Analytical Services: Service Tutorial
Analytical Services: Service Tutorial
Analytical Services: Service Tutorial
Service Tutorial
No.: P-069 Rev.: Draft Class.: internal Page 1 of 19
In refining processes used for edible oil production, the final deodorization step is particularly critical and
must be carefully controlled in order to avoid the formation of significant amounts of MCPD and Glycidol.
The deodorization step is performed to remove unwanted odors and bittering agents from the oil. Varying
the applied temperature during the deodorization process often merely changes the ratio of MCPD ester to
Glycidol ester formed, but does not eliminate the formation of these compounds.
While toxicological studies on rats have shown that 3-MCPD causes tumors, the effect of 2-MCPD is less
well known. 3-MCPD is labeled as a possible human carcinogen. In contrast Glycidol has already been
classified as a probable human carcinogen.
For the determination of 3-MCPD, Glycidol and their esters, several different methods have been
published. The two main approaches to determining the esters are the direct method using LC/MS and the
indirect methods using GC/MS.
The direct method has the disadvantage of having to deal with complex chemical compositions of the
esters formed: The fatty acid distribution and the formation of both monoesters and diesters result in a
wide variety of MCPD- and Glycidylesters being formed. This means that a lot of individual substances
have to be quantified in order to determine the total amount of the contaminants. The situation is further
complicated by the fact that quantification standards are unavailable.
When looking at the toxicologically relevant part, it should be considered that during the intestinal
resorption process, 3-MCPD-esters are split completely into free 3-MCPD. The Glycidol esters are also
quantitatively converted to free Glycidol in the human body. For all these reasons, the indirect methods
are currently being favored. The indirect methods basically all work according to the same principle. All
esters are split into free MCPD and Glycidol, which are derivatized and determined by GC/MS.
"...it is recommended to use the American Oil Chemists' Society standard methods"
AOCS Official Methods Cd 29a-13, Cd 29b-13 and Cd 29c-13 are three different methods that have been
found through international collaborative studies to produce statistically the same results, and therefore
are considered equivalent for use in the determination of 2- and 3-MCPD fatty acid esters and glycidyl
fatty esters in edible oils and fats.
Analytical Services
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No.: P-069 Rev.: Draft Class.: internal Page 3 of 19
The analysis is divided into two assays (A and B). The quenching reaction after the saponification step is
the main difference between the two. In assay A, the saponification reaction is stopped by adding an
acidic sodium chloride solution. Under these reaction conditions, free Glycidol is converted to 3-MCPD
and the combined amounts of 3-MCPD and Glycidol are determined as 3-MCPD. In assay B, the quenching
reagent is a chlorine free acidic salt solution. In this case free Glycidol is not converted to 3-MCPD. The 3-
MCPD amount in both samples is determined by GC/MS after derivatization with Phenylboronic acid. The
amount of Glycidol in the edible oil sample is determined as the difference between the 3-MCPD amounts
found in assays A and B, appropriately corrected using a conversion factor ("Transformation constant").
3. Apparatus
There are two versions available: a stand-alone workstation and an online system for mount on an Agilent
7890 GC.
The components which are unique for the stand-alone workstation are color-coded in green, for the
online system are color-coded in blue.
Agitator [100100-006-00]
6 x 4 mL inserts for Agitator [014856-006-00]
2 x Trayholder [100100-014-00]
4 x VT24-4 [020008-000-00]
2 x VT40g [015587-000-00]
4. Chemicals
All chemicals are not included in the scope of delivery.
4.1 Reagents
Reagent Intended use CAS-No.
