Introduction
3-MCPD is known to be a heat-induced contaminant that can be formed by
different food processing techniques such as heating (e.g. smoking,
roasting, baking, grilling) of various kinds of foods, (Hamlet, 2002).
2- and 3-monochloropropanediol fatty acid esters (2- and 3-MCPDE) as
well as glycidyl fatty acid esters (GE) are also heat-induced food
contaminants that are ubiquitously found in refined edible oils/fats,
(Zelinkova, 2006; Weisshaar, 2010; Kuhlmann, 2011). 3-MCPD has been
classified by the International Agency for Research on Cancer (IARC) as
possibly carcinogenic to humans (category 2B) (International Agency for
Research on Cancer, 2012) while glycidol has shown genotoxic properties
and was ranked as probably carcinogenic to humans (category 2A),
(International Agency for Research on Cancer, 2000). Animal experiments
as well as human studies have shown that digestion of 2-MCPDE, 3-MCPDE
and GE releases the core compounds completely or to large extent,
(Abraham, 2013; Appel, 2013; Abraham 2021). Correspondingly, 3-MCPD,
glycidol and their fatty acid esters are considered to have the
potential to show adverse health effects when ingested. The European
Food Safety Authority considered that in terms of exposure the fatty
acid bound compounds do show the same toxicity as the free analytes on
molar base, (EFSA CONTAM panel, 2016).
It appears evident that food emulsifiers might contain MCPDE as well as
GE if they are directly or indirectly derived from edible oils/fats when
heat treatment is applied as deodorisation or distillation during
manufacturing. Since 2018, the European Food Safety Authority (EFSA) and
the European Commission (EC) released calls for technical and
toxicological data on different classes of emulsifiers used as food
additives, (EFSA, 2018; EC, 2018a; EC, 2018b). These calls for data were
launched as a follow-up to EFSA’s re-evaluation of a number of food
emulsifiers, (EFSA, 2017a; EFSA, 2017b; EFSA, 2017c), in line with EU
Regulation 257/2010, (EU 2010). By these calls, analytical data and/or
the lowest technologically achievable levels of 3-MCPD/3-MCPD
esters/glycidol/glycidyl esters were requested.
From a regulatory perspective, EU Regulation 2020/1322, amending
Regulation 1881/2006, sets a maximum level of 1000 µg/kg for glycidol in
the form of glycidyl fatty acid esters in vegetable oils/fats placed on
the market for the final consumer, or for use as a food ingredient. In
oils/fats used for the production of baby food and cereal-based foods
for infants and young children, the maximum level for GE is 500 µg/kg.
With the same regulation, maximum quantities for the sum of free and
fatty acid bound 3-MCPD (analyte group) are set for the same foods for
which maximum levels for GE have been established. Due to the lower
3-MCPD formation potentials, a stricter maximum level (1250 µg/kg) for
the sum of 3-MCPD and 3-MCPD fatty acid esters, expressed as
3-MCPD is set for oils and fats from coconut, maize, rapeseed,
sunflower, soybean, palm kernel and olive oils and mixtures of oils and
fats with oils and fats only from this category. A higher maximum level
of 2500 µg/kg applies to other vegetable oils (including olive pomace
oils), fish oils and oils from other marine organism and mixtures of
oils and fats with oils and fats only from this category. For oil mixes
from both categories with quantitatively known ingredients, the
category-related maximum levels apply to the single oil or fat
components of the mix. In case of oil and fat mixes of unknown
composition, the higher maximum level of 2500 µg/kg applies. For
vegetable oils and fats, fish oils and other marine oils that are
destined for the production of baby food and processed cereal-based food
for infants and young children the maximum level for the sum of
3-MCPD and 3-MCPD fatty acid esters, expressed as 3-MCPD is set to 750
µg/kg. Finally, for infant formula, follow-on formula and foods for
special medicinal purposes for infants and young children as powders the
maximum level is addressed as 125 µg/kg for powders and as 15 µg/kg for
liquids, (EU, 2020). This Regulation does not apply directly to food
emulsifiers. However, there remains a correlation as food emulsifiers
might be used for the production of certain types of baby and young
children foods. Furthermore, from practical perspective, it can be
assumed that food control laboratories will analyse the lipid phase
extracted from compound foods in order to conclude, if the oils used for
the production of the corresponding foods match official regulation or
other limits set by producers or retailers. This approach might give
improper results in case that food emulsifiers contributed to the 3-MCPD
and glycidol levels of the extracted lipid fraction. Considering these
circumstances, the availability of a reliable analytical method for the
determination of 3-MCPD, glycidol and their derivatives in food
emulsifiers seems to be of high relevance.
