molecules

Article
Determination of Triacylglycerols by HTGC-FID
as a Sensitive Tool for the Identification of
Rapeseed and Olive Oil Adulteration
Ying Qian 1, * , Magdalena Rudzińska 1 , Anna Grygier 1 and Roman Przybylski 2
1 Poznań University of Life Sciences, Poznań, Wojska Polskiego 28, 60-637 Poznań, Poland;
[email protected] (M.R.); [email protected] (A.G.)
2 University of Lethbridge, 4401 University Drive West, Lethbridge, AB T1K 3M4, Canada;
[email protected]
* Correspondence: [email protected]; Tel.: +1-0048-618-487-276

Academic Editors: Alessandra Gentili and Chiara Fanali




Received: 27 July 2020; Accepted: 25 August 2020; Published: 26 August 2020

Abstract: Triacylglycerols (TGs) are the most common compounds in food lipids, accounting for 95%
of the weight of edible oils. The aim of this study was to scrutinize a procedure for quantitatively
assessing possible adulteration of olive and rapeseed oil through GC-FID analysis of TGs. The recovery
of TG standards ranged from 21% to 148%, and the relative response factor (RRF) ranged from 0.42 to
2.28. The limits of detection were in the range of 0.001 to 0.330 µg/mL, and the limits of quantitation
from 0.001 to 1.000 µg/mL. The validated method was used to determine the TGs in olive oil (OO),
refined rapeseed oil (RRO), and their blends. Eight TGs were detected in refined rapeseed oil, and
10 in olive oil. The addition of 1% of olive oil to rapeseed oil or vice versa can be detected using
this method. Three triacylglycerols were pinpointed as indicators of adulteration of rapeseed oil
with olive oil (PPO, PPL, PSO). The method described here can be used for controlling the quality of
these oils.

Keywords: triacylglycerols; gas chromatography; validation; identification; olive oil; rapeseed

oil; adulteration

1. Introduction
Triacylglycerols (TGs) are the most common compounds in food lipids, accounting for more than
95% of the weight of edible oils. The glycerol molecule is esterified with three fatty acids (Figure 1),
although monoglycerols and diacylglycerols may also be present. The monoesters and diesters are
often used in food applications as additives and emulsifiers. The TG composition of edible oils is rather
difficult to analyze, due to its complexity, with vegetable oils containing a wide range of different
fatty acids (FAs). However, such analysis is possible for mixtures of TGs with similar molecular
weights but different molecular structures, due to the three possible positions of the fatty acid on the
glycerol molecule.
Molecules 2020, 25, x FOR PEER REVIEW 2 of 11

O R1

R2 O O
O
O
O R3
Chemical structure
Figure 1. Chemical structure of triacylglycerol. R1,
R1, R2,
R2, R3
R3–acyl
– acylmoieties.
moieties.

A range
Molecules 2020, 25,of capillary
3881; columns has been
used for the determination www.mdpi.com/journal/molecules
doi:10.3390/molecules25173881 of triacylglycerols in edible
fats and oils. A medium polarity open-tubular fused silica TG CB-type capillary column (Chrompack,
São Paulo, Brazil) has been used to separate the TGs of several vegetable oils [4]. To analyze TG in
cocoa butter, an equivalent 100%-dimethylpolysiloxane phase (DB-1) capillary column can be used
Molecules 2020, 25, 3881 2 of 11

