This document describes an automated high throughput sample preparation system called LISSY-MCPD that can analyze 3-MCPD, 2-MCPD, and glycidyl ester in edible oils and fats using four different established methods. The system uses standard laboratory equipment and can process up to 480 samples per week depending on the specific method. It offers flexibility to use different extraction and analysis methods. Results from the automated system using an established method were comparable to manual preparation and had lower variation. The system is suitable for various food safety and quality control applications.

1. Automated High Throughput Analysis of 3-MCPD, 2-MCPD and
Glycidyl Ester in edible Oils and Fats, using the Methods AOCS
Cd 29a-13, Cd 29b-13, Cd 29c-13, EFSA / EN-779
Figure 2: a) 24 position extraction block
b) 24 position sonification station
c) 4 centrifuge baskets 6 positions each
Figure 1: LISSY-MCPD System, including fume hood and centrifuge
Figure 5: Combined SPE / LL extraction system for MOSH,
PAH, PCB, AOX, EOX
REFERENCES:
[1] AOCS Official Method Cd 29a-13 (2013)
[2] AOCS Official Method Cd 29b-13 (2013)
[3] AOCS Official Method Cd 29c-13 (2013)
[4] Wenzel T., Samaras V., Giri A., Buttinger G., Karasek L.,
Zelinkova Z. External Scientific Report:
Development and validation of analytical methods for the
analysis of 3-MCPD (both in free and ester form) and glycidyl
esters in various food matrices and performance of an adhoc
survey on specific food groups in support to a scientific opi-
nion on comprehensive risk assessment on the presence of
3-MCPD and glycidyl esters in food;
EFSA supporting publication 2015: EN-779
[5] European Commission Joint Research Centre Directorate
F – Health, Consumers & Reference Materials, Food & Feed
Compliance (F.5), 2016:
Standard Operating Procedure for the Simultaneous Determi-
nation of 3-MCPD, 2-MCPD and Glycidy Fatty Acid Esters in
Various Food Matrices by Derivatisation in Organic Phase
Glycidol 147,0 => 91,0 / 150,0 => 93,0
C(std)/C(is)
Y = 1,2871x - 0,0009
R2 = 0,9991
0,000 0,200
0,200
0,400
0,400
0,600
0,600
0,800
0,800
1,000
1,000
1,200
1,200
1,400
0,000
A(std)/A(is)
INTRODUCTION:
The presented automated high throughput sample preparation system
LISSY-MCPD performs four different well established methods to analyze
3-MCPD (free and ester form), 2-MCPD and glycidyl ester in oil and fat
samples. Dependent on the sample matrix or the analytical needs, the methods
can be changed from Cd 29a-13 [5] to Cd 29b-13 [2], to Cd 29c-13 [3], to
EFSA / EN-779 [4] or any combinations of the AOCS methods with the
EFSA / EN-779. Also other liquid / liquid extraction methods (LL) can be
established on the system. In upgraded versions, combinations of solid phase
extraction (SPE) and liquid / liquid extraction methods for MOSH, PAH, PCB,
AOX, EOX are possible.
The throughput of the single methods is dependent on the workflow itself:
Cd 29a-13 up to 192 workups per week
Cd 29b-13 up to 192 workups per week
Cd 29c-13 up to 480
EFSA / EN-779 up to 480 workups per week
SYSTEM DESCRIPTION:
In cooperation with a food quality and safety lab, we developed a high
throughput sample preparation system for the determination of 3-MCPD
(free and ester form), 2-MCPD and glycidyl ester in various food matrices. In
Table 1 the automated workflow of the AOCS Cd29 a-13 [1] according to the
JRC Standard Operating Procedure [5] is shown for the preparation of 24
workups in one run. The automated workflow is similar to the manual workflow.
Table 2 presents a possible weekly preparation schedule to get 192 results
within 5 days.
Since the LISSY-MCPD-System (Figure 1 and 2) is developed for a maximum
of flexibility in the use of different preparation methods, also the other
methods, Cd 29b-13 [2], Cd 29c-13 [3] and EFSA / EN-779 [4] can be used. The
throughput of the methods is dependent on the workflow itself. For example
with the Cd 29c-13 method, first GC result is available after around 4 hours, the
complete GC results of 72 workups are available at 11:00 if the workup was
started at 08:00 the day before, assuming an 8h working day (Table 3) and
3 GC systems.
The crucial point of automation is the question, whether the automated
workup performs as well as the manual method. In Figure 3 are the
chromatograms of a complex food matrix shown, worked up according IRC
Standard Operating Procedure [5], in Figure 4 the calibration curve of
gylcidyl. The results are comparable with the manual method and the
variation of the results is much lower in the automated workflow.
