., 2007, Lambropoulou et al., 2007),
nitrogen (Mukherjee et al., 1996, Cerkvenik et al., 2000, Nag et al., 2008) and argon-methane
mixture (Losada et al., 1996, Mukherjee et al., 1996, Mallatou et al., 1997). Most residue
methods employ splitless injection of 13 l of the sample extract (Hura et al., 1999,
Cerkvenik et al., 2000, Nawaz, 2003, Lambropoulou et al., 2007, Nag et al., 2008). Use of an
autosampler with low injection speed has allowed for introduction of large volume extracts
and resulted in better resolution. The detectors may be connected in parallel to allow results to
be obtained with only one injection (Ahmed, 2001).
Losada et al. (1996) has used a multiresidues method for determination of the
organochlorine pesticides (aldrin, dieldrin, endrin, p,p-DDT and DDT isomers (o,p-DDD,
p,p-DDD, p,p-DDE), -HCH, lindan (-HCH), heptachlor-epoxide,) in raw bovine milk,
provided by Association of Official Analytical Chemists (AOAC, 1990). In another study,
Mallatou et al. (1997) has used the general multiresidues method of AOAC (Association of
Official Analytical Chemists, 1984) for determination of organochlorine pesticides in cow
milk (lindan (-HCH), -HCH, p,p-DDE, methyl parathion). In his researches, Ghidini et al.
(2002) has used a multiresidues method provided by Di Muccio et al. (1988), which may
determine simultaneously 24 OCPs and 18 PCBs. Armendariz et al. (2004) has adapted and
validated a gas chromatographic multiresidues method for determination of organochlorine
pesticides in cow milk (aldrin, o,p-DDT, p,p-DDT and DDT isomers (o,p-DDD, o,p-DDE
and p,p-DDE), -HCH, -HCH, lindan (-HCH), heptachlor, heptachlor epoxide,
hexachlorobenzene, mirex). Multiresidue methods are used for determining simultaneously of
more than one residue in a simple analysis (Ahmed, 2001, Nawaz, 2003).
The monitoring of the organochlorine pesticide residues in cow milk is often at the
microgram per kilogram (g/kg) level or lower, so that the method used must present a very
high sensitivity, specificity, precision and accuracy. Martinez et al. (1997) achieved a limit of
detection by 0,0003 mg/kg (ppm) for -HCH, aldrin and chlordane, 0,001 mg/kg for -HCH,
lindan (-HCH), -HCH, dieldrin, heptachlor, heptachlor-epoxide, p,p-DDE, o,p-DDD, p,pDDD and p,p-DDT and 0,01 mg/kg for o,p-DDT. Cerkvenik et al. (2000) has realized a
limit of detection by 0,003 mg/kg for HCH, lindan and 0,005 mg/kg for DDT. Ghidini et al.
(2002) using the Di Muccio et al. (1988) method obtain a limit of detection by 0,0002 mg/kg
for more OCPs (aldrin, cis-chlordane, trans-chlordane, dieldrin, o,p-DDE, p,p-DDE, o,pDDT, o,p-DDD, p,p-DDD, endrin, -endosulfan, heptachlor, hexachlorocyclohexane (HCH , -HCH, -HCH, -HCH), HCB, octachlorstirene, trans-nonachlor, mirex).

1. For organochlorine pesticide residues determination in cow milk are mainly used
multiresidue methods, due to presence of more than one organochlorine pesticide in cow milk
or milk products samples.
2. For the identification and quantitative determination of the organochlorine pesticide
residues from cow milk is mainly used the gas-chromatography coupled with electron capture
detector (GC-EDC).


3. The sensitivity, precision and accuracy of the methods currently used are very high,
so that the organochlorine pesticide residues from cow milk may be detected at microgram
per kilogram fat (g/kg) level.




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