A number of different analytical methods are used for TG determination. Gas chromatography (GC)
is widely used due to its speed, convenience, and sensitivity [1]. High-temperature gas chromatography
effectively separates TGs at temperatures above 300 ◦ C [2]. Polyunsaturated fatty acids are prone
to thermal degradation at this temperature, which can distort their true composition [1]. TGs have
a high boiling point, and it is difficult to volatilize them [3]. For the purpose of high-temperature
TG analysis, special columns have been developed to assist analysis at temperatures above 250 ◦ C,
to analyze diluted TG using GC directly [3].
A range of capillary columns has been used for the determination of triacylglycerols in edible
fats and oils. A medium polarity open-tubular fused silica TG CB-type capillary column (Chrompack,
São Paulo, Brazil) has been used to separate the TGs of several vegetable oils [4]. To analyze TG in
cocoa butter, an equivalent 100%-dimethylpolysiloxane phase (DB-1) capillary column can be used [5].
The identification of milk fat in chocolate was performed on the basis of the TG composition determined
using the CP-TAP CB columns (Agilent Technologies, Santa Clara, CA, USA) [6]. Lili et al. (2011) used
(50%-phenyl)-methylpolysiloxane phase (DB-17ht) and 100%-dimethylpolysiloxane stationary phase
(DB-1ht) capillary columns to quantify monoacylglycerols (MAG), diacylglycerols (DAG), and TGs in
food lipids and oils. When the GC × GC-MS technique was used to determine TGs in fish oil, fused
silica capillary columns (SLB-5ms) and ethylene glycol (Supelcowax-10) were used [7,8]. Adulterants
of ghee were identified by analysis of triglycerides separated on a (5%-diphenyl)-dimethylpolysiloxan
(CP-Ultimetal SimDist CP7532) column [9]. Supelco and Restek suggested using fused silica
MET-Biodiesel or MXT-Biodiesel TG columns [10]. In addition, (5%-phenyl)-dimethylpolysi loxane
(ZB-5ht) Phenomenex, (5%-phenyl)-methylpolysiloxane (DB-5ht, Agilent Technologies, Santa Clara,
CA, USA), fused silica (Rtx-5SilMS, Restek, Bellefonte, PA, USA), and 5%-phenylmethyl polysiloxane
(VF-5ms, Varian, Palo Alto, CA, USA) can be used to determine TGs [11]. The Capillary fused silica
Rtx-65TG column by Restek is specially tested for triacylglycerols. The phase resolves TG by the degree
of unsaturation, as well as by carbon number (www.restek.com). This column has been used for the
determination of TGs in a range of dairy products [2].
Vegetable oils play an important role in the human diet. They are a source of essential fatty
acids, vitamins, phytosterols, tocopherols, and other antioxidants. Global production of vegetable oils
increased from 90.5 million tonnes in 2000–2001 to 207.5 million tonnes in 2019–2020. In 2018–2019, the
consumption of palm, soybean, and rapeseed oils was respectively 70, 57, and 28 million tonnes [12].
In addition to traditional vegetable oils produced in large quantities, olive oil is also very popular,
with a 2018–2019 world consumption of 3 million tonnes. The popularity of olive oil is associated
with its high antioxidant content and its reputation as an element of a healthy Mediterranean diet.
The main ingredients in olive oil are TGs, which can make up 98% of the content [4]. Olive oil has a
high price, making it a target for adulteration with less expensive oils and fats. The problem of olive
oil adulteration is not new but depends on the region or country where other cheap vegetable oils are
produced [12]. It has been found that the adulteration of olive oil with rapeseed oil cannot be detected
by measuring the refractive index, viscosity, or melting point [3]. However, triacylglycerol analysis by
gas chromatography could be a rapid and sensitive method for identifying the adulteration of olive oil
with rapeseed oil.
The aim of this study was to explore a method of quality and quantity analysis of TGs using
GC-FID, and to employ this to detect the adulteration of olive oil by rapeseed oil.

2. Results and Discussion

2.1. Optimization and Validation of the GC-FID System

Basic data on the chemical structure of the analyzed TGs are presented in Table 1 and Figure 1.
Figure 2 shows the chromatogram of the separated TGs. The carbon numbers of TG standards were
used to optimize the separation, and ranged from 48 (PPP) to 57 (NNN); the double bond number
was 0–6. The equivalent of carbon number (ECN) was calculated by the relation ECN = CN-2 × DB,
Molecules 2020, 25, 3881 3 of 11