Several officially validated analytical methods are available for the
parallel determination of ester-bound 3-MCPD and ester-bound glycidol in
edible oils & fats. The most common methods used seem to be the AOCS
Official Methods Cd 29a-13, Cd29b-13 and Cd29c-13 (AOCS, 2017a-c) which
also were adopted as ISO standards 18363-3, 18363-2 and 18363- 1 (ISO,
2015; ISO, 2018; ISO, 2017). AOCS Cd 29c-13 is also equal to the
standard C-VI 18 (10) of the German Society for Fat Research (DGF),
(DGF, 2011). The AOCS Cd29 / ISO 18363 / DGF C-VI approaches are
referred to as indirect methods that are based on chemical
transesterification so as not to determine the various single fatty acid
ester derivatives of 3-MCPD and glycidol but to release these core
components in their free form. The resulting highly reactive free
glycidol is stabilised by transformation into monobromopropanediol
(AOCS Official Methods Cd 29a, b-13) or into induced 3-MCPD (AOCS
Official Method Cd29c-13, Part A). At present, food emulsifiers are not
within the scope of these methods. However, while the determination of
(2-)3-MCPD or (2-)3-MCPDE does not include a basic chemical conversion
of the molecular core structures, there does not seem to be any apparent
theoretical indication that the validated methods cannot be used for
neutral food emulsifiers in general. Nevertheless, there is no practical
confirmation of this assumption so far and it might remain questionable
if the limits of quantification are the same as with oils and fats. The
determination of GE is even more critical as it has been reported that
using the AOCS Official Method Cd 29a-13 for mono- and diacylglycerides
can lead to GE overestimations, (Zelinkova, 2017). Furthermore,
unpublished communications of analytical laboratories and their
customers suggest that the validated indirect methods may give
unreliable GE results, such as false positives, overestimations or
inconsistent values. In addition, internal research by the author has
shown instances where using AOCS Official Method Cd 29b-13 to analyse
food emulsifiers resulted in GE levels that increased or decreased over
time while 3-MCPD levels remained constant (data not shown). In theory,
these effects might be caused by GE decomposition during storage/sample
preparation or from the occurrence of other unstable GE-simulating
compounds. In particular, melting samples for aliquot preparation has
been shown in individual cases to significantly accelerate GE
decomposition (data not shown). Vice versa , artefact formation
during sample preparation cannot be excluded as a possible source of GE
overestimation or false-positive results. In order to overcome these
drawbacks in the determination of GE in food emulsifiers, a new approach
has been developed that is based on a modification of the AOCS Official
Method Cd 29b-13. Beside minor modifications in terms of sample weight
and solvents used, the main difference to the original method is the
quantitative differentiation of MBPD isomers which seem to be formed in
a relatively constant ratio when glycidol reacts with acidified sodium
bromide solution. MBPD artefact formation through other compounds as
glycidol seems to be related to the 3-MBPD isomer but not to the isomer
carrying bromine in the 2-position. Correspondingly, the ratio of both
isomers is determined and in case of increased ratio 3-MBPD : 2-MBPD the
quantification of glycidol is carried out via 2-MBPD.
The reported study was undertaken to evaluate the applicability of this
new analytical approach for the quantitative determination of 2- and
3-MCPD and their fatty acid esters as well as glycidyl fatty acid esters
in food emulsifiers that are prone to contain these analytes and to
provide validation data accordingly.
Materials and Methods
Collaborative Study
On request of the European Food Emulsifier Manufacturer´s Association
(EFEMA), which initiated this study, participation was restricted to SGS
Germany GmbH and EFEMA members. Some participants were new to the field
of processing contaminants trace analysis, others had a high level of
expertise. Every laboratory received ten food emulsifier test materials
as blind duplicates, giving a total of 20 samples. A study protocol was
supplied by e-mail and as a print-out to accompany the test samples. In
addition to general instructions and advice on sample storage
conditions, every protocol included the specific method to analyse 2-
and 3-monochloropropanediol esters and glycidyl esters. The protocol
also included a goods receipt form, an EXCEL-based raw data evaluation
template and a result submission sheet. No training sample nor internal
standard materials or solutions were supplied. Participants had to
prepare internal standard stock and working solutions according to the
method SOP.
