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3-MCPD SamplePrep Solution

based on AOCS Cd 29c-13
1. Introduction
1.3-Monochloropropanediol (3-MCPD), 2-Monochloropropanediol (2-MCPD) and Glycidol are contaminants
that are present in a variety of food samples. These compounds are formed in fatty/salty foodstuffs
whenever high temperatures are applied during processing. As an example, significant amounts of MCPD-
and Glycidol fatty acid esters can be produced in the edible oil refining process, which can be divided into
distinct steps:

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.

International Agency for Cancer Research (IACR)

-> Group 2B: possibly carcinogen to humans
Tolerable daily intake: 2 µg/kg body weight

Only limited data yet, but some (partially

unpublished) data suggest high toxicity level

International Agency for Cancer Research (IACR)

-> Group 2A: probably carcinogen to humans
Tolerable daily intake: as low as reasonably
achievable
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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.

1.1 Overview of indirect methods

Method Comments
Slow acid transesterification at 40°C (16 h), liquid-liquid extraction, derivatization
AOCS Cd 29a-13
with phenylboronic acid.
(Unilever method)
Measures 2-MCPD, 3-MCPD and Glycidol in one assay.
Slow alkaline transesterification at -25°C (16 h), liquid-liquid extraction,
AOCS Cd 29b-13
derivatization with phenylboronic acid.
(SGS 3 in 1)
Measures 2-MCPD, 3-MCPD and Glycidol in two assays.
Fast alkaline transesterification at RT, liquid-liquid extraction, derivatization with
phenylboronic acid.
AOCS Cd 29c-13
Measures 2-MCPD and 3-MCPD in two assays. Glycidol calculated from difference
between both assays.
Fast alkaline transesterification at 5°C, liquid-liquid extraction, derivatization with
Zwagerman /
phenylboronic acid.
Overman method
Measures 2-MCPD, 3-MCPD and Glycidol in one assay.

EU-Commission Recommendation 2014/661/EU

"...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.
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2. 3-MCPD SamplePrep Solution

based on AOCS Cd 29c-13, DGF C-VI 18 (10), ISO 18363-1
This Sample Prep Solution, based on the GERSTEL MultiPurpose Sampler, provides completely automated
determination of 3-MCPD, 2-MCPD and Glycidol in edible oils based on the AOCS Cd 29c-13 method,
which again is very similar to the ISO 18363-1 and DGF C-VI 18 (10) methods. All of the mentioned
methods are based on differential determination of Glycidol and 3-MCPD.

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").

2.1 Overview of chemical reactions

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2.2 Transformation constant T for Glycidol

The transformation constant (T) describes the efficiency of the conversion from Glycidol to 3-MCPD
following the method used for Assay A.
The calculated amount of Glycidol (x) is plotted against the measured amount of 3- MCPD (y). A linear
regression of the type y = mx + b is performed, the reciprocal slope (1 / m) provides the conversion factor
(t). Multiplying the conversion factor (t) with 0.67 (theoretical value of the molar mass ratio of Glycidol
and 3-MCPD) results in the transformation constant (T).
The following example 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.

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.

3.1 Not included in scope of delivery

 Analytical balance (+/- 0.0001 g)
 Universal pH-Indicator or pH meter
 GC/MS system with CIS
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3.2 Included in scope of delivery

MPS robotic / roboticpro dualhead XXL 160 cm [100060-160-NS]
Firmware rev. 2.4.18019.1130 or equivalent
Stand-alone supports 429 mm [100102-097-00]
or
MPS robotic / robotic dualhead XXXL 200 cm [100061-200-NS]
Firmware rev. 2.4.18019.1130 or equivalent
GC mounting kit e.g. Agilent 7890 [100102-090-00]
Adjustable support [100102-990-00]
Bracing angle right [021133-101-00]

MAESTRO Software [014872-001-00]

Vers. 1.5.3.39

GERSTEL mVAP station with vacuum pump and condenser [021754-100-NS]

(MOXA box in case there is no RS-232 on the PC) [014967-500-NS]
4 mL inserts for mVap [021754-004-00]

Gripper (6 mm) for mVAP [019597-003-00]

GERSTEL Quick MIX - option for GERSTEL MPS robotic [020672-000-NS]

6 x 4 mL vial option for GERSTEL QuickMix [022036-104-00]

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]

3 x Tray set for 3 x 180 mL [020296-000-00]

9 x 180 mL solvent vials [020026-000-00]

1 x Standard wash station [100100-034-00]

1 x High flow fast wash [100100-296-00]

Holder for 1 L solvent bottles for fast-wash [021716-000-00]
Waste canister with safety cap [015503-000-00]
Silicon tube ID 6 mm x 1,5 mm [015994-002-00]
2 x Safety cap Gl 45 (to replace HF fast-wash caps) [015504-004-00]

1 x GERSTEL USM [100100-101-00]

1 x Gerstel PSM [100100-105-00]

