Journal of Chromatography A, 1218 (2011) 68786883

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Determination of chloropropanols in foods by one-step extraction and

derivatization using pressurized liquid extraction and gas
chromatographymass spectrometry
I. Racamonde, P. Gonzlez, R.A. Lorenzo ,1 , A.M. Carro ,1
Department of Analytical Chemistry, Institute of Research and Food Analysis University of Santiago de Compostela, Avda de las Ciencias s/n 15782 Santiago de Compostela, Spain

a r t i c l e

i n f o

Article history:
Received 15 June 2011
Received in revised form 1 August 2011
Accepted 4 August 2011
Available online 10 August 2011
Keywords:
Pressurized liquid extraction
Simultaneous extraction and derivatization,
GCMS
Bakery foods
Chloropropanols
BSTFA

a b s t r a c t
3-Chloropropane-1,2-diol (3-MCPD) and 1,3-dichloro-2-propanol (1,3-DCP) were determined for the rst
time in bakery foods using pressurized liquid extraction (PLE) combined with in situ derivatization and
GCMS analysis. This one-step protocol uses N,O-bis(trimethylsilyl)triuoroacetamide (BSTFA) as silylation reagent. Initially, screening experimental design was applied to evaluate the effects of the variables
potentially affecting the extraction process, namely extraction time (min) and temperature ( C), number
of cycles, dispersant reagent (diatomaceous earth in powder form and as particulate matter with high
pore volume Extrelut NT) and percent of ush ethyl acetate volume (%). To reduce the time of analysis
and improve the sensitivity, derivatization of the compounds was performed in the cell extraction. Conditions, such as the volume of BSTFA, temperature and time for the in situ derivatization of analytes using
PLE, were optimized by a screening design followed to a Doehlert response surface design. The effect of
the in-cell dispersants/adsorbents with diatomaceous earth, Florisil and sodium sulfate anhydrous was
investigated using a BoxBehnken design. Using the nal best conditions, 1 g of sample dispersed with
0.1 g of sodium sulfate anhydrous and 2.5 g diatomaceous earth was extracted with ethyl acetate. 1 g
of Florisil, as clean-up adsorbent, and 70 L of BSTFA were used for 3 min at 70 C. Under the optimum
conditions, the calibration curves showed good linearity (R2 > 0.9994) and precision (relative standard
deviation, RSD 2.4%) within the tested ranges. The limits of quantication for 1,3-DCP and 3-MCDP,
1.6 and 1.7 g kg1 , respectively, are far below the established limits in the European and American legislations. The accuracy, precision, linearity, and limits of quantication provided make this analytical
method suitable for routine control. The method was applied to the analysis of several toasted bread,
snacks, cookies and cereal samples, none of which contained chloropropanols at concentrations above
the legislation levels.
2011 Elsevier B.V. All rights reserved.

1. Introduction
3-Chloropropane-1,2-diol (3-MCPD) and 1,3 dichloro-2propanol (1,3-DCP), the best-known chloropropanols, are
contaminants detected in acid-hydrolyzed vegetable proteins
(acid-HVP) as well as in foods such as bakery products with
components that are not subjected to acid hydrolysis during their
manufacture [16]. 3-MCPD esters are now found widespread
in thermally processed foodstuffs [7]. Both 3-MCPD and 1,3-DCP
were recently set by IARC into group 2B as probably carcinogenic
to humans [810]. The Food Chemical Codex (FCC) limited the
presence of 3-MCPD and 1,3-DCP in acid-HVP to not more than 1

Corresponding authors. Tel.: +34 981 563 100; fax: +34 981 547 141.
E-mail addresses: rosaantonia.lorenzo@usc.es (R.A. Lorenzo), tuchi.carro@usc.es
(A.M. Carro).
1
These authors have contributed equally to this work.
0021-9673/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2011.08.004

and 0.050 g mL1 , respectively [11] and Commission Regulation

(EC) established maximum levels of 0.02 mg kg1 for free 3-MCPD
in HVP and soy sauce [12]. There is no EU regulation for the other
chloropropanols.
Protection of the consumer health requires, the development
of effective and convenient methodologies able to identify and
determine chloropropanols in foods both accurately and sensitively. Most of the sample preparation procedures are usually
performed through several steps using Extrelut NT adsorbent and
large volumes (up to 150 mL) of solvents [2,9,1320]. Pressurized liquid extraction (PLE) is a simple and reliable technique
for trace analytes, which has been successfully applied in different matrices such as vegetables [21,22] shes [23], fruits
[24,25] and other foods [2628]. Extraction and clean-up are
carried out in an easy one step [21,29,30]. Due to the polar
nature of chloropropanols, a derivatization step previous to gas
chromatography (GC) analysis is recommended [28]. Heptauorobutyrylimidazole (HFBI), phenylboronic acid (PBA), ketones