All participants were asked to perform analyses on each of the 20 test
materials strictly following the method protocol and to send back the
results within four weeks of receiving the samples, together with any
comments regarding observed difficulties or anomalies and differences of
the applied procedure to the study protocol. However, due to laboratory
limitations caused by the coronavirus pandemic starting in early 2020, a
delay in result submission was accepted. For the same reason, results
were included even if deviations to the study protocol were identified.
This related, in particular, to the use of other chromatographic
injections systems such as programmable temperature vaporisers (PTV). If
ester hydrolysis rates were defined by the study protocol as potentially
insufficient, non-repeated measurements were also accepted. Furthermore,
it was decided not to insist on the use of isotope-labelled free D2-MCPD
as an internal standard. No information was supplied for the specific
identity of the test materials prior to the study. However, in
individual cases, it could have been possible in theory to identify
pairs of blind duplicates by their visual appearance. Participants were
advised to contact the Study Director if they experienced any problem or
had questions about the method or the protocol. The study protocol did
not contain any specific recommendation to exclude results below the
individual laboratories limits of quantification (LOQs) as the intention
was to determine the method applicability range based on the statistical
evaluation. Mean values were derived if multiple data sets were
submitted. This is not in line with recommendations for official method
validation, but it should reflect that some of the participating
laboratories were new to this field of analysis. Results continued to be
submitted between February and June 2020.
Six laboratories from four countries (3 x Denmark, 1 x France, 1 x
Germany, 1 x Ireland) participated in this study and reported results.
However, due to the statistical limitations that would result from such
a small number of participants, one laboratory reported three data sets
that were generated independently at different times (start, middle and
end of the testing period) by different lab technicians using separate
workplaces and separately prepared standard working solutions.
Furthermore, measurements of the final sample extracts were carried out
using three single GC-MS instruments and the raw data was evaluated by
another person so that the analysts carrying out sample preparation had
no information on the quantitative results. The following laboratories
participated in the ring trial:
DuPont Nutrition Biosciences ApS, Brabrand, Denmark
Dupont Nutrition & Health, Grindsted, Denmark
Kerry EMEA Global Technology & Innovation Centre, Kildare, Ireland
Oleon Innovation, Compiègne Cedex, France
Palsgaard A/S, Juelsminde, Denmark
SGS Germany GmbH, Hamburg, Germany
Sample scheme and
preparation
Prior to the trial, 40 food emulsifier samples supplied by EFEMA were
tested for specific analyte levels, stability and homogeneity. For this
purpose, all samples were analysed three to six times over a period of
six to eight weeks, using the analytical method that would be used by
the participants of the multi-laboratory trial. As this approach
required the preparation of duplicate samples, 6 to 12 data sets were
available per sample. First, the results were checked for trends of
obviously increasing or decreasing analyte levels. No such trend was
observed for 2- and 3-MCPD esters. However, individual samples indicated
decreasing GE levels. Homogeneity was assumed to be satisfactory if the
observed repeatability of analyte results did not significantly exceed
the range the testing laboratory would usually observe for common oils
and fats.
Out of the set of 40 food emulsifiers, ten test materials were selected
to fulfil the following criteria:
- In terms of possible MCPDE/GE occurrence and EFSA call for data, the
test samples should represent the most important (sub-)classes of food
emulsifiers.
- The selected test materials should cover commonly occurring and high
analyte contents and at levels that might become important under
current and future regulation of 3-MCPD and GE in edible oils and
fats. Furthermore, the study aimed to include samples with different
ratios of GE:MCPDE.
- Special emphasis was placed on including blank samples to check for
possible GE artefact formation during sample preparation or
measurement.
- Sample homogeneity and analyte stability of the test samples should
have been shown to be satisfactory such that no significant issues
were assumed to occur for the duration of the trial. The acceptance
criterion was set as a pre-testing RSDr value below 10 % for all
analytes present at a level ≥ 0.1 mg/kg.
It was decided not to include spiked blank samples to determine trueness
as a previous in-house validation of the method revealed satisfying
results when analysing certified vegetable oils from previous
proficiency tests. In addition, good recoveries were achieved when
spiking food emulsifier blank samples. However, spiking food emulsifiers
that contained glycidol, which showed significantly deviating GE results
when comparing the non-modified and the modified AOCS Official Method Cd
29b-13, resulted in satisfactory spiking recoveries for both methods.
