ISO/TS 16465:2024

Technical
Specification
ISO/TS 16465
First edition
Animal and vegetable fats and
2024-01
oils — Determination of phthalates
in vegetable oils
Corps gras d’origines animale et végétale — Dosage des phtalates
dans les huiles végétales
Reference number
© ISO 2024
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Published in Switzerland
ii
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Part A - Determination of DEHP . 1
4.1 Principle .1
4.2 Reagents .1
4.3 Apparatus .3
4.4 Sampling .4
4.5 Preparation of the test sample .4
4.6 Procedure .4
4.6.1 Phthalates extraction from oil .4
4.6.2 Gas Chromatography .5
4.7 Quantitative determination .6
4.7.1 Calibration curve.6
4.7.2 Quantitative determination of DEHP .6
4.8 Precision of the method.7
4.8.1 Interlaboratory test .7
4.8.2 Repeatability .8
4.8.3 Reproducibility .8
4.9 Test report .8
5 Part B - Determination of eight phthalates. 8
5.1 Principle .8
5.2 Reagents .8
5.3 Apparatus .10
5.4 Sampling .11
5.5 Preparation of the test sample . 12
5.6 Procedure . 12
5.6.1 Phthalates extraction from oil . 12
5.6.2 Gas chromatography .16
5.7 Quantitative determination .17
5.7.1 Calibration curve.17
5.7.2 Quantitative determination of phthalate .18
5.8 Precision of the method.19
5.8.1 Interlaboratory test .19
5.8.2 Repeatability .19
5.8.3 Reproducibility .19
5.9 Test report .19
Annex A (informative) Results of an interlaboratory test — Part A - Determination of DEHP .20
Annex B (informative) Results of an interlaboratory test — Part B - Determination of eight
phthalates . .21
Bibliography .25

iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 11, Animal
and vegetable fats and oils.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
Technical Specification ISO/TS 16465:2024(en)
Animal and vegetable fats and oils — Determination of
phthalates in vegetable oils
1 Scope
This document specifies two methods for the quantitative determination of phthalates in vegetable oils by
gas chromatography-mass spectrometry (GC-MS):
— Part A for the determination of di-2-ethylhexyl phthalate (DEHP).
— Part B for the determination of eight phthalates: dimethyl phthalate (DMP), diethyl phthalate (DEP),
di-isobutyl phthalate (DIBP), dibutyl phthalate (DBP), benzylbutyl phthalate (BBP), di-2-ethylhexyl
phthalate (DEHP), di-isononyl phthalate (DINP), di-isodecyl phthalate (DIDP).
Both methods are applicable for all vegetable oils, including crude, refined and virgin.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes
requirements of this document. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
ISO 661, Animal and vegetable fats and oils — Preparation of test sample
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
4 Part A - Determination of DEHP
4.1 Principle
Phthalates are first extracted from oil with acetonitrile followed by dispersive-solid-phase extraction
clean-up (dispersive SPE), which is done to remove organic acids, excess water and other components using
a combination of primary secondary amine (PSA) sorbent and C18 bonded phase. Finally, the extract is
concentrated and analysed by gas chromatography-mass spectrometry (GC-MS).
4.2 Reagents
WARNING — Attention is drawn to national regulations that specify the handling of hazardous
substances, and users' obligations thereunder. Technical, organizational and personal safety
measures shall be followed.
Unless otherwise specified, use only reagents of recognized analytical grade.
4.2.1 Toluene, purity 99 % min.

4.2.2 Acetonitrile, trace organic analysis grade, purity 99 % min.
4.2.3 n-Hexane, trace organic analysis grade, purity 99 % min.
1)
4.2.4 Chromabond adsorbent Diamino (PSA), Chromabond adsorbent C18 .
NOTE These powders can be sources of contamination. To remove the contaminants, the powders can be purified
using Soxhlet extraction by extracting twice with 200 ml of n-hexane during a six-hour period.
4.2.5 n-Dodecane, purity 99 % min.
4.2.6 Standards
Table 1 provides the CAS number of phtalates.
Table 1 — CAS number of phthalates
2) ®
Phthalates Synonym CAS Registry Number
di-2-ethylhexyl phthalate DEHP 117-81-7
deuterated di-2-ethylhexyl phthalate DEHP-d4 93951-87-2
4.2.6.1 Solution of DEHP-d4 internal standard, mass concentration ρ = 50 µg/ml.
As an example, in a volumetric flask of 100 ml, weigh to the nearest mg, approximately 50 mg of DEHP-d4
and dilute to 100 ml of toluene and then proceed with a second dilution of this mixture of 10 ml → 100 ml
with toluene.
4.2.6.2 DEHP calibration solutions
As an example, in a volumetric flask of 100 ml, weigh to the nearest mg, approximately 50 mg of DEHP and
dilute to 100 ml of toluene (SM= 500 µg/ml). Then prepare:
— 20 µl of SM and dilute to 10 ml of acetonitrile: SF1 = 1 µg/ml
— 200 µl of SM and dilute to 10 ml of acetonitrile: SF2 = 10 µg/ml
— 2 ml of SM and dilute to 10 ml of acetonitrile: SF3 = 100 µg/ml
Then prepare a calibration range in acetonitrile following Table 2, using glass syringes washed 10 times
with toluene and five times with related solutions.
1) Chromabond sorbent C18 ref 730611 and Chromabond sorbent Diamino (PSA) ref 730611 from Macherey Nagel
(www .mn -net .com) have proved to be free of phthalate contamination. This information is given for the convenience of
users of this document and does not constitute an endorsement by ISO of these products. ®
2) CAS Registry Number is a trademark of the American Chemical Society (ACS). This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent
products may be used if they can be shown to lead to the same results.