2 x 10 µL syringe 10-Gn-Pr-T [100111-018-00]

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Replacement needles for 10 µL syringe [100111-505-00]

2 x 2500 µL syringe 2.5M-Gw-Er-T [100112-001-00]

Replacement needles for 2500 µL syringe [100112-500-00]

Screw vials 4 mL with round bottom [093640-113-00]

Screw caps for 4 mL vial magnetic [093640-103-00]

Screw vials 1.5 mL [093640-046-00]

Screw caps for 1.5 mL vial [093640-075-00]

Restek Rxi-17 Sil MS, 30 m x 0.25 mm x 0.25 µm [000090-395-00]

3.3 Consumables and replacements

10 µL syringe 10-Gn-Pr-T [100111-018-00]
Replacement needles for 10 µL syringe [100111-505-00]
Replacement plunger for 10 µL syringe [100111-800-00]

2500 µL syringe 2.5M-Gw-Er-T [100112-001-00]

Replacement needles for 2500 µL syringe [100112-500-00]
Replacement plunger for 2500 µL syringe [100112-801-00]

Screw vials 4 mL with round bottom [093640-113-00]

Screw caps for 4 mL vial magnetic [093640-103-00]

Screw vials 1.5 mL [093640-046-00]

Screw caps for 1.5 mL vial [093640-075-00]

PE push on caps for 180 mL solvent vials [093640-097-00]

Charcoal Filter 48g [015504-032-00]

PP fittings black for ID 3.2 mm tubing 5 each [015504-024-00]
Safety cap valve incl. Filter for up to 150 mL/min flow [015504-019-00]

Tubing for mVAP transport adapter, Package of 6 [018444-006-00]

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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)
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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.

5.2 Online system

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.
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5.3 Tray positions

The positions of the trays are identical for both versions. Install all modules accordingly.

5.4 MPS Initialization

Check firmware version. Import provided templates:
 “WashVialType.180SRg.xml”
 “VialType.FourCVMagc.xml”:
o Corrects needle penetration for Rack Item 4CVMagnetic to 44 mm and makes it editable
o for 4 mL vials for QuickMix, mVAP and Trays
 “RackTypeRectangle.VT24gc.xml”:
o Corrects position of needle penetration check
Changes on MPS Handheld:
 Set PreRinseLiner to 0 for each Wash of HF-FastWash
o Since changes to Rinse Duration are ignored (Bug in current Firmware) PreRinseLiner was
deactivated to save solvent
Create 9 Vials (Wash Vial) “Vial1” to “Vial9” for the reservoirs using “180SRg”,
Create a new Wash Module (Standard Wash Module) giving “Pos1@Standard Wash 1” to
“Pos4@Standard Wash 1” for positions 1 to 4 of the standard wash.

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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5.6 MAESTRO Software

Check MAESTRO version. Install MAESTRO as Instrument 1. Add MPS tuning to Maestro.ini:
[MPS Tuning]
Minimum Syringe Volume=0.05
Add the following extensions:
 PrepMaster
 mVapdivided
 StopAfterMixingTime
Configure the following plugins:
 quickMix
 Vacuubrand PC 3001
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Copy provided prp-files into Maestro PrepSequence folder:

 3-MCPDc_online_with_melting_import_x.x.x.prp
 3-MCPDc_standalone_with_melting_import_x.x.x.prp
 3-MCPDc_standalone_without_melting_import_x.x.x.prp
 3-MCPDc_pH_Value_x.x.x.prp
 3-MCPDc_Phaseseparation_x.x.x.prp
Install virtual trays:
 Copy the provided „VirtualTrays_1.ini” into folder:
o C: -> ProgramData -> Gerstel -> Maestro -> MasCfg
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5.7 Assignment of reagents and solutions to vials

Vial Reagent / Solution
Vial 1 Methanolic sodium hydroxide solution
Vial 2 NaCl solution
Vial 3 NaBr solution
Vial 4 n-Hexane
Vial 5 not used
Vial 6 not used
Vial 7 Isooctane
Vial 8 MTBE / Ethyl acetate solution
Vial 9 MTBE
Pos 1 Isooctane (Online version: wash of injection syringe)
Pos 2 Isooctane (Online version: wash of injection syringe)
Pos 3 Phenylboronic acid solution
Pos 4 3-MCPD-d5-1,2-bis-palmitoyl ester
HF Fast wash 1 Distilled water
HF Fast wash 2 Acetone
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5.8 HF Fast Wash

Adjust the flow rate according to the CTC Fast Wash Manual. Setpoint should be 200 µL/s.

