3-MCPD Esters - staging.files.cms.plus.com

“Indirect” Method for the Determination of
3-MCPD Esters: Hydrolysis Time and
Recovery Considerations for the Acid
Hydrolysis Method
J. D. Pinkston, D. P. Iannelli, T. R. Mertens
The Procter & Gamble Company,
Winton Hill Business Center,
6300 Center Hill Ave.,
Cincinnati, OH 45224
pinkston.jd@pg.com
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“3-MCPD esters”
3-Monochloropropane-1,2-diol Fatty Acid Esters
3-MCPD esters (3-MCPD esterified with 1 or 2 fatty
acids) are part of an emerging regulatory issue.
3-MCPD esters have been reported in refined fats and
oils by several groups (Svejkovská et al., 2004;
Weißhaar, 2008; Zelinková , Svejkovská , Velísek, &
Dolezal, 2006).
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Analytical Methods
Methods can be classified in two general groups:
“Indirect” methods: Esters are cleaved to a single
compound, “free 3-MCPD”, which is then
derivatized and determined by GC/MS.
(Divinova et al. Czech J Food Sci. 2004, 22, 182–189;
Weiβhaar, Eur. J. Lipid Sci. Technol. 2008, 110, 183-186;
Kuhlmann, Eur. J. Lipid Sci. Technol. 2011, 113, 335–344;
Ermacora & Hrncirik, JAOCS 2012, 89, 211-217.).
“Direct” methods: Major esters are determined
directly via LC/TOFMS or LC/MS/MS.
(Haines et al., JAOCS 2011, 88, 1-14.)
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Indirect Methods:
Advantages and Drawbacks
The indirect methods provide significant advantages:
Straightforward determination of one species by GC/MS
(widely available).
Excellent LODs and LOQs.
Only 2 standards (incl. 1 SIL internal standard) required.
Detects all 3-MCPD esters.
However, there are areas for potential improvement:
Hydrolysis in presence of trace Cl - may result in inaccurate,
higher results with indirect methods.
Glycidol esters may be converted to 3-MCPD during
sample prep --> overestimation of results.
Sample preparation is tedious.
Method does not differentiate between free 3-MCPD and/or
different 3-MCPD esters.
Details provided in Hrncirik et al., Eur. J. Lipid Sci. Technol.
2011, 113, 361-367.
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Indirect Methods:
Choices for Initial Cleavage of Esters
Base hydrolysis (methoxide):
Fast & straightforward (minutes)
Trace chloride and glycidyl esters can artificially increase
the level of 3-MCPD esters detected.
Free 3-MCPD is unstable under basic hydrolysis conditions,
decreasing S/N ratios and sensitivity (Hrncirik et al. Eur. J.
Lipid Sci. Technol. 2011, 113, 361-367.).
Enzymatic hydrolysis:
Few studies have been conducted (Hamlet & Sadd, Czech J.
Food Sci. 2004, 22, 259-262.).
Acid hydrolysis (H 2SO 4 in MeOH):
Longer hydrolysis times (hours)
Trace chloride can artificially increase the level of 3-MCPD
esters detected (though glycidyl esters are destroyed) .
Free 3-MCPD is stable under acidic hydrolysis conditions.
Acid hydrolysis method found to be more robust (Hrncirik et
al., Eur. J. Lipid Sci. Technol. 2011, 113, 361-367.).
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Implementation/Development of the Acid
Hydrolysis Indirect Method
Key attributes of Ermacora & Hrncirik’s indirect acid
hydrolysis method (JAOCS 2012, 89, 211-217):
Interference of high chloride levels (when present)
eliminated with simple H 2O extraction step.
No interference from glycidyl esters.
Authors suggest that time required for acid
transesterification can be reduced from 16 h to 4 h without
loss of accuracy, repeatability, or sensitivity.
SIL di-ester (PP-3-MCPD-d5) recommended as most
appropriate internal standard.
We found high recoveries of monoesters when this
method was applied. (Accuracy for diesters was
acceptable.)
We have studied a variety of aspects of the method,
including hydrolysis time.
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Key Points & Modifications to Acid Hydrolysis
Method by Ermacora & Hrncirik
Step in Method
Choice of internal
standard
Initial water wash
(optional in Ermacora &
Hrncirik)
Initial sample solvent
for water wash
Ermacora &
Hrncirik*
PP-3-MCPD-d5 No change
Recommended
to remove Cl -
P&G
Retained – no change, but used for
all samples.
THF MTBE - better separation of oil-rich
and water-rich phases in initial water
wash (MTBE evaporated and THF
added for subsequent steps)
Hydrolysis time 4 hrs 24 hrs (improved accuracy for
monoesters when I.S. is a diester)
Selected ion monitoring
ions
m/z 147 (m/z 150
for SIL internal
standard)
m/z 196 (m/z 201 for SIL I.S.) – m/z
147 is common “bleed” ion for
siloxane-based GC stationary
phases.
* - Ermacora & Hrncirik, JAOCS 2012, 89, 211-217.
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Abundance
170000
150000
130000
110000
90000
70000
50000
30000
10000
GC/MS Background Ions Considerations
Choice of SIM Ion: m/z 147
vs. m/z 196?