Molecules 2020, 25, x FOR PEER REVIEW 3 of 11

where CN is a carbon number and DB is the number of double bonds [13]; this ranged from 42 to 57.
The
The calculation
calculation of of the
the equivalent
equivalent carbon
carbon number
number isis aa powerful
powerful tool
tool for
for identifying
identifying TG TG [14].
[14]. Table
Table 11
presents TGs eluted from the column in order of increasing number of carbon
presents TGs eluted from the column in order of increasing number of carbon atoms, and of increasing atoms, and of
increasing
unsaturationunsaturation
for the samefor number
the sameofnumber
carbon of carbon
atoms. Inatoms.
terms Inof terms
the sameof theCN, same CN, theorder
the elution elution
of
order of TGs
TGs was was increased
increased by DB.
by DB. PPP PPP appeared
appeared first, duefirst, due to having
to having the lowestthe CN
lowest CNNNN,
of 48. of 48. with
NNN,CN with
57,
CN
was57,
anwas an uncommon
uncommon TG thatTG that eluted
eluted last. Thelast. The peaks
peaks of TGsofwithTGsthewith the CN
same sameand CNDBand DB
are are close,
very very
close, such
such as OOL asand
OOL andLLO,
LLS, LLS,and
LLO, and OOLn.
OOLn. Two sets Two setspositional
of TG of TG positional
isomers—OOP isomers—OOP
and OPO,andandOPO,
LLO
and LLO and LOL—were eluted together. The relative retention times (rrt)
and LOL—were eluted together. The relative retention times (rrt) were calculated using Equation (1) were calculated using
Equation
and ranged(1) and
fromranged
0.70 tofrom
0.80 0.70 to 0.80TGs,
for three for three
0.80 toTGs,
0.900.80 to for
also 0.90three
also for
TGs, three
andTGs,
moreand more
than than
0.90 for
0.90 for 11 standards
11 standards of TGs1).
of TGs (Table (Table 1).

Figure
Figure2.
2.GC-FID
GC-FIDchromatogram
chromatogramof
oftriacylglycerols
triacylglycerols (TG)
(TG) standards.
standards.

Forvalidation,
For validation,the theHTGC-FID
HTGC-FIDmethod method waswas used
used to determine
to determine the the
TGsTGs in edible
in edible oils. oils.
To thisToend,
this
end, solutions
solutions of 17 individual
of 17 individual standards
standards were prepared.
were prepared. Relative Relative
responseresponse
factorsfactors
(RRFs),(RRFs),
recovery, recovery,
limits
limits
of of detection
detection (LOD),(LOD),
limits of limits of quantitation
quantitation (LOQ),(LOQ),
precision,precision, and repeatability
and repeatability are presented
are presented in Tablein
Table 1. The recovery of TG standards ranged from 21% to 148%, and
1. The recovery of TG standards ranged from 21% to 148%, and RRF ranged from 0.42 to 2.28. TheRRF ranged from 0.42 to 2.28.
The limits
limits of detection
of detection were inwerethe in the range
range of 0.001 ofto0.001
0.330toµg/mL,
0.330 µg/mL,
while thewhile theoflimits
limits of quantitation
quantitation were in
were in the range of 0.001 to 1.000 µg/mL. The precision of the HTGC-FID
the range of 0.001 to 1.000 µg/mL. The precision of the HTGC-FID method was evaluated by assessing method was evaluated by
assessingand
intraday intraday
interdayandprecision
interdayby precision
analyzing by analyzing
TG solutions TGcontaining
solutions containing
1.25 µg/mL1.25 µg/mL
of each of each
standard,
standard, the
reporting reporting the retention
retention times, andtimes, and calculating
calculating the normalized
the normalized areas. Theareas. The intraday
intraday precision precision
on the
on the retention times of the TG was lower than 0.5%, and area precision
retention times of the TG was lower than 0.5%, and area precision was below 5% (data not showed). was below 5% (data not
showed).
The interday Theprecision
interdayon precision on thetimes
the retention retention
of thetimes of the was
standards standards was1.0%,
less than less than 1.0%,
and the peakandarea
the
peakless
was areathan
was 27%.
less than 27%.
In the In the reference,
reference, a wide arange wide ofrange of precision
precision was accepted,
was accepted, depending
depending on theon
the complexity of
complexity of the matrix.the matrix.
For the
For thestandards,
standards,solutions
solutionsofof TGTG calibration
calibration curves
curves werewere prepared
prepared in range
in the the range
of 0.5ofto0.5 to
10.0
10.0 mg/mL. Regression coefficients for standard curves ranged from 0.9900
mg/mL. Regression coefficients for standard curves ranged from 0.9900 for POO to 0.9980 for POP. for POO to 0.9980 for POP.
Molecules 2020, 25, 3881 4 of 11

Table 1. Chemical and validation parameters used for the analysis of TG by GC-FID method.