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

6.2 Checking vial penetration depth at phase boundary

To ensure that the vial penetration depth is set to max. 1 mm above the phase boundary, follow this
procedure:
 Fill two vials with 100 mg each of an edible oil (e.g. customer sample)
 Place vial 1 in position 1 of Raw “A”
 Place vial 2 in position 1 of Raw “B”
 Optional: replace standard solution in Pos 4 with toluene. A standard is not required for this check
 Start PrepSequence “3-MCPDc_Phaseseparation_x.x.x.prp” with vial range 1-1 using PrepMaster
 Wait for sequence to be finished
 Change rack 6 of the VT-40g trays to VT-24gc using the FourCVMagc rack item
 Manually place one of the finished vials to position 20 of rack 6 (VT-24gc)
 Check vial penetration at position 20:
o Use MPS handheld wheel to move needle tip to a position max. 1 mm above the phase
boundary
o Read value on the handheld display. The reading should be 39 mm
 In case of values <> 39 mm, the old value has to be replaced with the new one in one (or all) of
these PrepSequence files:
o 3-MCPDc_online_with_melting_import_x.x.x.prp
o 3-MCPDc_standalone_with_melting_import_x.x.x.prp
o 3-MCPDc_standalone_without_melting_import_x.x.x.prp
 The parameter “Vial Penetration=39.00” can be found in these ADD-methods:
o [Remove upper Layer]
o [Remove upper Layer 2nd]
o [MCPD Transfer]
 Change back rack 6 to VT-40g using the FourCVMagc rack item
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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 -

7.2 Analysis conditions GC

GC Model Agilent 7890B
Column Model Rxi-17Sil ms (Restek), 30 m x 0.25 mm x 0.25 µm
Mode Ramp flow:
1.2 mL/min (15 min)
10 mL/min per min to 3 mL/min
Pneumatics Mode Splitless
Pressure 71.844 kPa
Purge Flow to Split Vent 45 mL/min at 2 min
Gas Saver 35 mL/min at 5 min
Temps Initial 60°C (1 min)
Rate 1 20°C/min to 160°C (0 min)
Rate 2 10°C/min to 220°C (0 min)
Rate 3 25°C/min to 320°C (9 min)

7.3 Analysis conditions QQQ

MS Model Agilent 7010B
Source Type High Efficiency Source
Ionization EI
Electron Energy Tune Setting (70 eV)
Solvent Delay 6 min
Det. Setting Gain 20
Flows Collision Gas N2, 1.5 mL/min
Quench Gas He, 2.25 mL/min
Temps Source 230°C
Quad 1 150°C
Quad 2 150°C
Transfer 320°C
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7.3.1 Quantifier and qualifier ions for QQQ

7.4 Post run analysis

Open MassHunter QQQ Quantitative Analysis.

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. !)

7.4.3 Internal standard – globals setup

Assign relative ISTD (= deuterated internal standard for the AOCS method):
 Go to “Method” --> “Method Setup Tasks” --> “Globals Setup”
 Check the “Relative ISTD” mark
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7.4.4 Internal standard – ISTD setup

Setup ISTD:
 Go to “Method” --> “Method Setup Tasks” --> “ISTD Setup”
 Set ISTD Conc.

Exemplary calculation of ISTD:

 ISTD original concentration (e.g. from Toronto Chemicals) is 1000 µg/mL
 Recommended concentration for use in method is 5 ng/µL
(dilute original conc. 1:200 by using 50 µL original conc. in 10 mL Toluene)
 The MPS adds 150 µL of this ISTD Mix to each sample
 Conversion factor of 3-MCPD-d5-ester to 3-MCPD-d5 is 5.374
(MW 3-MCPD-d5-ester / MW 3-MCPD-d5)
 Amount of ISTD in each sample is:
150 µL x 5 ng/µL / 5.374 = 140 ng = 0.14 µg

7.4.5 Exemplary result window

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7.4.6 Exemplary sequence table

Two methods are required for preparing the sequence table for a single sample:
 The first method (here: “AOCS29c Test”):
o Uses the sample in “Vial n” from tray “Raw Sample A”, performs the sample prep steps
and stores the prepared sample in “Vial n” in tray “Final Sample A”
o Uses the sample in “Vial n” from tray “Raw Sample B”, performs the sample prep steps
and stores the prepared sample in “Vial n” in tray “Final Sample B”
o Injects from “Vial n”, tray “Final Sample A” and starts the GC/MS/MS run
 The second method (here: “AOCS29c Test_2”):
o Injects from “Vial n”, tray “Final Sample B” and starts the GC/MS/MS run
For the next sample repeat the two lines using “n+1”
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7.4.7 Exemplary chromatogram

Red Trace: Assay A
 3-MCPD peak represents the 3-MCPD content plus the Glycidol content of the sample
 Glycidol has been converted to 3-MCPD
 2-MCPD:
o not designated to be quantified in this AOAC method
o but using a deuterated 2-MCPD standard it can be quantified in either Assay A or B
(not part of this SPS)
Blue Trace: Assay B
 3-MCPD peak represents the 3-MCPD content only
 Glycidol has been converted to 3-MBPD and is not seen
 2-MCPD: see comments above