As already mentioned, the flexibility of the LISSY-MCPD system (Figure 1 and
2) allows also the use of nearly all LL extraction methods e.g. for MOSH, PAH,
PCB, AOX, EOX. If necessary the system can also be equipped with SPE and
Barcode sample identification.
An example for a very complex SPE / LL system is presented in Figure 5.
CONCLUSION:
• The presented automated sample preparation system
LISSY-MCPD is a high throughput system, Cd 29a-13 up to 192 per
week, Cd 29c-13 up to 480 per week
• LISSY-MCPD is flexible in the use of many different methods
• LISSY-MCPD is using standard lab-ware
• LISSY-MCPD can be used independent from GC, GC/MS
STEP MANUAL / ON SYSTEM ACTION
1 m Temper samples
2 m weigh samples into glass vials
3 m add internal standard
4 s Add THF
5 s Vortex
6 s Add NaBr aequous solution
7 s Vortex
8 s Incubation 50°C, 15 min
9 s Add NaHCO3 Solution
10 s Add n-heptane
11 s Vortex
12 s Centrifuge
13 s Transfer upper phase to new vial
14 m Evaporation
15 s Add THF
16 s Add H2SO4/MeOH Solution
17 s Vortex
18 m Derivatisation 40°C, 16h
19 s Add NaHCO3 Solution
20 s Vortex
21 s Evaporation
22 s Add Na2SO4 Solution
23 s Add n-heptane
24 s Vortex
25 s Discard upper phase
26 s Repeat previous 3 steps 23-25
27 s Add Ethylacetate
28 s Vortex
29 s Transfer upper phase to new vial containing solid NaCl
30 s Repeat previous 3 steps 27-29
31 s Repeat previous 3 steps 27-29
32 s Add phenylboronic acid solution
33 s Vortex
34 s Incubation in ultrasonic bath
35 s Transfer to new vial
36 m Evaporation
37 s Add n-heptane
38 s Vortex
39 s Centrifuge
40 s Transfer to GC vials
TIME MONDAY TUESDAY WEDNESDAY THURSDAY FRIDAY
08:00
„Workup
1-24“
„Workup
49-72“
„Workup
97-120“
„Workup
145-168“
08:30
Evap. ex-
tern
Evap. ex-
tern
Evap. ex-
tern
Evap. ex-
tern
09:00
09:30
„Workup
25-48“
„Workup
73-96“
„Workup
121-144“
„Workup
169-192“
10:00
Evap. ex-
tern
Evap. ex-
tern
Evap. ex-
tern
Evap. ex-
tern
10:30
11:00
11:30
12:00
12:30
13:00
13:30
14:00
Workup 49-72 Workup 97-120 Workup 145-168
14:30
15:00
Workup 1-24
15:30
Workup 72-95 Workup 121-143 Workup 169-191
16:00
16:30
Workup 25-47
17:00
17:30
18:00
TIME WORKUP 1-24 WORKUP 25-48 WORKUP 49-72
08:00 Workup
09:00
10:00
11:00 GC time 24x Workup
12:00
13:00
14:00 GC time 24x Workup
15:00
16:00
17:00 GC time 24x
18:00
19:00
20:00
21:00
22:00
23:00
00:00
01:00
02:00
03:00
04:00
05:00
06:00
07:00
08:00
09:00
10:00
11:00
12:00
Figure 4: Glycidyl calibration curve
Figure 3: GC MS/MS chromatogram of a complex food matrix after fat extraction
Table 1: Automated Cd 29a-13 method according JRC Standard Operating Procedure [5] Table 2: Cd 29a - 13 workup schedule for one week
Table 3: Time line for Cd 29c-13
3 MCPD
1,00mg / kg
2 MCPD
0,42mg / kg
Glycidol
0,10mg / kg
Fritz Kaiser, Stefan Baumgärtel, Michael Ott, Zinsser Analytic GmbH, Eschborn, Germany
Zinsser Analytic GmbH
Schwalbacher Str. 62 . 65760 Eschborn
Tel.: +49 6196 58693 0 . info.zinsser-analytic@gardnerdenver.com
www.zinsser-analytic.com