I. Racamonde et al. / J. Chromatogr. A 1218 (2011) 68786883

and trimethylsilyl (TMS) derivatives are used for this purpose

[3,8,9,14,16,20,31,13].
The aim of this work was to develop a rapid and accurate
extraction method (PLE) for gas chromatography/mass spectrometry (GC/MS) analysis of 3-MCPD and 1,3-DCP in bakery
products. The simultaneous extractionderivatization by N,Obis(trimethylsilyl)triuoroacetamide (BSTFA) and clean-up into
the PLE cell was optimized using experimental designs. To the best
of our knowledge, this method has been not previously applied to
the analysis of chloropropanols.
2. Experimental
2.1. Reagents and materials
Chemicals and reagents were supplied as follow: 1,3dichloropropanol 98%, 3-monochloro-1,2-propanediol 98%,
1,3-dichloropropanol-d5 (d5 -1,3-DCP) (98.1% atom D) and 3monochloro-1,2-propanediol-d5 (d5 -3-MCPD) (98.1% atom D) used
as surrogate internal standard (IS) by C/D/N Isotopes (PointeClaire, Quebec, Canada). Stock solutions of each individual analyte
at 5000 g mL1 and a mixture of all were prepared in ethyl
acetate and were stored at 18 C. N,O-bis(trimethylsilyl) triuoroacetamide/trimethylchlorosilane (BSTFA/TMCS) 99:1 by
Supelco (Bellefonte, PA, USA); ethyl acetate (Chromanorm) by
VWR-Prolabo (Mollet del Valls, Barcelona, Spain).
Extrelut NT and Celite 545 (0.01-0.04 mesh) were from Merk
(Darmstadt, Germany) and sodium sulfate anhydrous 99% by Panreac (Barcelona, Spain). Diatomaceous earth (DE) in powder form,
Florisil (60100 mesh), sea sand (5070 mesh), silica gel (70230
mesh) and activated basic aluminum oxide were supplied by
Aldrich (Madrid, Spain).
Syringe lters (Millex GV, 13 mm, and 0.22 m) were from Millipore (Billerica, MA, USA) and cellulose lters (20 mm diameter)
for PLE cells from Restek (Bellefonte, PA, USA).
2.2. Samples
Samples of various toasted breads (low salt toast, whole meal
toast) corn and wheat breakfast cereals, two kinds of snacks and a
our based biscuit were bought at a local supermarket. The samples
used in the optimization of sample preparation step were triturated
and homogenized in an electric mill and dried at 60 C to remove the
moisture and stored at room temperature in a desiccator until use.
The food samples were spiked with different amounts of working
standard solutions in order to prepare the samples for the different
studies.
Approximately 25 g of sample were placed in a beaker with a
broad base and covered with 50 mL of ethyl acetate spiked with
chloropropanols standards to obtain the necessary concentration
in food for each analyte. The spiked sample was allowed to airdry in the dark for three days and stored at room temperature until
extraction, in order to simulate the normal interaction between the
food and the target compounds.
2.3. Gas chromatography determination
All analysis was performed using an Agilent 7890A gas
chromatograph (Wilmington, DE, USA) coupled to an inert
MSD triple axis mass spectrometer (Agilent 5975C) equipped
with a split/splitless 7693 injector. Chromatographic separation was achieved using an HP-5MS fused silica capillary
column (30 m 0.25 mm i.d, lm thickness 0.25 m of 5%-phenylmethylpolysiloxane) from Agilent Technologies. Helium (purity
Spain) was employed as the
99.999%, Carburos Metlicos, A Coruna,
carrier gas with a constant ow of 1.2 mL min1 . The samples were

