GERSTEL Application Note No.

191, 2017

Fully Automated Determination of 3-MCPD and Glycidol in Edible

Oils by GC/MS Based on the Commonly Used Methods
ISO 18363-1, AOCS Cd 29c-13, and DGF C-VI 18 (10)

Dominik Lucas, Andreas Hoffmann, Carlos Gil

GERSTEL GmbH & Co. KG, Eberhard-Gerstel-Platz 1, 45473 Mülheim an der Ruhr, Germany

KEYWORDS
3-MCPD, Glycidol, edible oil, lab automation, ISO converted into 3-MCPD. Following derivatization, the
18363-1, AOCS Cd 29c-13, DGF C-VI 18 (10) 3-MCPD amounts in both samples are determined by
GC/MS as Phenylboronic acid (PBA) esters. Assay
ABSTRACT B is used to determine the amount of 3-MCPD in the
3-MCPD and Glycidol and especially their fatty acid sample while assay A provides the combined amounts
esters are process contaminants that are formed, for of 3-MCPD and Glycidol. The amount of Glycidol is
example, when edible oils and fats are refined. At determined as the difference between the assay A and
least some of the above-mentioned substances are assay B results.
classified as potential human carcinogens, a fact which The work presented here involves an automated
has prompted the introduction of rules and regulations evaporation step as prescribed in the abovementioned
that specify tolerable daily intake values and maximum official methods. This ensures that for most matrices,
levels in edible oils. Different analytical methods are the required limits of detection can be reached using
available for the determination of these compounds. a single quadropole mass spectrometer (MSD). A
These methods follow two different strategies: further important aspect of the evaporation step is that
Direct determination or, more commonly, indirect it removes excess derivatization reagent, which could
determination of the contaminants. otherwise build up in the GC/MS system and influence
This AppNote describes a solution for fully system stability.
automated determination of 3-MCPD and Glycidol in It is demonstrated that method ISO 18363-1,
edible oils based on the reliable indirect method DGF which is equal to both method AOCS Cd 29c-13
C-VI 18 (10), similar to the ISO 18363-1 and AOCS and method DGF C-VI 18 (10), can be automated
Cd 29c-13 methods that are essentially identical. using the GERSTEL MPS. The obtained results show
The edible oil sample is divided into two parts good correlation with reference data. The excellent
(assays A and B). Both are saponified using a standard deviations achieved for the complete sample
Sodiumhydroxymethanol solution, but different preparation and analysis workflow speak in favor of
quenching methods are used. In assay A, free Glycidol automation.
is converted to 3-MCPD using acidic quenching
conditions in the presence of chloride. In contrast, for
assay B, the quenching reagent is an acidic chloride
free salt solution, in which free Glycidol is not
GERSTEL Application Note No. 191, 2017

INTRODUCTION
3-Monochloropropanediol (3-MCPD), 3-MCPD. The Glycidol esters are also quantitatively
2-Monochloropropanediol (2-MCPD) and Glycidol converted to free Glycidol in the human body. For
are contaminants that are present in a variety of food all these reasons, the indirect methods are currently
samples. These compounds are formed in fatty/salty being favored. The indirect methods basically all work
foodstuffs whenever high temperatures are applied according to the same principle. All esters are split into
during processing. As an example, significant amounts free MCPD and Glycidol, which are derivatized and
of MCPD- and Glycidol fatty acid esters can be determined by GC/MS.
produced in the edible oil refining process, which can In this AppNote a Sample Prep Solution based on
be divided into distinct steps as outlined in figure 1. the GERSTEL MultiPurpose Sampler is presented
that provides completely automated determination
Degumming
Deodorization
of 3-MCPD and Glycidol in edible oils based on
Bleaching
Dewaxing
the DGF C-VI 18 (10) method, which again is very
similar to the ISO 18363-1 and AOCS Cd 29c-13
methods. All of the mentioned methods are based on
differential determination of Glycidol and 3-MCPD.
Figure 1. Refining process for production of edible The analysis is divided into two assays (A and B).
oils. The quenching reaction after the saponification step
In refining processes used for edible oil production, the is the main difference between the two. In assay A,
final deodorization step is particularly critical and must the saponification reaction is stopped by adding an
be carefully controlled in order to avoid the formation acidic sodium chloride solution. Under these reaction
of significant amounts of MCPD and Glycidol. The conditions, free Glycidol is converted to 3-MCPD and
deodorization step is performed to remove unwanted the combined amounts of 3-MCPD and Glycidol are
odors and bittering agents from the oil. Varying the determined as 3-MCPD. In assay B, the quenching
applied temperature during the deodorization process reagent is a chlorine free acidic salt solution. In this
often merely changes the ratio of MCPD ester to case free Glycidol is not converted to 3-MCPD. The
Glycidol ester formed, but does not eliminate the 3-MCPD amount in both samples is determined by
formation of these compounds. GC/MS after derivatization with Phenylboronic acid.
While toxicological studies on rats have shown that The amount of Glycidol in the edible oil sample is
3-MCPD causes tumors, the effect of 2-MCPD is less determined as the difference between the 3-MCPD
well known. 3-MCPD is labeled as a possible human amounts found in assays A and B, appropriately
carcinogen. In contrast Glycidol has already been corrected using the conversion factor.
classified as a probable human carcinogen.
For the determination of 3-MCPD, Glycidol and EXPERIMENTAL
their esters, several different methods have been Instrumentation. The automated sample preparation
published. The two main approaches to determining was performed on a GERSTEL MultiPurpose Sampler
the esters are the direct method using LC/MS and the (MPS robotic, DualHead version). One key module
indirect methods using GC/MS. The direct method of the solution is the GERSTEL QuickMix, which
has the disadvantage of having to deal with complex performs the vigorous shaking required during
chemical compositions of the esters formed: The fatty the liquid/liquid extraction steps. Furthermore the
acid distribution and the formation of both monoesters method ISO 18363-1, which is equal to the AOCS
and diesters result in a wide variety of MCPD- and Cd 29c-13 method and the DGF C-VI 18 (10) method,
Glycidylesters being formed. This means that a lot of requires evaporative concentration of the samples
individual substances have to be quantified in order during derivatization. This step is automated using the
to determine the total amount of the contaminants. GERSTEL mVAP and provides the significant added
The situation is further complicated by the fact benefit of removing excess derivatization reagent,
that quantification standards are unavailable. When which could otherwise build up in the GC/MS system
looking at the toxicologically relevant part, it should and influence system stability. The sample was injected
be considered that during the intestinal resorption via a Cooled Injection System CIS 4 (GERSTEL) and
process, 3-MCPD-esters are split completely into free transferred to the column (Restek Rxi-17 Sil ms, 30 m,