These unexpected findings revealed the infeasibility of GE spiking for
the purpose of verifying method trueness in this case.
Table 1 presents the samples included in this method validation
study.
Amongst the selected samples, the focus was set very much on the EU food
additive E 471 due to the assumption that mono- and diacylglycerides
(MAG and DAG) are most likely to contain relevant levels of MCPDE and
GE. The intention was to include as many different subclasses of E 471
as possible. For this reason, half of the E 471 test materials were
distilled monoacylglycerides (DMG), the other half was mixtures of MAG
and DAG (MDG). Furthermore, both groups contained emulsifiers derived
from different classes of fatty acids, namely saturated, monounsaturated
and polyunsaturated fatty acids.
Based on the pre-testing results, the “Ink” sample was chosen as an E
471 material that contains no analyte or only a minor quantity
(< 0.1 mg/kg). This choice was made to verify that the applied
method does not carry the risk of significant analyte overestimations or
false-positive results by artefact formation during sample preparation
or measurement. “Scissors” represented DMG, containing low analyte
amounts below the maximum levels that are set for oils and fats by
recent EU regulation. With this sample, the glycidol content was
expected to range below or at the limit of quantification (LOQ) of the
method. Conversely, “Orange” served to reflect a DMG material with
3-MCPD and GE contents above regulatory limits. The test material
“Green” exhibited a large excess of 3-MCPD in comparison to glycidol,
while “Peach”, in contrast, contained significantly more glycidol than
3-MCPD. The “Monday” MDG sample was expected to represent comparably
low but quantifiable analyte levels below the regulatory limits while
“March” contained glycidol as a major analyte not too far above the 1
mg/kg level and had a negligible MCPD content. The sample named “Olive
Tree” contained minor amounts of MCPD and the highest glycidol level of
the selected test specimens. This choice was made to obtain information
on the analytical range of the method. Finally, the test materials
“Caravaggio” and “Berlioz” served as single representatives of two
classes of polyglycerol esters, namely E 475 and E 476. “Caravaggio”
did not contain any of the target analytes in significant amounts.
However, this sample showed positive but inconsistent GE findings when
applying the non-modified quantification approach of the official method
AOCS Cd 29b-13. Similar effects were observed with the test materials
“Scissors”, “Green” and “Monday”. During pre-testing, none of the
investigated E 476 polyglycerol polyricinoleate samples contained
significant amounts of any of the target analytes. In this regard,
“Caravaggio” and “Berlioz” served as blank samples to check that no
false-positive findings could occur when using the tested method.
To prepare aliquots, samples that were powders, pellets, flakes or
liquid were transferred directly into 200 mL screw cap plastic vessels
that were labelled with the corresponding anonymous sample code. Test
materials listed in Table 1 as “(solid)” were molten and
transferred in liquid form. A minimum amount of 25 g was obtained for
most of the sample aliquots. However, the quantities were lower in
certain cases due to a limited availability of the corresponding
original sample. Prior to sample filling the vessels were tested to
ensure they were free of glycidol and MCPD. All samples were stored
frozen (-25 °C +/- 3 °C) prior to distribution to the participants. Dry
ice was used as a cooling agent during transit.
Principle of the test
method
For the determination of free and bound 2-MCPD, free and bound 3-MCPD
and bound glycidol as free 2-MCPD, free 3-MCPD and free 3-MBPD
respectively, two aliquots (A and B) of the sample are spiked with
surrogate standards (D5 -2-MCPD,
D5 -3-MCPD,
D5 -glycidylester in assay A and
D5 -2-MCPD-1,3-diester,
D5 -3-MCPD-1,2-diester in assay B) and dissolved
in tertiary butyl methyl ether (t BME). Both assays are
processed in parallel. The addition of a diluted solution of sodium
hydroxide or sodium methoxide in methanol at low temperatures will
release free 2-MCPD, free 3-MCPD and free glycidol over a period of 8 h
to 16 h. This reaction is stopped by adding an excess of sodium bromide
in acidic solution. Under acidic conditions, free glycidol reacts with
inorganic bromide to form 3-MBPD and 2-MBPD. Undesired non-polar
compounds in the sample are removed by multiple extraction of the
aqueous phase with t BME and iso -hexane. The analytes,
together with the surrogate standards, are transferred into an organic
phase by multiple extraction of the aqueous phase with diethyl ether,
ethyl acetate or a mixture of both solvents. Derivatisation of the diol
analytes to form dioxaborolane derivatives is carried out in the organic
phase by reaction with phenylboronic acid (PBA). In order to remove
excess amounts of PBA, the sample extract is subsequently placed over a
small amount of anhydrous sodium sulfate and evaporated to dryness under
a stream of nitrogen before the soluble residue is finally resorbed iniso -octane and measured by GC/MS, applying separate thermal
ramping of the injector and the GC column.