6879

injected in the splitless mode, and then the splitter was opened
after 2 min. The GC oven temperature were programmed as follows:
initial temperature 50 C for 2 min; then increased at 3 C min1 to
100 C and nally from 100 to 280 C at a rate of 40 C min1 (kept
for 10 min). The injector temperature was hept constant at 280 C.
The total run time of analysis was 33 min.
The mass spectrometer was operated in electron impact (EI),
70 eV of ion energy, with a 10 min solvent delay. The temperatures of the interface, EI ionization source and the quadruple mass
analyzer were set at 280 C, 230 C and 150 C, respectively.
Data acquisitions were carried out over the mass range m/z
60400, in SIM mode (Single Ion Monitoring) to maximize sensitivity. To identify the compounds 445 L of 1 g mL1 standard
solution of each individual analyte was introduced into an amber
vial, 55 L of derivatization reagent was added, the mixture was
shaken vigorously in a vortex and heated to 60 C for 70 min for the
derivatization reaction. Then 1 L was injected in the CG/MS to nd
the higher m/z and characteristic ions for each compound. Specic
conditions for each analyte are listed in Table 1. Quantication was
accomplished by the relative areas vs. internal standards, which
were added to samples at the spiked step. For analyzing chloropropanols by gas chromatography if is necessary to carry out this
kind of reaction in order to modify their low volatility and high
polarity and improve the sensibility [8,31]. Trimethylsilyl (TMS)
derivatives were prepared according to a previous work dealing
with the BSTFA derivatization of chloropropanols [32]. The reaction
was carried out with standard solutions in ethyl acetate, demonstrating the suitability of this solvent to accomplish derivatization.
The TMS derivatives were stable for more than one month.
2.4. Extraction and derivatization procedure
Extractions were accomplished using a pressurized liquid
extractor ASE 200 from Dionex (Sunnyvale, CA, USA), equipped
with a 24-sample carousel, 11 mL stainless steel cells, and 40 mL
collection vials. Two cellulose lters (Dionex) were placed at the
bottom of the PLE cell And aA layer of the adsorbent clean-up (0.6 g
of Florisil) was placed on the lters. 1 g sample was mixed with
1.5 g of anhydrous sodium sulfate (drying agent) and 2 g of DE (dispersant agent), using a mortar and a pestle, and the mixture was
introduced into the cell lling the remaining volume with clean
sand (5070 mesh particle size, SigmaAldrich). Then, another cellulose lter was placed on the top. In all experiments, isotopically
labelled analogues (30 ng mL1 ) were added as surrogate standards
to each sample before extraction. The extraction pressure was set
to 1500 psi and ethyl acetate was employed as extraction solvent.
The cell was tightly closed and placed into the carousel of the ASE
system. Extractions were performed by preheating the cell before
lling with the solvent (preheat method). Analytes were recovered in one extraction cycle of 5 min, at 130 C. The ush volume
of ethyl acetate was 80% and the purge time was set to 90 s. The
PLE extract (ca. 20 mL), was collected in vials sealed with rubber
septa, evaporated using a gentle stream of nitrogen in a Turbo Vap
II concentrator (Zymark, Hopkinton, MA, USA) and adjusted to a
nal volume of 1 mL. 445 L of extract was derivatized with BSTFA
as described in Section 2.1 and 1 L was analyzed by GC/MS.
In the simultaneous extractionderivatization experiments,
70 L of BSTFA was added to the sample before the addition of
the drying agent (0.1 g of anhydrous sodium sulfate) to avoid the
susceptibility towards hydrolysis of silylation agents [29,33] and
the dispersion sorbent (2.5 g of DE). 1 g of Florisil, was added as
clean-up adsorbent. Thereafter, the PLE procedure was carried out.
Analytes were recovered in one extraction cycle of 3 min, at 70 C.
The ush volume and the purge time were as previously described.
The PLE extracts were collected and evaporated in a Turbo Vap II
concentrator and adjusted to a nal volume of 1 mL. The extract

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I. Racamonde et al. / J. Chromatogr. A 1218 (2011) 68786883

Table 1
Retention times (tR ), quantier ion and qualier ions for the chloropropanols by GCMS.

Quantier ion (m/z)

Qualier ion (m/z)
Retention time (min)

