Quantitative Analysis of Phthalates Plasticizers in Traditional Egyptian Foods (Koushary and Foul Medams), Black Tea, Instant Coffee and Bottled Waters by Solid Phase Extraction-Capillary Gas Chromatography-Mass Spectroscopy
Mahmoud A. Mohamed
Abdallah S. Ammar
In the present study, method of solid-phase extraction
followed by capillary gas chromatography coupled to mass spectrometry
(SPE-GC-MS) was used for quantitative analysis of trace levels of phthalates
in the most tow Egyptians traditional food (foul medams and koushary)
and drinks (black tea and instant black coffee) and bottled water samples.Method performance was evaluated in terms of accuracy, linearity,
limits of detection and recovery. Also the practical application of extraction
and analysis method was explained.
to cite this article:
Mahmoud A. Mohamed and Abdallah S. Ammar, 2008. Quantitative Analysis of Phthalates Plasticizers in Traditional Egyptian Foods (Koushary and Foul Medams), Black Tea, Instant Coffee and Bottled Waters by Solid Phase Extraction-Capillary Gas Chromatography-Mass Spectroscopy. American Journal of Food Technology, 3: 341-346.
Foodstuffs packed in plastics is the potential migration
of plastic additives such as plasticizers, stabilizers, antioxidants and
residual monomers from the plastic packaging material into the contained
foodstuffs (Goulas et al., 1998). Phthalate esters are the major
class of plasticizers permitted by FDA for use in food-contact plastics.
Phthalate esters are synthetic compounds widely used as polymer additives
in plastics, rubber, cellulose and styrene industry, to improve their
softness and flexibility. They are present in many consumer products including
children toys, cosmetics, personal care products, blood bags, organic
solvents, packaging, paper coatings and insecticides, etc. (Agency for
Toxic Substances and Disease Registry, 2000; David et al., 2001).
Due to widespread use, phthalates are considered as ubiquitous
environmental pollutants. Since their physical rather than chemical incorporation
in the polymeric matrix, phthalates easily migrate into foods, beverages
and drinking water from the packaging or bottling material or manufacturing
processes, being ingested and absorbed into the body, as well as during
blood transfusions from PVC blood bags (Balafas et al., 1999; Wahl
et al., 1999). Also highest levels tend to be found in fatty foods,
such as milk and dairy products, fish, meat and vegetable oils (Schettler,
Phthalates are suspected to be endocrine disrupting chemicals
exhibiting carcinogenic action (McKee et al., 2004). Due to their
potential risks for human health and environment, several phthalates has
been listed as priority substances by many national and international
regulatory organizations. The European Union (EU) published a candidate
list of substances with evidence or potential endocrine disrupting action,
which includes di-n-butyl phthalate, butyl benzyl phthalate and Di-2-Ethylhexyl
Phthalate (DEHP). Since DEHP is the most widespread phthalate produced
and used, it was incorporated in the list of priority substances in the
field of water policy established by EU and the World Health Organization
(WHO), has established a guideline value of 8.0 μg L-1
in fresh and drinking waters (European Union Council, 2001; WHO, 2003).
Capillary gas chromatography with flame ionization, electron
capture or mass spectrometry detection and high performance liquid chromatography
with diode-array and tandem mass spectrometry detection prior to sample
preparation methodologies, i.e. liquidliquid extraction and solid-phase
extraction, have been purposed for the determination of phthalates in
several types of matrices such as water (US Environmental Protection Agency,
2001; Cortazar et al., 2002), milk (Calafat et al., 2004),
urine (Silva et al., 2004), serum and plastics (Li et al.,
2004) and packing materials (Balafas et al., 1999). All above methods
are highly performance, but very expensive and need more time for many
steps of each method.
Dietary intake from contaminated food is likely to be
the largest single source of phthalate exposure in the general population.
Phthalate levels in food, however, are widely variable and data may not
reflect current exposure levels. Maximal daily intake estimates were 0.48
μg/kg/day for di-n-butyl phthalate, 4.9-18 μg/kg/day for bis
(2-ethylhexyl) phthalate and 0.11-0.29 μg/kg/day for butyl benzyl
phthalate (Schettler, 2006).