Common background ion in
siloxane-based column and
septum “bleed” peaks in
GC/MS
77
91
104
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210
m/z-->
147
EI Mass Spectrum of the
PBA Derivative of 3-MCPD
196
M ·+
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Abundance
1600
1400
1200
1000
800
600
400
200
Choice of SIM Ion, m/z 147 vs. m/z 196?
0
Time (min)
9.92 9.96 10.00 10.04 10.08 10.12 10.16
Abundance
0.33 ppm (3.0 µmolar) standard Sample near LOD (~0.04 ppm (0.4 µmolar)
280
240
200
160
120
80
40
m/z 147
m/z 196
interferent
0 9.92 9.96 10.00 10.04 10.08 10.12 10.16
Time (min)
Abundance
1300
1100
900
700
500
300
100
0 9.92
Time (min)
9.96 10.00 10.04 10.08 10.12 10.16
Abundance
170
165
160
155
150
145
140
m/z 147
m/z 196
interferent
135
Time (min)
9.92 9.96 10.00 10.04 10.08 10.12 10.16
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Accuracy/Recovery vs. Hydrolysis Time
CSO* spiked with PP-3-MCPD at 56.2 µmolar (=6.21 ppm “free 3-MCPD”)
or with P-3-MCPD at 59.9 µmolar (=6.62 ppm “free 3-MCPD”)
Monoester (w/ diester IS)
CSO* spiked with P-3-MCPD,
PP-3-MCPD-d5 internal standard
Hydrolysis Time (hr) % recovery
4 225
7 143
16 149
24 124
Monoester (w/ monoester IS)
CSO* spiked with P-3-MCPD,
P-3-MCPD-d5 internal standard
Hydrolysis Time (hr) % recovery
7 116
* - Fresh Refined Cotton Seed Oil,
total 3-MCPD esters: 3.29 µmolar
(=0.36 ppm “free 3-MCPD”)
Diester (w/ diester IS)
CSO* spiked with PP-3-MCPD,
PP-3-MCPD-d5 internal standard
Hydrolysis Time (hr) % recovery
4 108
7 109
16 115
24 115
Recoveries acceptable at any
time for monoester with
Rate of hydrolysis of
monoester IS, and for diester
monoester faster than that of
with diester IS.
diester, yielding high recovery
for monoester at shorter
hydrolysis times.
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Abundance
310
300
290
280
270
260
250
240
Limit of Quantitation/Limit of Detection
Selected ion monitoring (SIM)
chromatogram of lowest standard
m/z 196.0
LOQ = 0.11 ppm
S/N ≈ 8
110 pg injected, splitless injection
Estimated LOD (S/N ≈ 3) at 0.04 ppm
11.90 11.95 12.00 12.05 12.10 12.15 12.20 12.25 12.30
Time (min)
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m o l
µ
60
58
56
54
52
50
48
46
44
Precision
Protocol for measuring repeatability & intermediate
precision:
7 aliquots of working reference material vegetable oil
prepared on 3 separate days by 1 analyst. Each sample (1
per aliquot) determined in triplicate.
Overview:
A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C A B C injection
1 2 3 4 5 6 7 1 2 3 4 5 6 7 1 2 3 4 5 6 7 aliquot
1 2 3 day
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Variability by component:
Precision - Results
Component % of Total Variability
Day-to-day 66.2
Injection-to-injection (within
aliquot)
31.6
Aliquot/sample prep (within day) 2.2
Instrumental repeatability:
RSD: 3.6% (from 21 sets of triplicate injections)
Aliquot/sample prep repeatability:
RSD: 0.94% (from 3 sets of 7 aliquots)
Intermediate precision, day-to-day:
RSD: 5.2% (from 3 days)
Intermediate precision, total (1 analyst):
RSD: 6.3%
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Analyte response/Internal standard response
7.000
6.000
5.000
4.000
3.000
2.000
1.000
-1.000
Linearity & Range
Calibration Curve for Total 3-MCPD Esters, Acid Hydrolysis
y = 0.0572x - 0.0028
R² = 0.9999
0.000
0.0 20.0 40.0 60.0 80.0 100.0 120.0
Standard Concentration (µmolar)
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Update on Direct Method
We provided an update on our direct LC/MS/MS method for
3-MCPD esters at the last AOCS national meeting
(Pinkston & Stoffolano, 102 nd AOCS National Meeting and
Exposition, May 1-4, 2011, Cincinnati, OH).
Our method provided some advantages over a similar
LC/TOFMS method described by Haines et al. (JAOCS
2011, 88, 1-14.), notably little need for instrument
cleaning.
However, unresolved interferences required the use of a
standard addition method (with tedious sample
preparation).
We are investigating a simple silica SPE step which may
remove much of the interferent.
We are working to develop a more standard analytical
method incorporating the SPE cleanup with LC/MS/MS.
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Summary
We have implemented an indirect method for total 3-
MCPD esters in vegetable oils in our laboratory based
upon the acid hydrolysis method of Ermacora & Hrncirik.
Notable differences we recommend:
Use of MTBE as initial sample solvent for water wash.
24-hr hydrolysis time
GC/MS SIM ion: m/z 196
Acknowledgements
Thanks to Shuo Wang, Jim Jordan, Pete Stoffolano,
Jaque Heisey, Katrin Schutte, Dick DePalma, and
Debbie Ewald for their contributions.
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