The alkali-catalysed transesterification at low temperature minimises
the undesired transformation of 3-MCPD into glycidol that occurs to a
significant extent at room temperature. Nevertheless, in the event that
large amounts of 3-MCPD are present, even a minor transformation into
glycidol could artificially increase the glycidol results from assay A.
In order to achieve the correct glycidol results, assay B is used to
quantify the undesired 3-MCPD–glycidol transformation by determining
the amount of D5 -glycidol generated from
D5 -3-MCPD-diester in the sample preparation.
The corresponding transformation ratio is used to correct the glycidol
value derived from assay A. 3-MCPD has shown to be converted slightly
faster via glycidol into 3-MBPD than D5 -3-MCPD
via D5 -glycidol into
D5 -3-MBPD. Consequently, an isotopic factor has
to be determined empirically and considered for the quantitative
determination of the amount of glycidol that has been generated
accidentally from the non-labelled 3-MCPD by alkaline treatment in assay
A.
Quantification of the analytes is carried out by internal one-point
calibration using the corresponding D5 -esters
as surrogate standards. Peak integration is carried out for
corresponding mass-to-charge ratios. Therefore, no external calibration
is necessary. Likewise, no analyte recoveries have to be considered.
However, the cleavage rates of MCPD mono- and diesters might be
different and as only D5 -MCPD-diesters serve as
internal standards, ester cleavage should have proceeded on a large
scale. Therefore, the degree of ester cleavage is monitored by
calculating the differences in 3-MCPD results between assay A and B. In
order to avoid glycidol overestimations due to artefact formation from
bromide with reactive compounds that might be present in food
emulsifiers, quantification of glycidol via 2-MBPD is carried out for
control and correction purposes. The essential step in glycidol
quantification by this method is to exclude values resulting either from
3-MBPD or from 2-MBPD determination when one of the two results is
greater than 10 % of the mean value of both results. The exact
procedure as provided for the trial is given as Annex A . A few
editorial changes to the original protocol are included as a result of
participant input during the trial.
Statistical Evaluation
In order to include as much data in the statistical evaluation as
possible, it was decided not to exclude results that might have been
classified as being below the individual laboratories limit of
quantification. Instead, the methodological limits of quantification
were derived as method applicability levels by statistical analysis of
the submitted data.
Data sets were evaluated statistically following ISO 5725-2:1994 as far
as possible, (ISO, 1994). In this regard, numerical outlier testing was
performed by first applying Cochran´s test and then Grubbs´s test.
Cochran´s test helps to identify outliers and stragglers in terms of
repeatability. It identifies a significant deviation in duplicate sample
results from the average distribution of duplicate sample results. To
determine outliers and stragglers in terms of reproducibility, the
highest and lowest values were checked as single results as well as
result couples by using Grubbs´s test. Statistical evaluation included
the determination of mean values, absolute and relative repeatability
and reproducibility, reproducibility limits and HorRat values, (Horwitz,
2006). Method applicability for the quantitative determination of
ester-bound glycidol and free and ester-bound 2- & 3-MCPD was confirmed
to ensure that for a particular combination of analyte level and sample
type the corresponding relative repeatability (RSDR) did
not exceed 30 % and the corresponding HorRat R-value was within the
normal range 0.5 ≤ HorRat R ≤ 1.5 or in a higher but acceptable range
1.5 < HorRat R ≤ 2. In general, the exclusion of one outlier
or straggler within the dataset (n = 8) was accepted. If two out of
eight results had to be excluded, method applicability was confirmed
only if the corresponding HorRat R value was found to be in the normal
range 0.5 ≤ HorRat R ≤ 1.5. Using a similar procedure, HorRat r values
were derived and evaluated for repeatability. The corresponding data is
not shown as no results were excluded purely due to insufficient
reproducibility.