d5 -1,3-DCP-TMS

1,3-DCP-TMS

d5 -3-MCPD-TMS

3-MCPD-TMS

154
93.185
11.75

151
93.190
12.00

116
239
17.45

119
244
17.85

was ltered by means of a syringe lter with a pore size of 0.22 m

before GC injection.
2.5. Statistical analysis
Experimental designs were applied for optimizing the extraction method and to analyze the simultaneous effect of the main
factors affecting PLE and to evaluate the one-step extraction and
derivatization procedure. The basic and descriptive statistics and
the experimental design generation and analysis were performed
using NemrodW2000 (D. Mathieu, J. Nony, R. Phan-Than-Luu, NemrodW, Ver. 2000, LPRAI, Marseille, 2000) as the software package.
3. Results and discussion
3.1. Preliminary experiments
PLE preliminary assays were performed on fortied samples
using the general conditions described in EPA 3545 [34]: 1.5 g of
anhydrous sodium sulfate, one cycle of 5 min at 100 C, 1500 psi,
solvent ush of 60% and 90 s purge time. The adsorbents for cleanup were selected on the basis of the literature on PLE extraction of
contaminants from food matrixes [21,23,29,30]. One-step extraction and cleanup by PLE was evaluated. Anhydrous sodium sulfate
was added to the sample for eliminating the co-extracted water
[29,35]. 2 g of Extrelut NT, Celite or DE, with similar chemical
composition based on SiO2 but different mesh size, were initially
evaluated as dispersant agents to identify the combination that provided the cleanest extracts. The best results were obtained with DE
or Extrelut NT (data provided in electronic supporting information
le 1A) and were studied in a subsequent screening design. Different adsorbents were compared: 1 g of Florisil, a mixture of activated
0.4 of graphitized carbon (GCB) and 0.6 g of Florisil, 1 g of aluminum
oxide and 1 g of silica gel (data provided in electronic supporting
information le 1B). Aluminum oxide retained 3-MCPD. A good
extraction was not obtained without adsorbent or with GCB/Florisil.
The best results were obtained with 1 g of Florisil.
3.2. PLE conditions
An asymmetric screening design of the 22 33 //12 [36] type was
used to study the inuence of the extraction time (1, 3 and 5 min)
and the temperature (60, 100 and 130 C), the number of cycles (one
or two), the ush volume of ethyl acetate (40, 60 and 80%) and the
dispersant (DE or Extrelut NT) to obtain an efcient extraction and
cleanup. The effect of the pressure was studied because it generally has a negligible effect on the extraction yield [33]. 1 g of bread
spiked with 1,3-DCP and 3-MCPD at a concentration of 0.5 g mL1
in the nal extract was used. The results obtained were examined
with the aid of different graphic tools supplied in the software
package, such as delta weight plots [36] which allowed the relative effects of a level change on the response variable (peak area)
to be compared. The effects are shown as bars (data provided in
electronic supporting information, le 2). Extraction time was statistically signicant (p < 0.05) and 80% of the ush volume was only
statistically (p < 0.05) signicant for the extraction of 3-MCPD, but
any level of % Flush volume lead to similar results for the 1,3-DCP.
Better responses were achieved for both chloropropanols when

high levels of extraction temperature or DE were used. Since large

differences in responses between 1 and 2 cycles were not found,
one extraction cycle was preferred. The selected conditions for the
PLE were established as follows: one cycle, 80% ush of volume,
DE as dispersant, 130 C for 5 min and 1 g of Florisil as adsorbent.
Then PLE extracts were derivatized and analyzed by GC/MS.
3.3. Simultaneous PLE extractionderivatization
The post-extraction derivatization procedure involved excessive sample handling and increased the time of analysis due to
the conditions required for derivatization (60 C and 70 min). Thus
we developed a derivatization-extraction procedure, adding the
derivatization agent (BSTFA) directly into the PLE cell, taking advantage of the pressure and temperature conditions of PLE to reduce
the derivatization time and to achieve the GCMS analysis of the
extract without any further step.
3.3.1. Screening design
Working under the PLE conditions xed in Section 3.1, other factors potentially affecting the extractionderivatization efciency
were studied. An asymmetric screening design 21 32 //9 was used
for the study of the BSTFA volume (25, 50 and 70 L), the temperature (75, 90, 100 C) and the time (10 and 15 min) for PLE
extractionderivatization [36]. The graphics of the total effects [36]
are shown in Fig. 1. The high BSTFA volume provided, for both
chloropropanols much better results than the lower values; consequently the level was xed as 70 L. Higher BSTFA volume had
a negative effect due to a signicant increase of the background
signal in the chromatogram. Lower levels of temperature and time
cause a greater positive effect on the response, possibly because the
derivatization reaction should take place during the rst moments
of contact with the sample and the analytes, and furthermore the
derivatization agent can degrade at high temperatures. Therefore,
these factors were studied in more detail through a response surface methodology (RSM).
3.3.2. Response surface Doehlert design
The experiments were carried out according to aDoehlert matrix
made up of 9 experiments, including three central points [35].
Five levels for extraction time (3, 6, 8, 11 and 13 min) and three
levels for extraction temperature (46, 63 and 80 C) were considered. Data were evaluated by ANOVA. Since common behavior for
the extraction of both chloropropanols is expected the optimum
path of the response surface [36] was considered for the optimization (data provided in electronic supporting information le 1).
The optima conditions were temperature = 78 C and time = 5 min.
To reach the maximum, the time must have lower values while
the temperature must tend towards higher values. For 3-MCPD
the maximum response corresponds to temperature = 70 C and
time = 3 min. However, a more convenient solution is to use the
global desirability function to obtain the optimum operational
conditions for both compounds. Fig. 1C shows the 2D plot of
global desirability function. Response surface showed the maximum desirability at high temperature and low time period. In this
zone, desirability was close to 1. The optimal compromise values
were 3 min of static time at 70 C.