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 GERSTEL Application Note No. 191, 2017

Figure 2. GERSTEL MPS Workstation used for automated sample preparation of edible oils prior to GC/MS
determination of 3-MCPD and Glycidol.

di = 0.25 mm, df = 0.25 μm) using programmed step is repeated twice to remove matrix. Free 3-MCPD
temperature vaporization. For separation and detection is extracted by 600 μL MTBE/EtAc (3/2 v/v). The
a 7890 GC coupled to a 5977 MSD was used (both extract is collected in a new 2 mL vial pre- filled with
Agilent Technologies). Sodium sulfate as drying agent. After adding 30 μL of
Phenylboronic acid the sample is evaporated to dryness
Materials. 3-MCPD-d5-1,2-bis-palmitoylester, in the GERSTEL mVap module. The Phenylboronic
3-MCPD-1,2-bis-palmitoylester, 3-MCPD, Glycidyl acid derivates are redissolved in Isooctane and
stearate, Sodium hydroxide in Methanol- Solution, transferred to a new vial with μ-vial insert ready for
Acetic NaBr-Solution (600 g/L), Acetic NaCl-Solution injection. The fact that Phenylboronic acid is not
(600 g/L), Phenylboronic acid, MTBE/EtAc-Solution very soluble in Isooctane helps reduce the amount of
(3/2 v/v), Hexane, Isooctane, Water, Acetone, Toluene derivatization agent injected. The evaporation step is
therefore used both to increase the sensitivity of the
Sample Preparation. One analysis is comprised of analysis and also to remove excess Phenylboronic acid
two assays (A and B). For each assay, 100 mg of oil in order to protect the MSD.
are weighed in 4 mL screw cap vials and placed onto
the MPS. After adding 250 μL of MTBE and 100 μL Analysis conditions.
ISTD-solution, the sample is shaken vigorously in the MPS: 3 μL injection volume
GERSTEL QuickMix module. For the saponification PTV: baffled liner, deactivated
of MCPD- and Glycidyl esters, 350 μL of a MeOH/ solvent vent
NaOH solution is added. The sample is shaken slowly 40°C (0 min); 12°C/s; 300°C (5 min)
for 10 minutes. The assay A reaction is quenched with Column: 30 m Rxi-17 sil ms (Restek)
600 μL of acidic NaCl Solution while a chlorine di = 0.25 mm df = 0.25 μm
free NaBr solution is used for assay B to avoid the Pneumatics: He, constant flow = 1 mL/min
formation of additional MCPD from Glycidol. Oven: 50°C (2 min); 10°C/min; 200°C (0 min)
The following preparation steps are similar for 20°C/min; 300°C (5 min)
both assays. After the addition of 600 μL Hexane, MSD: Selected ion monitoring SIM
the sample is vigorously shaken and incubated for 10 3-MCPD: 196/198/147 amu
minutes. The sample is again shaken vigorously and 3-MCPD-d5: 201/203/150 amu
the organic Hexane layer is dispensed to waste. This

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GERSTEL Application Note No. 191, 2017