Table 2 — Example of preparation for DEHP calibration
Volume (µl) [Concentration] Solution of
[Concentration]
Volume of
DEHP-d4 DEHP-d4 Total
DEHP
acetoni-
internal volume
C
cal-dph
C trile to be
SF1 SF2 SF3 SM
cal-ph
standard (µl)
µg/ml added (µl)
µg/ml
(µl)
G0 0 - - - - 4 80 920 1 000
G1 0,02 20 - - - 4 80 900 1 000
G2 0,05 50 - - 4 80 870 1 000
G3 0,1 - 10 - - 4 80 910 1 000
G4 0,5 - 50 - - 4 80 870 1 000
G5 1 - - 10 - 4 80 910 1 000
G6 2 - - 20 - 4 80 900 1 000
G7 5 - - - 10 4 80 910 1 000
G8 10 - - - 20 4 80 900 1 000
Depending on the apparatus used, it is not possible to be linear from G0 to G8. In this case, it is necessary to
increase the volume of acetonitrile of recovery in order to be linear over the greatest possible range while
being able to see the level G1.
4.3 Apparatus
Glassware used for the determination shall be thoroughly cleaned at 550 °C during 6 h, such as Pasteur
pipette, vial, conical glass sample vial, 10 ml and 15 ml capacity.
4.3.1 Conical glass sample vials, 10 ml capacity.
4.3.2 Conical glass sample vials, 15 ml capacity.
4.3.3 Glass syringe, 10 µl, 20 µl, 50 µl, 100 µl, 250 µl and 500 µl capacity.
4.3.4 Automatic evaporator, for 10 ml tube (optional), recommended operating conditions: temperature
of the water bath = 40 °C, nitrogen pressure = 5 psi.
4.3.5 Conical glass sample vials, 2 ml capacity.
4.3.6 Gas chromatograph, suitable for use with capillary column, equipped with an injector split-splitless
or equivalent device, a temperature-programmable oven and mass detector with electron ionization source
(ionization energy of 70 eV) and SIM (single ion monitoring) mode.
4.3.7 Data acquisition system, with the possibility of manual integration.
4.3.8 Capillary column, capable of being programmed up to 400 °C ("high temperature" type) for which
the following characteristics are advised: 95 % dimethyl/5 % diphenyl polysiloxane stationary phase,
length of 15 m, internal diameter of 0,25 mm, film thickness of 0,1 µm or length of 30 m, internal diameter of
0,25 mm, 0,25 µm film thickness.
4.3.9 Microsyringe, 5 µl to 10 µl capacity, suitable for split less injection in gas chromatography.
4.3.10 Analytical balance, reading accuracy 0,001 g.
4.3.11 Pasteur pipette, in glass.