I. Racamonde et al. / J. Chromatogr. A 1218 (2011) 68786883

6881

Fig. 1. Graphics of total effects obtained using a screening design 21 32 //9, for 3-MCPD (A) and 1,3-DCP (B). Global desirability response surface plot using a Doehlert design
(C) and a BoxBenhken design (D).

3.3.3. Response surface BoxBenhken design

Based on results reported in the previous section, an adjustment of the proportions of the amounts of dispersant/adsorbent in
the cell was carried out due their potential inuence in the analytes recovery and in the dispersion of the matrix. The amounts of
Florisil (0.1, 0.8 and 1.5 g), DE (0.1, 1.5 and 2.9 g) and anhydrous
sodium sulfate (0, 0.25 and 0.5 g) were included in a BoxBenhken
design with 11 experiments [37]. After ANOVA evaluation, only
the effect of Florisil was statistically signicant (p < 0.01) for 1,3DCP. High levels of DE and medium-high levels of Florisil provided
good responses for both chloropropanols, while medium-low levels
of anhydrous sodium sulfate led to high extractionderivatization
efciency for both analytes. Considering the optimum path of
the response surface for both chloropropanols (data provided in
electronic supporting information, le 2), the optima conditions
correspond to 2.5 g and 2.6 g of DE for 1,3-DCP and 3-MCPD, respectively and 1.1 g of Florisil and 0.11 g of anhydrous sodium sulfate,
for both analytes. When the global desirability function was applied
to conrm these conditions, similar results were obtained with
the most favorable dispersant/adsorbent amounts for both compounds. The response surface for global desirability is shown in
Fig. 1D. The optimum of global desirability was equal to 1.0. The
maximum D value obtained with 1 g of Florisil, 2.5 g DE and 0.1 g
anhydrous sodium sulfate.
3.4. Validation of the extractionderivatization method by PLE
The instrumental linearity was evaluated in the range
490 ng mL1 (including six concentration levels in triplicate),
considering the area of the peaks relative to internal standards using isotopically labelled analogues as surrogate standards.

Concentration of the IS along the calibration curve was maintained constant at 30 ng mL1 for d5 -1,3-DCP and for d5 -3-MCPD.
The response functions were found to be linear with determination coefcient (R2 ) higher than 0.999. Limits of detection (LODs) and quantication (LOQs) of the overall method
(extractionderivatization by PLE following GCMS) were calculated as the concentrations giving a signal-to-noise ratio of 3 or
10 (S/N = 3 or 10), respectively according to the European norm EN
14573 [19] and the Eurachems recommendations [38]. LODs were
0.5 g kg1 for both chloropropanols while LOQs were 1.6 g kg1
for 1,3-DPC and 1.7 g kg1 for 3-MCPD. These values are lower
than the quantication limit of 3 g kg1 for both chloropropanols
in soy sauce and other food products reported by Abu-El-Haj et al.
[39] using a clean-up alumina column, dichloromethane extraction,
heptauorobutyryl anhydride derivatization, and isotope dilution
GCMS. Moreover, the obtained limits are much lower than those
established in the legislation [11,12] and than the limits proposed
by the European Commission for the ofcial control of the levels of
3-MCPD in food samples (LOD < 5 g kg1 and LOQ <10 g kg1 )
[40]. On the other hand, in order to evaluate the precision of
the proposed method, six independent analyses were performed
using spiked toasted bread samples at two concentration levels (20 ng g1 and 2 g g1 for both chloropropanols). The results
obtained showed relative standard deviation (RSD%) of 2.4 and
0.9% for 20 ng g1 and 2 g kg1 , respectively for 1,3-DCP and 1.4
and 1.03% for 20 g kg1 and 2 mg kg1 ,respectively, for 3-MCPD.
These values are fully compatible with the requirements established by the European Commission for 3-MCPD [40]. Accuracy
studies, expressed as % of recovery, were carried out by applying
the optimized extractionderivatization by PLEGCMS method to
the extraction of a real bakery samples. Due to the possible matrix

