APPLICATION NOTE

Gas Chromatography

Author:
Liaw Soon Thien B.Sc.

PerkinElmer Inc.
Malaysia

Analysis of 3-MCPD
(3-Chloro-1,2-Propanol) and Introduction
Chloropropanol are formed in
Glycidyl Ester using Clarus hydrolyzed vegetable protein (HVP)
produced by hydrochloric acid (HCl)
GC SQ8 Mass Spectrometer hydrolysis of proteinaceous
by-product from edible oil extraction,
such as soybean meal, rapseed meal
and maize gluten. The form of chloroponanol that most commonly found in food is
3-MCPD (3-chloro-1,2-propanediol), although others may also be present, including
2-MPCD (2-chloro-1,2-propanediol), 1,3-DCP (1,3-dichloro-2-propanol), and 2,3-DCP
(2,3-dichloro-1-propanol).
In palm oil or other edible oil production, the study has been found that chlorination of
acylglycerols can occur during deodorisation of edible oils, resulting in the formation of
fatty acid esters of 3-chloropropane-1, 2-diol. Fatty acid esters of glycidol [or glycidyl
esters (GE)] have been detected in refined oils. It has been observed that both GE and
MCPD esters are found in particularly high abundance in refined palm (Elaeis guineensis)
oil and palm-based fractions.
 Figure 1. Structures of 3MCPD, 3MCPD Esters, Glycidol and Glycidyl Esters.

Glycidyl esters (GE) are process contaminants generated during Sample Preparation Outline
the deodorisation step of edible oil refining. Several reports in Samples of Edible Oil label as EA 6395, FA 6641 and SA 5756
the literature aim at associating GE formation with the formation respectively has been prepared according to following protocol
of MCPD esters through common intermediates. GE is found in and analyze by GCMS.
high abundance in refined palm oil. Palm oil is unique in that it
contains high number of diacylglycerols (DAG, 4–12%).
Officially, there are three methods in AOCS for the testing of the 100 mg of oil/fat sample
2-MCPD ester, 3-MCPD esters and glycidyl esters utilising GCMS
for edible oils and fats. There are three different methods that
Addition of internal standards + Tetrahydrofuron (THF)
have been found through international collaborative studies to
produce statistically the same results, and therefore are these
methods are equivalent for use in the determination of 2- and Conversion of GE to 3-MCPD* monoesters
3-MCPD fatty acid esters and glycidyl fatty acid esters in the in bromide salt
edible oils and fats.
In this application note, AOCS Cd 29a-13 is focused. Glycidyl Conversion of 2- and 3-MCPD esters and 3-MCPD
ester is converted to 3-monobromopropanediol (3-MBPD) esters into free form in acid methanolic solution
monoesters in acid solution containing a bromide salt. 3-MBPD
esters, together with 2- and 3-MCPD esters, are then converted
into the free (non-esterified) form in acid methanolic solution. The Extraction of fatty acid methyl esters
fatty acid methyl esters generated during the reaction are extracted
from the samples; and 2- and 3-MCPD as well as 3-MBPD, are the Derivatization of 2- and 3-MCPD, and
derivatized with phenylboronic acid prior to GC-MS analysis. 3-MBPD with Phenylboronic acid (PBA)
This method is applicable for determination of 2- and 3-MCPD
fatty acid esters and glycidyl fatty acid ester (as a sum of all the
Analyzed by GCMS
respective esters) in edible oil/fats.
Figure 2. Need Caption.
Standard Preparation
All the stock standard and working standard are prepare
according to the step stated in the AOCS Cd 29a-13 method.

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Table 1. Need Caption. Table 2. Need Caption.
GC Condition Setting of SQ8 Mass Spectrometer
Column Elite-5MS (30 m x 0.25 mm ID x 0.25 um df) Inlet Line Temperature 200 °C
80 °C; hold for 1.0 min; Source Temperature 200 °C
Ramp 1: 10 °C/min to 170 °C;
Oven Temperature Program Electron Energy 70 eV
hold for 15.0 min
(Total GC run = 25 minutes) Multiplier Voltage 1840 (auto-tune)
Injector Temperature 200 °C Scan Mode SIR
1 μl, split ratio 50:1
Injection Volume
(after 1 min via timed event) Table 3. Need Caption.
Injection port type PSSI (splitless injection) SIR Parameters Setup
Carrier Gas Helium Solvent Delay 8.5 min
Flow Rate 1 mL/min Dwell Time 0.02 sec
Inter Scan Delay 0.01 sec
Quantifier Ion

Phenylboronic derivative of 3-MCPD = 147 m/z over STD Phenylboronic derivative of 3-MCPD d5 ion 150 m/z
Phenylboronic derivative of 2-MCPD = 196 m/z over ISTD Phenylboronic derivative of 3-MCPD d5 ion 201 m/z
Phenylboronic derivative of 3-MBPD (glycidyl-P) = 147 m/z over ISTD Phenylboronic derivative of 3-MBPD d5 ion 150 m/z

Figure 3. Typical selected ion chromatogram of PBA derivative of 3MCPD, 2MCPD and 3MCPD-d5 and 3MBPD-d5 working Standard.

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 Standard Calibration Curve
Working standard range from 0.03 to 0.93 ug (2MCPD and 3-MCPD) and 0.06 to 2.13 ug (GE), spike with 0.39 ug internal standard of
3-MCPD d5 and 0.62 ug Internal standard of 3-MCPD d5 was prepared to generate a multiple level ISTD calibration curve as below.

Table 1. Calculation on result of 3 edible oil samples for 2MCPD, 3MCPD and GE analysis.
3-MCPD
3-MCPD (ug)
(ug/g=mg/kg=ppm)
Concentration
Sample from Establish ISTD Concentration After
Sample
Weight, Calibration Curve, Divide by Sample
Marking
W (in g) Y= 1.29794 X + Weight, W
0.0141139
EA 6395 0.1051 0.04 0.380589914
FA 6641 0.1040 0.04 0.384615385
SA 5756 0.1078 0.03 0.278293135

2-MCPD
2-MCPD (ug)
(ug/g=mg/kg=ppm)
Figure 4. Standard Calibration Curve for 3-MCPD . Concentration
Sample from Establish ISTD Concentration After
Sample
Weight, Calibration Curve, Divide by Sample
Marking
W (in g) Y= 2.84102 X + Weight, W
0.0110154
EA 6395 0.1051 0.01 0.095147479
FA 6641 0.1040 0.01 0.096153846
SA 5756 0.1078 0.01 0.092764378

Glycidol 3-MCPD Glycidol 3-MCPD

(ug) (ug/g=mg/kg=ppm)
Concentration
Sample from Establish ISTD Concentration After
Sample
Weight, Calibration Curve, Divide by Sample
Marking
W (in g) Y= 1.31620 X + Weight, W
0.0114177

Figure 5. Standard Calibration Curve for 2-MCPD. EA 6395 0.1051 0.01 0.095147479
FA 6641 0.1040 0.02 0.192307692
SA 5756 0.1078 0.01 0.092764378

Conclusion and Discussion

It is concluded that Clarus 690 with SQ 8 is capable to detect PBA
derivative of 3-MCPD and GE in oil sample and can fulfil the requirement
of AOCS Cd 29a-13. It has shown good linearity with R-square of >0.99
and good sensitivity to sub-ppb level and able to meet requirement of
AOCS Cd 29a-13.

Figure 6. Standard Calibration Curve for GE.

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