Methanol Methanolic sodium hydroxide solution 67-56-1
Acetone Syringe wash 67-64-1
Toluene Solvent for standards 108-88-3
MTBE / Ethyl acetate solution (Extraction)
MTBE (2-Methoxy-2-methylpropane) 1634-04-4
Phenylboronic acid solution (Derivatization)
Ethyl acetate MTBE / Ethyl acetate solution (Extraction) 141-78-6
n-Hexane Extraction solvent 110-54-3
Isooctane (Trimethylpentane) Injection solution 540-84-1
Sodium bromide solution
Distilled water Sodium chloride solution 7732-18-5
Syringe wash
Sodium hydroxide
1310-73-2
or
Methanolic sodium hydroxide solution
Sodium methoxide solution
124-41-4
(recommended)
Sodium chloride Sodium chloride solution 7647-14-5
Sodium bromide (chlorine-free !!!) Sodium bromide solution 7647-15-6
Phenylboronic acid Phenylboronic acid solution (Derivatization) 98-80-6
Sodium bromide solution
Phosphoric acid, conc. 7664-38-2
Sodium chloride solution
If not otherwise specified, all reagents shall have at least p.a. quality
4.2 Solutions
Solution Intended use
20 g/L sodium hydroxide in methanol. Pestle the sodium hydroxide
pellets before preparing the solution
Methanolic sodium hydroxide solution
or
Dilute sodium methoxide solution to 25 g/L
200 g/L sodium chloride in water + approx. 3 mL conc. phosphoric
acid. pH has to be adjusted, so that a mixture of 600 µL sodium
NaCl solution
chloride solution with 200 µL methanolic sodium hydroxide solution
results in a pH of 1-3
600 g/L sodium bromide in water + approx. 3 mL conc. phosphoric
acid. pH has to be adjusted, so that a mixture of 600 µL sodium
NaBr solution
bromide solution with 200 µL methanolic sodium hydroxide solution
results in a pH of 1-3
MTBE / Ethyl acetate solution 3 / 2 (v/v)
Phenylboronic acid solution 24 mg in 10 mL MTBE
3-MCPD-d5-1,2-bis-palmitoyl ester Standard solution in toluene (recommended: dilute to 5 µg/mL)
Analytical Services
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No.: P-069 Rev.: Draft Class.: internal Page 9 of 19
4.3 Standards
Standards Intended use CAS-No.
3-MCPD-d5-1,2-bis-palmitoyl ester Quantification standard 1185057-55-9
3-MCPD-1,2-bis-palmitoyl ester For method development and validation 51930-97-3
Glycidyl stearate For method development and validation 7460-84-6
3-MCPD For method development and validation 96-24-2
5. Installation
5.1 Stand-alone workstation
Install USM with 10 µL syringe in left head, add 6 mm Gripper. Install PSM with 2500 µL syringe in right
head, add 10 mL magnet.
5.5 Teaching
Teaching of all modules. Please note:
mVAP: only left tower
mVAP: teach all 6 Head Slots
QuickMix: three-point teaching
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No.: P-069 Rev.: Draft Class.: internal Page 11 of 19
6. Adjustments
6.1 Checking of NaCl- and NaBr-solutions
To ensure that a mixture of 600 µL sodium chloride / sodium bromide solution with 200 µL methanolic
sodium hydroxide solution results in a pH of 1-3, follow this procedure:
Place 2 empty vials in positions 1 and 2 of the QuickMix
Start PrepSequence “3-MCPDc_pH_Value_x.x.x.prp” with vial range 1-1 using PrepMaster
Wait for sequence to be finished
Measure pH value in the resulting solutions in vials 1 and 2 using pH indicator paper or a pH meter
o Vial 1: In case pH is > 1-3 add phosphoric acid to the sodium chloride solution and start
again
o Vial 2: In case pH is > 1-3 add phosphoric acid to the sodium bromide solution and start
again
7. Analysis
7.1 Analysis conditions inlet
Model CIS 4
Liner Type Glass liner CIS4, baffled, non deactivated [013882-010-00]
Temps Initial 60°C (0 min)
Rate 1 12°C/min to 280°C (6 min)
Rate 2 -
7.4.1 Datafiles
Load datafiles:
Go to “File” --> “New Batch”
Choose directory where datafiles were stored
Import datafiles
7.4.2 Method
Load quantification method:
Go to “Method” --> “Open” --> “Open Method from Existing File…”
Choose the method file provided (please note version-no. !)