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I. Racamonde et al. / J. Chromatogr. A 1218 (2011) 68786883

Table 2
Extraction recoveries (n = 5 replicates) in fortied (20 ng g1 ) bakery samples by extractionderivatization by PLEGCMS. Results of the determination of 1,3-DCP-TMS and
3-MCPD-TMS in bakery samples (n = 4 replicates) are calculated for dry weight.
Sample*

1,3-DCP-TMS

Corn cereals (8.12%)

Snacks 1 (7.46%)
Toasted low salt bread
(8.06%)
Biscuits (4.45%)
Snack 2 (8.06%)
Wheat cereals (7.46%)
Toasted whole meal
bread (7.05%)

3-MCPD-TMS

Concentrations of 1,3-DCP
nonspiked bakery samples
(concentration, ng g1 SD)

Concentrations of 3-MCPD
nonspiked bakery samples
(concentration, ng g1 SD)

% Recovery

RSD%

% Recovery

RSD%

91.8
97.8
94.1

5.2
1.2
1.5

93.7
109
86.2

1.6
3.2
1.6

37.0 2.9
43.9 1.8
23.4 1.5

3.6 0.2
11.8 1.3
<LOQ

94.1

6.3

91.7

2.0

12.8 0.4
n.d.
13.4 0.1
99.28 2.9

2.8 0.1
6.8 0.7
n.d.
4.5 0.1

n.d. = not detected.

*
% moisture.

effect quantitative measurements in real samples usually require

to apply the standard addition method. For this reason, three
replicates of different bakery products (breakfast cereals, snacks,
toasted bread and biscuits samples) were analyzed using standard
addition method and IS at a concentration of 40 g kg1 for both
chloropropanols. The recovery was evaluated at a concentration
of 20 g kg1 (n = 5). Previous analyses of these samples showed
the presence of some of the target compounds and these initial
concentrations were taken into account to calculate the recoveries.
The recoveries were between 86.2% and 109% (Table 2) complying
with the requirements of the European Commission for methods to
analyze 3-MCPD in food samples [39]. The precision was also evaluated, and the RSD% values were lower than 6.3% with an average
value of 2.8%.
3.5. Application of the method to real samples
To further demonstrate the utility and performance of the proposed methodology, different bakery products, including breakfast

4. Conclusions

A
counts

100%

260

93.0

75%

220

50%

180

1,3-D C P -T MS

151.0

25%

140

185.0

0%
90

100

110

130

150

170

190 m/z

60
20
9.50

10.00 10.50 11.00 11.50 12.00 12.50 13.00 minutes

116.0
100%

75%

counts
160
140
120
100
80
60
40

cereals, toasted bread, snacks and biscuits, were analyzed in quadruplicate and quantication was accomplished using the standard
addition method (Table 2). The presence of chloropropanols was
evidenced in the samples analyzed, but the levels found did not
exceed the limit regulated for soy sauces and related products
[11,12]. These results are in agreement with the fact that the moisture content of bakery products inuences the chloropropanol
formation process, since glycerol appears to be a precursor in foods
with a low water content (<15%) [41]. Values shown in Table 2,
are in the same order of magnitude as those obtained for bakery
products reported by Hamlet [8] and Leon et al. [20]. Although
the information on 1,3-DCP in retail foods is scarce at present,
data from different reports suggest that 1,3-DCP may be present
in retail ready-to-eat processed foods including bakery products
without 3-MCPD and that can be related with the presence of
dimethylamineepichlorohydrin copolymer used in sugar rening
[42] at levels substantially higher than those of 3-MCPD [8]. Fig. 2
shows the SIM GCMS chromatogram obtained for a snack sample.
The spectrum obtained by MS conrmed the identity of the analytes
detected in the sample (Fig. 2).