RESULTS AND DISCUSSION

Figure 3. SIM-Chromatogram m/z 198 : Top: Virgin olive oil used as blank oil. Middle: Edible oil sample
assay B (3-MCPD). Bottom: Edible oil sample assay A (3-MCPD + Glycidol).
The first step for determination of 3-MCPD and The linearity of the method was verified by analyzing
Glycidol based on the indirect ISO 18363-1, AOCS Cd virgin olive oil spiked at five different levels. This was
29c-13, or DGF C-VI 18 (10) methods is to evaluate performed for both assays. In figure 5, the excellent
the efficiency of the conversion from Glycidol to linearity (R2> 0.9998) achieved for both assays from
3-MCPD following the method used for Assay A. 0.12 – 1.9 mg/kg is shown.
Figure 4 shows the amount of 3-MCPD formed as
a function of the amount of Glycidol (in the form of
Glycidyl stearate) in a spiked blank oil at five different
levels. A linear regression of the type y = mx + b is
performed, the reciprocal slope (1 / m) provides the
conversion factor (t).

Figure 4. The amount of 3-MCPD formed as a Figure 5. Linearity study for 3-MCPD assay B (top)
function of the amount of Glycidol at five different and Glycidyl assay A (bottom), 0.12-1.9 mg/kg each.
levels. A linear regression of the type y = mx + b is
performed, the reciprocal value of the slope (1 / m) Three different edible vegetable oil samples were
provides the conversion factor (t). analyzed and the results compared with the provided
reference values. These kind of edible oils do not
produce large amounts of 3-MCPD- and Glycidyl
esters. Therefore they are in the low level range for
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 GERSTEL Application Note No. 191, 2017

CONCLUSIONS
3-MCPD and Glycidol contamination. Table 1 shows In this work, we have shown that method ISO 18363-1
the results from assay B, listing the amount of 3-MCPD can be automated using the GERSTEL MPS and that
determined in three different edible oil samples as well the results obtained correlate well with reference data.
as the reference values. This method is similar to two other frequently used
Table 1. 3-MCPD amount found in three different methods: AOCS Cd 29c-13 and DGS C-VI 18 (10).
edible oils in mg/kg. The excellent relative standard deviations achieved for
Amount [mg/kg]
the complete process including GC/MS analysis speak
3-MCPD in favor of the presented automation solution.
Reference Automated
The work presented here involves an automated
Oil 1 0.77 0.68
evaporation step as prescribed in the abovementioned
Oil 2 0.68 0.63 official methods. This ensures that for most matrices,
Oil 3 0.27 0.29 the required limits of detection can be reached using
a single quadropole mass spectrometer (MSD). A
For a given edible oil sample, the difference between
further important aspect of the evaporation step is that
the results for assays A and B multiplied by the
it removes excess derivatization reagent, which could
previously determined conversion factor is used to
otherwise build up in the GC/MS system and influence
calculate the amount of Glycidol in the sample. In table
system stability.
2, the amounts obtained using this method are listed
along with reference values.
OUTLOOK
Table 2. Glycidol amount found in three different The described automation steps are not limited to the
edible oils in mg/kg. presented method. Such methods have alredy been
Amount [mg/kg] tested for derivatization methods like the recently
Glycidol Reference Automated presented 3 in 1 approach, and can be adapted for
Oil 1 0.14 0.12 that method with similar performance. The presented
Oil 2 0.44 0.31 method has the advantage of being able to analyze
Oil 3 0.11 0.06 a sample for Glycidol, 3-Monochloropropanediol
(3-MCPD) and additionally 2-Monochloropropanediol
To demonstrate the good repeatability of the automated
(2-MCPD), all in a single run.
sample preparation method, five samples of the
In addition to extracting and determining MCPD
same edible oil were analyzed undergoing individual
and Glycidol esters, the described automation platform
sample preparation and analysis. Table 3 shows the
can also extract and determine PAHs from edible oils
repeatability based on the entire sample preparation
using automated solid phase extraction combined with
procedure and the subsequent GC/MS analysis.
GC/MS determination.
Table 3. Repeatability for 3-MCPD and Glycidol
(n=5 samples). LITERATURE
Amount [mg/kg] [1] h t t p : / / w w w. b f r. b u n d . d e / d e / f r a g e n _ u n d _
# 3-MCPD Glycidol antworten_zur_kontamination_von_
1 0.72 0.34
lebensmitteln_mit_3_mcpd___2_mcpd__und_
glycidyl_fettsaeureestern-10538.html
2 0.63 0.34
[2] DGF C-VI 18 (10)
3 0.66 0.31
[3] AOCS Official Method Cd 29b-13
4 0.69 0.32 [4] ISO 18363-1:2015 Animal and vegetable fats
5 0.68 0.37 and oils -- Determination of fatty-acid-bound
Mean 0.68 0.33 chloropropanediols (MCPDs) and glycidol by
SD 0.03 0.02 GC/MS -- Part 1: Method using fast alkaline
RSD % 5.00 6.44 transesterification and measurement for 3-MCPD
and differential measurement for glycidol
For 3-MCPD, a relative standard deviation of 5 %
was calculated. The relative standard deviation for the
amount of Glycidol is 6.44 %.
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