Koshary and Foul medams are commonly consumed in Egypt
as traditional foods and served hot and mostly in plastics packages.
In the present study, solid phase extraction followed
by capillary gas chromatography coupled to mass spectrometry (SPE-GC-MS)
was used as simple, accurate and economic method for quantitative analysis
of trace levels of phthalates in the most tow Egyptians traditional food,
foul medams and koushary and traditional drinks, black tea and instant
black coffee and bottled water which has increasing in consumption in
recent years. The performance of the method was evaluated in terms of
accuracy, linearity, limits of detection and recovery percentage.
MATERIALS AND METHODS
Phthalate Esters standard individual and Mix (500 μg mL-1
each in methanol) including Dimethyl Phthalate (DMP), Diethyl Phthalate
(DEP), di-n- butyl phthalate (DBP), Butyl Benzyl Phthalate (BBP), bis(2-ethylhexyl)
adipate (DEHA), bis(2-ethylhexyl) phthalate (DEHP), were purchased from
Supelco (Bellefonte, PA, USA). HPLC-grade methanol (MeOH, 99.9%), were
obtained from Fluka Chemie AG (Buchs, Switzerland). Ultra-pure water was
prepared from Milli-Q water purification systems (Millipore, Bedford,
MA, USA). An Oasis HLB glass (6 mL; 0.5 g) cartritges were purchased from
Waters Inc., Milford, USA.
Egyptians traditional food (koushary and foal medams) were obtained
in one day from different local restaurants located in Giza city, one
sample set was packed in plastic bags and other sample set was packed
in glass dishes. Bottled water samples were obtained from different markets
and the expiration date was within 6 months. Black tea and instant
black coffee were prepared in laboratory, one sample set in plastic cups
and other set in glass cups.
Food samples (1000 g) were homogenized with 500 mL of ultra-pure water
for 15 min, then the mixture was centrifuged at 10000 g during 20 min,
then supernatant was recovered and extracted with solid phase cartridge.
Water samples (1 L) directly extracted with solid phase cartridge. One
liter (5 cups) of black tea or coffee were directly extracted after 5
min of preparation.
Five hundred milliliter sample (supernatant or water) was filtrated
through the SPE cartridge at flow rate 1.0 mL min-1 by vacuum
using a vacuum manifold. After this, cartridges were flushed with 2x20
mL aliquots of ultra-pure water followed by reapplication of the vacuum
for 15 min to remove residual water. Each cartridge was eluted with 2
mL methanol and the eluent was concentrated to <1 mL by evaporation
under a stream of N2 gas and then adjusted to 1.25 mL (1 g)
with methanol and stored until analysis. A laboratory blank prepared by
SPE of 1 L of ultra-pure water was included with each sample set. Also
spiked sample at level 10 μg L-1 was included to evaluate
GC-MS Instrument Operating Conditions
GC-MS analysis were performed on a HP 6890 Series gas chromatograph
coupled to a HP 5973 mass selective detector (HP Technologies, USA). The
GC analysis was performed on a HP-5MS (80 m x 0.25 mm I.D., 0.25 μm
film thickness) capillary column (5% diphenyl, 95% dimethylpolysiloxane;
HP, USA). Injection system having a septumless sampling head was used
and helium as carrier gas was maintained in the constant pressure mode
and the inlet pressure was 35 psi. The oven temperature was programmed
from 60 °C (3 min) to 120°C at a rate of 5 °C/min and from
120 to 230 at rate of 8 °C/min and final temperature held for 10 min
The MS was operated in Selected-Ion Monitoring (SIM) mode and several
groups having target ions were monitored at different time windows defined
by the corresponding retention times. Two ions of each phthalate were
chosen, according to the mass spectra characteristic features obtained
in the full-scan mode (from 35 to 550 m/z and by comparison with the NIST
library reference spectral bank and data recording and instrument control
were performed by the HP ChemStation software (HP Technologies, USA).
Each of the measurements described was carried out in at least three
replicate. Unpaired t-tests were performed using the dada analysis in
the spreadsheet program (Microsoft, 2003). The probability level used
in evaluating test statistics was p = 0.05, 0.01.