TG CN DB ECN rrt RRF Recovery (%) LOD (µg/mL) LOQ (µg/mL) Precision (%) Repeatability (%) RSD (%)
LLL 54 6 42 0.97 0.44 21 0.001 0.001 2.8 8.0 1.80
LLO 54 5 44 0.95 1.47 148 0.083 0.251 20.0 52.6 3.53
LOL 54 5 44 0.95 0.73 74 0.169 0.511 7.0 19.5 2.63
OOLn 54 5 44 0.96 0.42 36 0.330 1.000 17.7 49.6 11.74
OOL 54 4 46 0.94 1.11 56 0.109 0.330 18.0 50.3 4.46
LLS 54 4 46 0.95 0.85 41 0.140 0.424 2.26 6.3 0.73
PPL 50 2 46 0.79 1.11 73 0.125 0.379 7.3 20.7 1.84
PPP 48 0 48 0.70 0.86 79 0.140 0.421 2.2 6.3 0.73
OOP 52 2 48 0.85 1.39 140 0.088 0.267 0.3 0.7 3.64
OPO 52 2 48 0.86 1.24 125 0.181 0.549 0.5 1.5 2.87
OOO 54 3 48 0.93 2.28 102 0.053 0.16 26.7 74.8 3.23
PPS 50 0 50 0.77 1.72 108 0.001 0.001 20.7 57.9 3.33
PSO 52 1 50 0.85 1.52 74 0.170 0.515 22.6 63.4 4.11
OOS 54 2 50 0.92 1.72 99 0.067 0.202 0.2 0.6 0.57
SSO 54 1 52 0.91 1.00 61 0.001 0.001 8.2 23.0 2.27
SSS 54 0 54 0.91 1.00 90 0.010 0.010 2.1 20.1 1.92
NNN 57 0 57 1.00 1.00 100 0.117 0.354 16.1 45.1 4.45
CN: Carbon numbers; DB: Double bond numbers; ECN: Equivalent of carbon number; RRF: Relative response factor; rrt: Relative retention time. Repeatability is expressed as the relative
standard deviation of TGs; RSD: Relative standard deviation; LOD: Limits of detection; LOQ: Limits of quantitation.
Molecules 2020, 25, 3881 5 of 11
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2.2. 2.2.
Identification
2.2.
of TGs
Identification
Identification
in in
of TGs
of TGs
Olive Oil
Olive Oiland
in Olive
Refined
andand
Oil Refined Rapeseed
Rapeseed
Refined
OilOil
Oil
Rapeseed
TheThelogarithms
Thelogarithms of of
logarithms standards’
standards’ relative
of standards’relative retention
retention
relative retentiontimes
times (rrt)
(rrt)
times were
were
(rrt) were used
used toto
used todevelop
develop
develop the
thethe
plot plot
used
plot used
used for
forfor
identification
identification [15,16]
[15,16] (Figure
(Figure 3).
3). Firstly,
Firstly, the
the saturated
saturated original
original standards
standards were
werelocated on
located
identification [15,16] (Figure 3). Firstly, the saturated original standards were located on the graph the
on graph
the graph
on on
on the thethe
doubledouble
doublebonds
bonds bonds number
number number(DB)
(DB) equals
equals
(DB) zero
zero
equals axis.
axis.
zero Secondly,
Secondly,
axis. Secondly, the
the monoacid
themonoacid
monoacidoriginal standards
original
original standards
standards were were
were
positioned
positioned on the
on theongraph,
positioned graph, and
andand
the graph, these
these points
points
these were
pointswere connected
wereconnected by
connected by lines.
bylines.Then, the
lines.Then,
Then,TGs
thetheconstituting
TGsTGs of two
constituting
constituting of twoof
two types
types
types ofoffatty
offattyacids
fatty acidswere
acids werelocated
were located
locatedover thethe
over
over line connecting
theline
lineconnectingthethe
connecting two corresponding
two
the corresponding
two monoacid
correspondingmonoacid TGs. TGs
TGs.
monoacid TGs
TGs.
with three
with different
three differentfatty acids
fatty were
acids placed
were in
placed the
in space
the between
space between lines connecting
lines the
connecting monoacid
the monoacid TGs.TGs.
TGs with three different fatty acids were placed in the space between lines connecting the monoacid
Following
Following this, thethe
this, points of unknown peaks were placed on on
thethe
graph andand
identified (Figure 4). 4).
TGs. Following this, the points
pointsofofunknown
unknownpeaks peaks were
wereplaced
placed graph
on the graph identified
and (Figure
identified (Figure 4).