4.3.12 Centrifuge, capable of attaining at least 3 000 min−1, suitable for 10 ml tubes.
4.3.13 Pipettes, capable of pipetting volumes up to 10 ml.
4.4 Sampling
A representative sample should have been sent to the laboratory. It should not have been damaged or
changed during transport or storage.
Sampling is not part of the method specified in this document. A recommended sampling method is given in
[1]
ISO 5555.
Analyse only sample aliquots packaged in glass bottles with suitable lids (the sample contact material from
the lid should be, for example, polytetrafluoroethylene (PTFE), aluminium, rubber but not plastic nor soft
seal) to avoid further contamination. Plastic packaging is not suitable.
Due to the presence of phthalates in the environment, the analysis of these compounds requires precautions
throughout the analysis:
— avoid contact with plastic material,
— test the solvents/materials used for phthalates,
— a blank sample should be analysed every four samples,
— glassware that cannot be baked needs to be rinsed with a suitable solvent (acetone),
— avoid contamination from the septum of the injection vials.
4.5 Preparation of the test sample
Prepare the test sample in accordance with ISO 661.
4.6 Procedure
4.6.1 Phthalates extraction from oil
Weigh to the nearest mg, in a conical glass sample vial of 10 ml, 1 g of oil and add 40 µl of DEHP-d4 solution
at 50 µg/ml (4.2.6.1). Shake with a vortex for 15 s.
Add 6 ml of acetonitrile and shake with a vortex for 1 min (or 5 min with an automatic vortex).
Centrifuge the conical glass for 2 min at 3 000 rpm.
Transfer the upper layer in a conical glass of 15 ml containing 400 mg of C18 (4.2.4) and 400 mg of PSA
(4.2.4).
Shake with a vortex for 1 min.
Transfer the upper layer in a conical glass of 10 ml.
Add 50 µl of n-dodecane.
Evaporate acetonitrile at 40 °C to reach 500 µl ± 50 µl.
There remains a droplet of fat at the bottom of the tube from the dodecane.
Shake with a vortex for 15 s (or 2 min with an automatic vortex).
Centrifuge the conical glass for 2 min at 3 000 rpm.
Transfer the solvent layer avoiding the fat droplet in an injection vial and inject into GC-MS.

In order to measure the level of contamination of the laboratory, prepare a blank sample. For blank sample,
add only 40 µl of DEHP-d4 internal standard solution (4.2.6.1) in a conical glass sample vial of 10 ml and
follow the same procedure as for classic sample. (Values of 0,05 mg/kg for DEHP should not be exceeded).
In order to avoid contamination from the septum of the vial (DMP), use a septum with polytetrafluoroethylene
(PTFE), otherwise a small piece of aluminium sheet between the cork and the vial can be used.
NOTE Depending on the apparatus used, the concentration of the extract can be modified and adapted according
to the system.
4.6.2 Gas Chromatography
4.6.2.1 Gas chromatography setup
Install the column (4.3.8) in the gas chromatograph (4.3.6) and check the working conditions by injecting the
solvent, acetonitrile. The baseline should be straight with a small positive drift. If the drift is high, proceed
to condition the column, for a negative drift check the connections of the column.
If the column is used for the first time, it is necessary to condition the column by heating it in the column
oven using a temperature gradient up to 370 °C (depending on the oven temperature chosen for the analysis)
in 4 h, maintain the temperature for 2 h.
4.6.2.2 Working conditions for gas chromatography analysis
The working conditions as shown in Table 3 and Table 4 have proved to be adapted to the analysis.
These conditions can be adjusted in accordance with the characteristics of the gas chromatograph and the
mass detector apparatus and the column.
Table 3 — GC conditions
Column DB5 HT (15 m – 0,25 mm – 0,10 µm film thickness or 30 m – 0,25 mm – 0,25 µm film
thickness)
Injector Liner, tapered, deactivated liner with glass wool
Injection port temperature 350 °C
Injection temperature Splitless in pressure pulse (12 psi) for 1 min
program
1 min after the injection, split at 150 ml/min
3 min after the injection, split at 20 ml/min
Injection volume 1,0 µl
Oven temperature 50 °C for 1 min, ramp at 15 °C/min to 100 °C, ramp at 40 °C/min to 290 °C, ramp at 60 °C/
min to 350 °C, hold for 2 min
Carrier gas Helium/hydrogen constant flow rate, 1,2 ml/min
Syringe solvent clean-up Acetonitrile
Table 4 — MS conditions
Ion source temperature 300 °C
Quadrupole temperature 150 °C
Transfer line temperature 325 °C
Phthalates Quantifier ion Qualifier ion Qualifier ion Qualifier ion
Parameters for SIM mode DEHP 149 167 279 132
DEHP d4 153 171 283 136
Check that the volume of acetonitrile when injected at 350 °C is not greater than the volume of the liner.

4.7 Quantitative determination
4.7.1 Calibration curve
The calibration curve of DEHP is of type y = a * x
A C
ph cal-ph
=a*
A C
dph cal-dph
The calibration curve is determined as follows:
AC *
ph cal-dph
a=
AC *
dphcal-ph
where
a is the slope;
A is the peak area of DEHP in the calibration solution;
ph
A is the peak area of the DEHP-d4 internal standard in the calibration solution;
dph
C
is the concentration of the DEHP-d4 internal standard in the calibration solution, in micrograms
cal-dph
per millilitres (see Table 2);
C
is the concentration of DEHP in the calibration solution, in micrograms per millilitres (see Table 2).
cal-ph
4.7.2 Quantitative determination of DEHP
AV ** C
 