50%

3-MC P D -T MS

25%

239.0

0%

110 130 150 170 190 210 230 250 m/z

The suitability of the PLE technique for the extraction of chloropropanols from bakery foods has been demonstrated for rst time.
The use of DE as sample dispersant and Florisil as clean-up sorbent, combined with simultaneous in-cell derivatization, allowed
to obtain a ready to inject GC extract providing recoveries, better
than the derivatization out of the cell in a longer step. The main
advantages of the proposed methodology are the rapid and automated sample pre-treatment and the reduction of the sample size
and handling. The sensitivity of the method is suitable for the legislation requirements. The results obtained will be useful for future
studies aimed to determine the presence of chloropropanols in
foods, in the total dietary intake, and their persistence and toxicity
taking into account that it is unknown whether they are transformed into the human body and degraded or subjected to other
processes. Application of the method to other matrices, such as
biological ones, would also provide an interesting insight into the
uptake and bioaccumulation of chloropropanols in tissues or body
uids.
Acknowledgments

15.50 16.00 16.50 17.00 17.50 18.00 minutes

Fig. 2. Mass chromatograms and mass spectra of snacks 1 extract containing 1,3 DCP
(A) and 3-MCPD (B) in measured concentration of 43.9 g kg1 and 11.8 g kg1 ,
respectively.

This research was supported by the Project 10TAL209005PR

Direccin Xeral de I+D (Xunta de Galicia, SPAIN) and FEDER. The
authors thank Professor R. Phan-Tan-Luu of the University Paul
Czanne of Aix-Marseille III (France) for providing the software

I. Racamonde et al. / J. Chromatogr. A 1218 (2011) 68786883

NEMROD-W.I. Racamonde gratefully acknowledges her FPI grant

from the Spanish Ministry of Education and Science.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.chroma.2011.08.004.
References
[1] J. Velisek, J. Davidek, J. Hajslova, V. Kubelka, G. Janicek, B. Mankova, Z. Lebensm,
Unters For. 167 (1978) 241.
[2] C.G. Hamlet, P.A. Sadd, D.A. Gray, J. Agric. Food Chem. 52 (2004) 2059.
[3] C.A. Van Bergen, P.D. Collier, D.D.O. Cromie, R.A. Lucas, H.D. Preston, D.J. Sissons,
J. Chromatogr. 589 (1992) 109.
[4] S.W.C. Chung, K.P. Kwong, J.C.W. Yau, A.M.C. Wong, Y. Xiao, J. Food Compos.
Anal. 21 (2008) 569.
[5] C. Crews, P. Brereton, A. Davies, 18 (2001) 271.
[6] C. Crews, P. Hough, P. Brereton, D. Harvey, R. MacArthur, W. Matthews, Food
Addit. Contam. 19 (2002) 22.
[7] ILSI Europe Process Related Compounds/Natural Toxins Task Force and Risk
Assessment of Chemicals in Food Task Force in association with the European Commission (EC) and the European Food Safety Authority (EFSA). 3-MCPD
esters in food products. Summary Report. Brussels, 2009.
[8] C.G. Hamlet, in: J.S.H.Z. Gilbert (Ed.), Bioactive Compounds in Foods, Blackwell,
Oxford, 2001, pp. 323357 (ch. 12).
[9] Safety evaluation of certain food additives and contaminants/prepared by
the sixty-seventh meeting of the Joint FAO/WHO Expert Committee on
Food Additives (JECFA), Geneva 2007, WHO Food Additives Series, No. 58,
pp. 209238.
[10] Y. Grosse, R. Baan, B. Secretan-Lauby, F. El Ghissassi, V. Bouvard, L. BenbrahimTallaa, N. Guha, F. Islami, L. Galichet, K. Straif, Lancet Oncol. 12 (2011) 328.
[11] Food Chemical Codex, Acid Hydrolysates of Proteins, First Supplement to the
4th ed., Institute of Medicine, 1997, pp. 14.
[12] Commission Regulation (EC) No. 1881/2006 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off. J. Eur. Union L364,
524.
[13] P. Brereton, J. Kelly, C. Crews, S. Honour, R. Wood, A. Davies, J. AOAC Int. 84
(2001) 455.
[14] H. Runnqvist, S.A. Bak, M. Hansen, B. Styrishave, B. Halling-Sorensen, E. Bjoerklund, J. Chromatogr. A 1217 (2010) 2447.
[15] X. Xu, Y. Ren, P. Wu, J. Han, X. Shen, Food Addit. Contam. 23 (2006) 110.
[16] M. Huang, G. Jiang, B. He, J. Liu, Q. Zhou, W. Fu, Y. Wu, Anal. Sci. 21 (2005) 1343.
[17] C.G. Hamlet, P.G. Sutton, Rapid Commun. Mass Spectrom. 11 (1997) 1417.
[18] R. Schuhmacher, J. Nurmi-Legat, A. Oberhauser, M. Kainz, R. Krska, Anal. Bioanal.
Chem. 382 (2005) 366.