Figure 3. GC-FID
Figure
Figure chromatograms
3. GC-FID chromatograms
3. GC-FID ofofTG
chromatograms TG in
in (A)
of TG (A) olive
olive
in (A) oiloil
oil
olive (OO)
(OO) and
and
(OO) (B)
(B)
and refined
refined
(B) rapeseed
rapeseed
refined oil (RRO).
oil oil
rapeseed (RRO).
(RRO).

Figure
Figure 4.4.Plot
Figure Plot for
4. Plot identification
forfor
identificationof of
identification unknown TGsTGs
of unknown
unknown in the
TGs inintested
the refined
tested
the rapeseed
refined
tested oil (RRO)
rapeseed
refined and
oil (RRO)
rapeseed oil olive
and
(RRO) oil
olive
and (OO).
oilolive
(OO).
oil (OO).
Molecules 2020, 25, 3881 6 of 11

2.3. Determination of TGs in Blended Olive Oil and Refined Rapeseed Oil
The validated method was used to determine the TGs in olive oil (OO), refined rapeseed oil (RRO),
and their blends. The results are shown in Table 2, and the GC chromatograms of TG in olive oil and
refined rapeseed oil are presented in Figure 3.
Eight TGs were detected in the refined rapeseed oil, and 10 in the olive oil (Table 2). The main
TG in both oils was OOO, which was made up of 46% of RRO and 52% of OO, respectively. The next
TGs in RRO was OOL (21%) and OOLn (13%), while OO contained OOP (25%) and OOL (9%). OOP,
POL, POLn, LLO, and OOS were both detected in RRO (6%, 6%, 3%, 4%, 1.5%, respectively) and OO
(25.4%, 6%, 2%, 1%, respectively). Three TGs (PPO, PPL, and PSO) were determined in OO (2%, 1%,
1%, respectively) but were not detected in RRO, and one TG OOLn determined in RRO (21%) was not
found in OO. PPO made up 2% of olive oil, and PPL and PSO made up 1%. The amount of OOLn in
RRO was 13%. These results agree with the literature data [4,14,17]. PPO, PPL, and PSO were detected
in olive oil, palm oil, and peanut oil [4], but were not identified in rapeseed oil [14].
OOLn was identified in pine seed oil (8%) and soybean oil (2%), but was absent from olive,
sunflower, sesame, corn, wheat germ, rice bran flax seed, melon, and pomegranate seed oil [4,16,18].
These TGs could serve good indicators for identifying blends of RRO and OO.
Most of the literature data on triacylglycerols in edible oils are based on their distribution by ECN,
and describe the percentage of these groups [14,19,20]. The percentage composition of TGs depends
on the number of compounds detected. In the tested RRO, triacylglycerols with ECN48 dominated
(52%), followed by ECN46 (27%), ECN44 (20%), and ECN50 (1%). TGs with ECN48 were the main
compounds in olive oil (79%), while ECN46, ECN50, and ECN44 made up 16%, 4%, and 1%. TGs with
ECN48 made up 62%–72% of high oleic rapeseed oil (OOO + GLO), 43%–49% of medium oleic acid
rapeseed oil (OOO + GLO), and 25%–27% of low oleic high erucic rapeseed oil (OPO + GLP + OOO +
GLO + ErLnP + GGLn) [14]. The composition of extra-virgin olive oil ranged from 42% (OOP + OOO)
to 50% of TGs with ECN48 [17,20].
The determination of individual TGs in rapeseed and olive oils allows the identification of key
TGs that may be distinguishing features of adulterated oils. When 1% of OO was added to RRO, peaks
of PPO, PPL, and PSO were detected at 0.8, 0.9 and 0.2 mg/g, (0.08%, 0.10%, and 0.01%), respectively
(Table 2). The content of OOP increased from 47.4 mg/g in RRO (5%) to 71.5 mg/g (8%) in blended
oil. When 2.5% OO was added to RRO, PPO content increased to 3.9 mg/g (0.44%), PPL to 1.9 mg/g
(0.21%), PSO to 1.4 mg/g (0.16%), and OOP to 82.4 mg/g (9.24%). The contents of these TGs increased
in proportion to the fraction of OO in RRO.
When RRO was added to OO, the level of OOLn was used as an indicator of adulteration.
We observed an increase in the content of this TG in blends from 2.2 mg/g (1% RRO/OO) to 4.1 mg/g
(2.5% RRO/OO), 6.0 mg/g (5% RRR/OO) to 12.2 mg/g (10% RRO/OO) (Table 2). Changes in the content
of other TGs in the blended oils were not unambiguous, and can only suggest the adulteration of olive
oil by rapeseed oil.
When OO was adulterated by soybean, sunflower, or corn oils, the absolute value of the differences
between the theoretical and experimental ECN42 (LLL) contents was the most effective means of
detecting even low levels of adulteration [20,21]. The content of linoleic acid in rapeseed oil is
much lower than in soybean, sunflower, or corn oils, and LLL was not detected in our experiment.
The greatest differences between the theoretical and experimental content of TG in the blended oils
were detected for LLO and ranged from 3% to 9%.
Molecules 2020, 25, 3881 7 of 11