RESULTS AND DISCUSSION
Six phthalate esters were selected for the present
study, since they prove to be the most common widespread phthalate contaminants
(Scientific Committee on Food, 1999). A standard mixture (10 mg L-1)
was analyzed by capillary GC-MS in the full-scan mode to record the mass
spectral fragmentation pattern of each compound which were chosen to attain
the best response in the selective ion detector mode acquisition and good
sensitivity and selectivity. The instrumental calibration was performed
with standard mixtures ranging from 15 to 100.0 μg L-1,
(r2>0.85) for the six phthalates, using the corresponding
target ion abundances. To evaluate the instrumental precision, sensitivity
was checked through the limit of detection (LOD) between 40 to 100 ng
L-1 was measured daily (Table 1). The result
of spiked water matrices (n = 3) at the 10 μg L-1 level,
producing excellent recoveries (>85%) and good selectivity and sensitivity
was observed when comparing with blank assays for almost all compounds
under study (Fig. 1). The laboratory blank is very important
since the contamination is a major problem in the analysis of phthalates,
especially from unclean plastic-
||Retention time (Rt), selected ions (m/z), linear range
(μg L-1), limit of detection (LOD (ng L-1),
correlation coefficient (r2) and average of recovery percentage
(% R) of selected compounds
|Linear range based on 6 levels of concentration, Recovery
percentage based on spiked sample at level 100 μg L-1
||The comparison between the responses (abundance) of
six phthalates in spiked sample with blank sample
||Levels (Mean±SD, n = 5) of phthalate (μg
L-1 for water, tea and coffee and μg kg-1
for koushary and foul medams) detected in real samples
|a: In plastic bag or cup, b: In glass dish or cup, nd:
containing glassware, organic solvents and many items
in laboratory settings and even from septum in injection system (Polo
et al., 2005). In present study, any contact with plastic material
was avoided and before use, all laboratory glassware was properly washed
several times with acetone and ultra-pure water. Furthermore, phthalate
contamination from the septum bleeding was avoided during the present
study, extraction cartridges were glass. The mass spectra of blank sample
showed in Fig. 1.
The LODs determined in present study were low enough
to detect phthalate contamination, comparable with previous reports in
literature in water samples (Polo et al., 2005) and other food
samples (Calafat et al., 2004). Data of real samples recorded in
Table 2, from the data, six phthalates were detected
in all sample but not in the same sample. The DBP and BBP were the most
abundant phthalate in bottled water samples, All levels were below the
restricting limits set by the international regulatory organisms (European
Union Council, 2000).
Data in Table 2 showed that foul medams
served in plastic bag had higher DEHA content than black tea (significantly
at p = 0.05), also served in plastic cup (0.33 and 0.23 μg L-1,
respectively). Foul medams had high temperature, long contact time with
plastic bag and high fat (oil) content, where these parameters increase
the migration of DEHA and oil act as un-polar media for migration of DEHA
(un-polar). Schettler (2006) found that the migration of the compound
did occur, that it increased with length of contact time and temperature
and the direct contact between the film and foods with a high fat content
at the surface. All plasticizers are poorly water soluble substances.
This is especially true for DEHP, DEHA which show true water solubility
in the lower μg L-1-range. The results in Table
2 showed that DEHP and DEHA didn`t detected in bottled water because
they are very poorly water soluble substances. All DEHP and DEHA detected
levels were significantly (p = 0.01) lower than the tolerance daily intake
of 50 μg kg-1 body weight (Scientific Committee on Food,
Based on the data of method performance, solid phase
extraction followed by capillary gas chromatography coupled to mass spectrometry
could be used for analysis of trace levels of phthalates with high accuracy
and recovery addition to ease of use and. Egyptian food, tea and coffee
which selected in present study usually served hot in plastic materials
either cups or dishes contained phthalates. Generally, the diet has been
considered the prime source of phthalates exposure in the population.
According to the above mentioned results, foods should be prepared, served
and packaged in a glass or polyolefin containers.
This research was supported by a grand from the
US-Egypt Joint Board on Scientific and Technological Cooperation. Author
thanks Prof. Dr. Wael Bazaraa, Professor of Food Science Technology for
his advising and promotion
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