ph dphdph
C = - C
 
ph blank
 
AM ** a
dph
 
where
a is the slope;
A is the peak area of DEHP;
ph
A is the peak area of the DEHP-d4 internal standard;
dph
C is the concentration of the DEHP-d4 internal standard solution, in micrograms per millilitres;
dph
V is the volume of the DEHP-d4 internal standard solution, in millilitres;
dph
C is the concentration of DEHP, in milligrams per kilogram;
ph
C is the concentration of DEHP in the blank, in milligrams per kilogram (see 4.6.1);
blank
M is the mass of the test portion, in grams.
If the volumes of the internal standard solution indicated in this method are respected, and if the same
solution of DEHP-d4 internal standard is used both for the calibration solutions and for the sample
preparation, the following simplified calculation formulae can be used for the determination of the
calibration curve and the quantification of phthalate:

4.7.2.1 Calibration curve
The calibration curve for DEHP is of type y = a * x
A
ph 40
=am**
cal-ph
A 80
dph
The calibration curve is determined as follows:
A *2
ph
a=
Am *
dphcal-ph
where
a is the slope from the calibration, using the same DEHP-d4 internal standard solution for the
samples and for the calibration;
A is the peak area of DEHP in the calibration solution;
ph
A is the peak area of the DEHP-d4 internal standard in the calibration solution;
dph
m
is the mass of DEHP in the solution calibration, in micrograms.
cal-ph
NOTE The coefficient of 2 is added in the formula to consider the ratio of the amount of internal standard between
the calibration solutions and the samples (80 µl of the internal standard solution for the calibration and 40 µl of the
internal standard solution for the samples).
4.7.2.2 Quantitative determination of DEHP
A
 
ph
C = - C
 
ph blank
 
AM ** a
 dph 
where
C is the concentration of DEHP, in milligrams per kilogram;
ph
a is the slope from the calibration, using the same DEHP-d4 internal standard solution for the
samples and for the calibration;
A is the peak area of the DEHP;
ph
A is the peak area of the DEHP-d4 internal standard;
dph
M is the mass of the test portion, in grams;
C is the concentration of DEHP in the blank, in milligrams per kilogram (see 4.6.1).
blank
4.8 Precision of the method
4.8.1 Interlaboratory test
Details of the test and the precision of the method are summarized in Annex A. The values derived from this
interlaboratory test will possibly not be applicable to concentration ranges and matrices other than those
given.
4.8.2 Repeatability
The absolute difference between two independent single test results, obtained with the same method on
identical test material in the same laboratory by the same operator using the same equipment within a short
interval of time, shall in not more than 5 % of cases exceed the value of r given in Table A.1.
4.8.3 Reproducibility
The absolute difference between two single test results, obtained with the same method on identical test
material in different laboratories by different operators using different equipment, shall in not more than
5 % of cases exceed the value of R given in Table A.1.
4.9 Test report
The test report shall contain at least the following information:
a) all information necessary for the complete identification of the sample;
b) the sampling method used, if known;
c) the test method used, with reference to this document, i.e. ISO/TS 16465:2024;
d) all operating details not specified in this document, or regarded as optional, together with details of any
incidents occurred when performing the method, which can have influenced the test result(s);
e) the test result obtained;
f) if the repeatability has been checked, the final quoted result obtained.
5 Part B - Determination of eight phthalates
5.1 Principle
Phthalates are first extracted from oil with acetonitrile followed by dispersive-solid-phase extraction
clean-up (dispersive SPE) which is done to remove organic acids, excess water and other components
using a combination of primary secondary amine (PSA) sorbent and C18 bonded phase. Finally, the extract
is concentrated and analysed by gas chromatography-mass spectrometry (GC-MS). For refined oil, the
acetonitrile extract is directly concentrated without using the dispersive-SPE purification step.
5.2 Reagents
WARNING — Attention is drawn to national regulations that specify the handling of hazardous
substances, and users' obligations thereunder. Technical, organizational and personal safety
measures shall be followed.
Unless otherwise specified, use only reagents of recognized analytical grade.
5.2.1 Toluene, purity 99 % min.
5.2.2 Acetonitrile, trace organic analysis grade, purity 99 % min.
5.2.3 n-Hexane, trace organic analysis grade, purity 99 % min.
3)
5.2.4 Chromabond adsorbent Diamino (PSA), Chromabond adsorbent C18 .
3) Chromabond sorbent C18 ref 730611 and Chromabond sorbent Diamino ref 730611 from Macherey Nagel
(www .mn -net .com) have proved to be free of phthalate contamination. This information is given for the convenience of
users of this document and does not constitute an endorsement by ISO of these products.
...