Table 2. Triacylglycerol levels in refined rapeseed oil, olive oil, and their blends (mg/g).

Blended Oils OO/RRO Blended Oils RRO/OO

TGs RRO OO
1.0% 2.5% 5.0% 10.0% 1.0% 2.5% 5.0% 10.0%
PPO Nd 20.8 ± 2.8 0.8 ± 0.3 3.9 ± 0.8 4.3 ± 0.3 7.9 ± 0.3 18.4 ± 0.8 17.0 ± 0.5 15.0 ± 0.8 12.4 ± 0.6
PPL Nd 9.3 ± 4.1 0.9 ± 0.2 1.9 ± 0.6 2.1 ± 0.1 2.7 ± 0.1 8.5 ± 0.4 8.4 ± 0.3 8.1 ± 0.5 7.4 ± 0.4
PSO Nd 6.7 ± 0.8 0.2 ± 0.1 1.4 ± 0.5 1.9 ± 0.1 5.8 ± 0.3 7.6 ± 0.3 6.0 ± 0.4 4.1 ± 0.5 3.9 ± 0.2
OOP 47.4 ± 2.1 254.2 ± 8.2 71.5 ± 3.7 82.4 ± 2.3 105.7 ± 4.3 124.7 ± 5.6 246.5 ± 8.7 235.0 ± 7.1 212.0 ± 9.6 190.9 ± 10.1
POL 53.3 ± 2.6 60.6 ± 2.5 48.3 ± 2.3 50.1 ± 2.0 54.2 ± 3.0 42.4 ± 2.0 55.2 ± 2.7 49.0 ± 2.3 43.3 ± 2.1 43.7 ± 1.9
POLn 26.9 ± 1.4 1.6 ± 0.5 26.1 ± 1.7 24.3 ± 1.6 23.0 ± 1.1 18.9 ± 0.9 2.8 ± 0.1 4.9 ± 0.2 6.4 ± 0.2 11.7 ± 0.3
OOS 13.7 ± 4.1 33.3 ± 1.1 11.7 ± 0.8 14.5 ± 0.8 20.2 ± 1.4 23.3 ± 1.1 33.2 ± 1.4 31.7 ± 1.8 29.7 ± 1.2 22.5 ± 0.9
OOO 424.1 ± 10.1 516.5 ± 11.1 429.3 ± 9.6 430.2 ± 9.3 443.8 ± 8.6 470.0 ± 10.8 515.6 ± 8.8 507.8 ± 9.8 504.9 ± 10.6 500.2 ± 9.9
OOL 194.6 ± 8.3 88.5 ± 6.4 183.3 ± 7.7 155.4 ± 7.5 137.7 ± 5.3 121.5 ± 6.2 83.6 ± 5.7 91.0 ± 4.5 107.6 ± 5.2 114.3 ± 4.6
OOLn 117.8 ± 5.7 Nd 116.8 ± 5.1 97.3 ± 3.8 80.8 ± 2.1 68.2 ± 3.1 2.2 ± 0.1 4.1 ± 0.3 6.0 ± 0.4 12.2 ± 0.6
LLO 34.9 ± 2.2 8.4 ± 0.6 32.6 ± 0.9 30.7 ± 1.1 28.3 ± 1.1 24.9 ± 0.9 8.7 ± 1.1 9.4 ± 0.5 9.9 ± 0.5 12.7 ± 0.6
RRO: Refined rapeseed oil; OO: Olive oil.
Molecules 2020, 25, 3881 8 of 11