Results and Discussion
Tables 2.1 - 2.3 display summaries of the statistical
evaluation. The laboratory order corresponds to the time of result
submission.
In general, the applied method showed a satisfactory applicability for
the determination of glycidyl fatty acid esters and the sum of 3-MCPD
and 3-MCPD fatty acid esters in all tested food emulsifiers. Only the
samples that contained no analyte or non-quantifiable minor analyte
contents as evidenced during pre-testing caused an inacceptable number
of results to be excluded or the reproducibility relative standard
deviation to exceed the accepted maximum value of 30 % and/or the
corresponding HorRat R value to be larger than 2. These findings were
expected in terms of blank or semi-blank samples and might be considered
to be an indication that the method is generally unlikely to generate
false-positive results. This applies to the results for glycidol1) in the samples “Ink” and “Scissors”, to 2- &
3-MCPD 2) in sample “March” and to all analytes in
both polyglycerol samples “Caravaggio” and “Berlioz”. Considering
that a data set of eighty individual results was available for every
analyte, the number of excluded outliers or stragglers differed. Twelve
glycidol results were excluded, eight 3-MCPD results and four 2-MCPD
results respectively. Furthermore, the results indicate that method
applicability was achieved for much lower 2- and 3-MCPD contents in
comparison to glycidol where the LOQ was estimated to range between 0.10
mg/kg and 0.20 mg/kg. These differences seem to be understandable for
several reasons. Amongst the tested analytes, glycidol is the most
unstable one and, using the tested method, it undergoes a chemical
reaction that changes its core structure. The corresponding chemical
reaction of epoxy-ring opening and MBPD formation is certainly a source
of error as it might be influenced by the pH value, other nucleophiles
or the possible presence of other reactive components. Moreover,
glycidol reacts to produce the two isomers 2-MBPD and 3-MBPD, which are
both considered for quantitation. Thus, the signal intensities are
lowered correspondingly which results in greater measurement
uncertainty. A further impact on repeatability and reproducibility of
the glycidol determination can be assumed from the undesired conversion
of 3-MCPD into induced glycidol. This effect is measured and corrected
within the tested method, but these steps can also be considered with
evidence to expand the result variation.
Results for glycidol
At method applicability levels between 0.22 mg/kg and 10.35 mg/kg, the
HorRat values for glycidol ranged between 0.7 and 1.4, showing good
correlation to the expected normal range of 1 +/- 0.5. The corresponding
reproducibility relative standard deviations (RSDR)
showed values from 10.1 % to 29.0 % while the relative repeatability
(RSDr) ranged from 3.9 % to 24.9 % and did not exceed
the acceptable maximum level of 30 %. As expected, the
RSDR and RSDr values increase in general
with decreasing analyte concentrations. The HorRat value was only higher
than expected at 1.7 for “Olive Tree”, which contained 28.20 mg/kg
glycidol, the highest level among the test materials. Nevertheless, it
was still in the acceptable range < 2. At the same time, the
corresponding RSDR value of 16.1 % was not close to the
critical limit of 30 %. The reason for the HorRat value being higher
than commonly found for this concentration range, might be that the
analyte level exceeded the default one-point calibration maximum level
of 5 mg/kg almost by a factor of six. In addition, one lab submitted
results that were roughly 30 % lower than the average for all mean
glycidol contents > 1 mg/kg. This deviation was not big
enough to become an outlier, but it had a relatively high impact on the
method reproducibility due to the small number of participants. As the
repeatability relative standard deviation for glycidol in the “Olive
Tree” sample was quite low as expected (RSDr = 1.9 %),
this indicates that the relatively high deviation in results between the
participants might be caused by variations in the concentrations of the
calibration standards used. The second impact on the reproducibility of
glycidol-determination is given by the relative amount of 3-MCPD present
in parallel with glycidol. As pointed out in the previous paragraph, a
high ratio of 3-MCPD : glycidol should negatively impact the precision
of the glycidol determination. This effect becomes obvious when
comparing the results of the two samples “Green” and “Monday”, which
both showed almost the same amount of glycidol. “Green” contained
3-MCPD in a more than eightfold excess and consequently, repeatability
and reproducibility of the glycidol results were close to the exclusion
limits (RSDr = 24.9 %; RSDR = 29.0 %).