6883

[19] DIN EN 14573 Foodstuffs-Determination of 3-Monochloropropane-1,2-Diol by

GC/MS, 2005, Beuth Verlag, Berlin.
[20] N. Leon, V. Yusa, O. Pardo, A. Pastor, Talanta 75 (2008) 824.
[21] D. Garcia-Rodriguez, A.M. Carro-Diaz, R.A. Lorenzo-Ferreira, R. Cela-Torrijos, J.
Chromatogr. A 1217 (2010) 2940.
[22] M. Barriada-Pereira, M.J. Gonzalez-Castro, S. Muniategui-Lorenzo, P. LopezMahia, D. Prada-Rodriguez, E. Fernandez-Fernandez, Talanta 71 (2007) 1345.
[23] P. Haglund, S. Sporring, K. Wiberg, E. Bjoerklund, Anal. Chem. 79 (2007) 2945.
[24] A. Juan-Garcia, G. Font, C. Juan, Y. Pico, Food Chem. 120 (2010) 1242.
[25] G. Sagratini, J. Manes, D. Giardina, Y. Pico, J. Agric. Food Chem. 54 (2006) 7947.
[26] S. Sporring, C. von Holst, E. Bjoerklund, Chromatographia 64 (2006) 553.
[27] E. Rodriguez, F.N. Villoslada, M.C. Moreno-Bondi, M.D. Marazuela, J. Chromatogr. A 1217 (2010) 605.
[28] J.A. Mendiola, M. Herrero, A. Cifuentes, E. Ibanez, J. Chromatogr. A 1152 (2007)
234.
[29] T. Tanaka, T. Hori, T. Asada, K. Oikawa, K. Kawata, J. Chromatogr. A 1175 (2007)
181.
[30] E. Bjoerklund, S. Sporring, K. Wiberg, P. Haglund, C. von Holst, Trends Anal.
Chem. 25 (2006) 318.
[31] T. Wenzl, D.W. Lachenmeier, V. Goekmen, Anal. Bioanal. Chem. 389 (2007) 119.
[32] A.M. Carro, R.A. Lorenzo, R. Cela, XV Meeting SEQA, San Sebastin (Spain), 2009.
[33] X. Cao, G. Song, Y. Gao, J. Zhao, M. Zhang, W. Wu, Y. Hu, Chromatographia 70
(2009) 661.
[34] EPA 3545 method http://www.dionex.com/en-us/webdocs/68591-EPA-3545ASE.pdf (accessed 10.08.10).
[35] V. Yusa, G. Quintas, O. Pardo, P. Marti, A. Pastor, Food Addit. Contam. 23 (2006)
237.
[36] M.D. Lewis, G.A. Phan-Tan-Luu, R Pharmaceutical Experimental Design, Marcel
Dekker, New York, 1999.
[37] R.H. Myers, D.C. Montgomery, C.M. Anderson-Cook, Response Surface Methodology, 3 ed., Wiley, New Jersey, 2009.
[38] The tness for purpose of analytical methods: a laboratory guide to method
validation and related topics. EURACHEM Guide, rst English ed 1. 0-1998, LGC,
Teddington Ltd. http://www.eurachem.org/ (accessed 10/03/10).
[39] S. Abu-El-Haj, M.J. Bogusz, Z. Ibrahim, H. Hassan, M. Al Tufail, Food Control 18
(2006) 81.
[40] Commission Regulation 333/2007/EC of 28th March Laying down methods of
sampling and analysis for the ofcial control of the levels of lead, cadmium,
mercury, inorganic tin, 3-MCPD and benzo (a) pireno in foodstuffs. Off. J. Eur.
Union, L 88, 2007.
[41] P. Brereton, C. Crews, S. Hasnip, P. Reece, J. Velisek, M. Dolezal, C. Hamlet, P.
Sadd, D. Baxter, I. Slaiding, R. Muller, The Origin and Formation of 3-MCPD in
Foods and Food Ingredients, Food Standards Agency, UK, 2005.
[42] S.A. Masten 1,3-Dichloropropanol [CAS No. 96-23-1], Review of toxicological Literature. A report prepared for National Toxicology Program
(NTP), National Institute of Envoronmental Health Sciences (NIEHS),
National Institute of Health, U.S. Deparment of Health and Human Services. NTP/NIEHS, North Calorina http://ntp-server.niehs.nih.gov/ntp/htdocs/
Chem Background/ExSumPdf/dichloropropanol.pdf (accessed 26.07.11).