3. Materials and Methods

3.1. Materials
All solvents of analytical grade were purchased from Sigma-Aldrich (Steinheim, Germany). Standards
of the triacylglycerols 1,2-linoleoyl-3-oleoyl-sn-glycerol (OLL), 1,3-palmitoyl-2-oleoyl-sn-glycerol (POP),
1,3-palmitoyl-2-linoleoyl-sn-glycerol (PLP), trinonadecanoyl-glycerol (NNN), at over 99% purity, were
obtained from Sigma-Aldrich (St. Louis, MO, USA). Trioleoyl-glycerol (OOO), 1,2-oleoyl-3-sn-palmitoyl-
glycerol (OOP), 1,2-palmitoyl-3-linolein-sn-glycerol (PPL), 1,3-oleoyl-2-palmitoyl-sn-glycerol (OPO),
1,2-palmitoyl-3-stearoyl-sn-glycerol (PPS), 1,2-oleoyl-3-stearoyl-sn-glycerol (OOS), 1,2-stearoyl-3-
oleoyl-sn-glycerol (SSO), 1-palmitoyl-2-stearoyl-3-oleoyl-sn-glycerol (PSO), 1,2-oleoyl-3-linoleoyl-
sn-glycerol (OOL), 1,2-linoleoyl-3-oleoyl-sn-glycerol (LLO), 1,2-oleoyl-3-linolenoyl-sn-glycerol (OOLn),
1,3-linoleoyl-2-oleoyl-sn-glycerol (LOL), tristearoyl-glycerol (SSS), tripalmitoyl-glycerol (PPP),
and 1,2-linoleoyl-3-steareroyl-sn-glycerol (LLS), at over 99% purity, were purchased from Larodan
(Solna, Sweden). Refined olive pomace oil produced by Primadonna (Poland) and refined rapeseed oil
produced by ZT Kruszwica (Warsaw, Poland) were purchased from a supermarket in Poland.

3.2. Sample Preparation

Solutions of 17 individual TG standards (PPP, PPS, PPL, PSO, OOP, OPO, SSO, OOS, OOO, OOL,
LLS, LLO, LOL, SSS, OOLn, LLL, and NNN) were prepared. Briefly, 10 mg of each TG standard was
dissolved in 10 mL of dichloromethane. Then 1 mL of each prepared solution was placed in a 2 mL
autosampler vials, and 1 µL was injected and separated by gas chromatography.
We prepared 7 solutions of TG standards with concentration ranging from 0.05 to 10 mg/mL to
determine linearity.
Before blending, the refined rapeseed oil (RRO) and olive oil (OO) underwent TG analysis.
The blends consisted of 1%, 2.5%, 5%, and 10% additions of RRO to OO and OO to RRO. 30 mg
of each oil (RRO, OO, and the blends) was weighed out in 10 mL volumetric flasks, and 3 mg of
trinonadecanoyl-glycerol (NNN) was added to all samples as an internal standard (IS). The oils
were then dissolved in 10 mL dichloromethane, and 1 mL of the solution was placed in 2 mL vials.
All samples were prepared in triplicate.

3.3. HTGC-FID Conditions

Solutions of standards and oils were injected by autosampler (Thermo Scientific AI 1310) with a
split ratio of 1:30 into a Thermo Scientific Trace 1300 gas chromatograph equipped with an RTX-65TG
capillary column (30 m × 0.25 mm i.d. 0.1 µm; Restek Corp., Bellefonte, PA, USA) and flame ionization
detector. The GC oven temperature was programmed to rise from 250 ◦ C to 360 ◦ C at 4 ◦ C/min, before
being held for 25 min. The injection port was held at 360 ◦ C. Hydrogen (99.99%) was used as the carrier
gas at a flow rate of 1.5 mL/min.