In comparison, “Monday”, which contained a 2.7-fold excess of 3-MCPD
compared with glycidol, had more satisfactory RSDr and
RSDR values for glycidol (RSDr = 4.8 %;
RSDR = 19.3 %). The best reproducibility for glycidol
results (RSDR = 10.1 %) was found for “March”, which
had an elevated glycidol level of 1.4 mg/kg and contained no significant
amounts of 3-MCPD. A data summary is displayed in Table 3 .
Results for 3- and
2-MCPD
For 3-MCPD method applicability levels above 0.06 mg/kg and for 2-MCPD
concentrations above 0.02 mg/kg, all data showed to be valid with no
RSDR value exceeding 30 % or any HorRat R value
> 2. The HorRat R values varied from 0.5 to 1 within a
range of 0.06 mg/kg to 2.86 mg/kg (3-MCPD) and from 0.5 to 0.8 within a
range 0.02 mg/kg to 0.72 mg/kg (2-MCPD). These values are slightly
better than expected but they are not in the range < 0.5 which
could be classified as being questionably good. This might be due to the
results being averaged, but also due to internal one-point calibration,
which might enhance reproducibility as it excludes any possible error
caused by external calibration. The RSDr and
RSDR values for 3-MCPD were between 1.5 % and 15.6 %
and between 7.8 % and 18.2 %, respectively. The RSDrresults for 2-MCPD were calculated to be 2.6 % to 20.8 % while the
RSDR values showed to be 10.0 % to 23.1 %. The study
revealed a surprisingly high sensitivity for 3-MCPD and an even greater
sensitivity for 2-MCPD. In fact, 2-MCPD occurs commonly in most refined
oils and fats in amounts that are approximately one third of the
corresponding 3-MCPD content. This ratio was also found in the food
emulsifier samples included in this trial and consequently, 2-MCPD was
always present in significantly smaller concentrations as 3-MCPD. At the
same time, the lowest number of result outliers was related to 2-MCPD.
Taking into account the lower 2-MCPD levels, the data can be interpreted
as an indication that repeatability and reproducibility were slightly
better than expected for this analyte in comparison to 3-MCPD. The
reason for this could be varying background levels of 3-MCPD, a common
issue due to possible 3-MCPD contamination of consumables such as
laboratory glassware, plastic devices, packaging materials etc. In
contrast, the same issue has rarely been reported for 2-MCPD. Background
contamination might have a negative impact on the method performance,
especially at these low concentration levels. However, no serious
evaluation can be made based on the present results as no specific
advice on how to deal with background levels was given in the method
SOP. Another impact on the method performance was indicated by the
comparably high RSDr and RSDR values for
3-MCPD in “Olive Tree”, which contained glycidol in an approximately
eightyfold excess to 3-MCPD and in a two hundred and fiftyfold excess to
2-MCPD. The reason for this effect, which occurred more significantly
with 3-MCPD than with 2-MCPD, might be the large amounts of glycidol
present. Another possibility is that traces of chloride being present in
the sample itself or existing as is a common impurity of the reagents or
consumables used during sample preparation, might react with GE or the
free glycidol released during interesterification to form small
quantities of induced 3-MCPD. Naturally, this effect is hardly
reproducible and would therefore impact the precision of the method.
Conclusions
A method for the determination of free and ester-bound 2- and 3-MCPD as
well as ester-bound glycidol in glycerol-based food emulsifiers was
successfully validated in a collaborative trial. The method that appears
as a modification of AOCS Official Method Cd 29b-13 (ISO 18363-2) showed
applicability for the determination of the aforementioned analytes in
food emulsifiers classified as E 471, E 475 and E 476. The limits of
quantification, expressed as method applicability are 0.22 mg/kg for
glycidol being present as GE, 0.06 mg/kg for total 3-MCPD and 0.02 mg/kg
for total 2-MCPD.
Acknowledgments
We thank the following collaborators for their participation:
Helle Lochmann & Annette Tjørnelund Jensen, DuPont Nutrition
Biosciences ApS, Brabrand, Denmark
Katja Kjelgaard Baumann & Johnny Wittendorf Madsen, Dupont Nutrition &
Health, Grindsted, Denmark
Adrian Burke & Julie Vichier, Kerry EMEA Global Technology &
Innovation Centre, Kildare, Ireland
Ramzi Sellik, Oleon Innovation, Compiègne Cedex, France
Mikkel Bach Skovsgaard & Britta Japp, Palsgaard A/S, Juelsminde,
Denmark
References