3.4. Validation
The parameters used to validate the method were: Fitting an analytical curve and determining its
linearity, recovery, limit of detection (LOD), limit of quantitation (LOQ), precision, relative standard
deviation (RSD), and repeatability. These parameters were calculated by peak area and evaluated
according to AOCS Official Methods Ce 5b-89 (2009) and Cd 11c-93 (2009) [15,22].
For linearity, 7 solutions of each TG standard in the range of 0.05 to 10.0 mg/mL were prepared
and injected 3 times per concentration level. The linearity of the method for each TG was evaluated by
determining the coefficient (r2 ) after the construction of the analytical curves. The precision of the
method was determined as the relative standard deviation from 6 replicates of the prepared standard
mixture at 0.05 mg/mL concentration.
The repeatability of the method was evaluated to determine the content of TG in the mix of
standards, using trinonadecanoyl-glycerol (NNN) as the internal standard (IS). The procedure was
Molecules 2020, 25, 3881 9 of 11

carried out on different days to obtain the intermediate precision. The relative standard deviation
(RSD) was calculated according to equation RSD (%) = Standard deviation/Mean × 100%.
Spike recovery was calculated by using 10 mg of each commercial standard mixed in 1 mL
dichloromethane. NNN was added as an internal standard to the calculate peak area of each standard.
The recovery (%) = Final amount detected/Amount spiked × 100%.
Limits of detection (LOD) and limits of quantitation (LOQ) were calculated for each TG using
17 standards at the lowest concentration of 0.05 mg/mL. LOD was estimated as the ratio of blank
signal to TG standard signal at the lowest concentration that was reliably distinguished from the blank
sample and for which detection was feasible. LOQ was the lowest concentration of the compound for
which quantitation was acceptable. LOQ was calculated for all TG standards at 3 times the limit of
detection obtained for each TG.

3.5. Calculation of Relative Response Factors (RRF)

Trinonadecanoyl-glycerol (NNN) was selected as the internal standard to determine the TG
content, because NNN was not present in edible oils. The relative response factors (RRS) were
calculated for all the analyzed standards of TGs, obtained by relating the area and concentration of
each standard to the concentration and area of the IS.

3.6. Identification of TG
In line with Pacheco et al. (2014) and AOCS Official Method Ce 5b-89 (2009), Equation (1) was
used to calculate the relative retention time, which relates the retention time (rt) of an analyte to solvent
and IS retention time.
rtanalyte − rtsolvent
rrt = (1)
rtIS − rtsolvent
rrt: Relative retention time; rtanalyte : Retention time of TAGs; rtsolvent : Retention time of dichloromethane;
rtIS : Retention time of internal standard.

4. Conclusions
This work has shown that the HTGC-FID method can be used to identify blending of rapeseed oil
with olive oil at the level of 1%. The method described is a fast one-step method (28 min) involving only
dilution of the oil in a solvent, and it allows separation and quantitation of 20 individual TGs. For TGs
typical of edible oils, the method’s recovery, precision, and repeatability provide reliable indications of
oil blending. When olive oil is added to rapeseed oil, or when olive oil is adulterated with rapeseed oil
at the level of 1%, this method can be used for quality control of these oils. Three triacylglycerols were
identified as indicators of the addition of olive oil to rapeseed oil (PPO, PPL, PSO). The presence of
OOLn indicated the adulteration of olive oil with refined rapeseed oil.

Author Contributions: Methodology and validation: Y.Q. and M.R.; writing and original draft: M.R., A.G. and
Y.Q.; writing and review: R.P. and M.R.; conceptualization: M.R. and R.P. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the National Science Centre, Poland grant number 2018/31/B/NZ9/00602.
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations
Triacylglycerols (TG); monoacylglycerols (MAG); diacylglycerols (DAG);1,2-linoleoyl-3-oleoyl-sn-glycerol
(OLL); 1,3-palmitoyl-2-oleoyl-sn-glycerol (POP); 1,2-palmitoyl-3-oleoyl-sn-glycerol (PPO), 1,3-palmitoyl-2-
linoleoyl-sn-glycerol (PLP); trinonadecanoyl-glycerol(NNN); trioleoyl-glycerol (OOO); 1,2-oleoyl-3-sn-
palmitoyl-glycerol (OOP); 1,2-palmitoyl-3-linolein-sn-glycerol (PPL); 1,3-oleoyl-2-palmitoyl-sn-glycerol (OPO);
1,2-palmitoyl-3-stearoyl-sn-glycerol (PPS); 1,2-oleoyl-3-stearoyl-sn-glycerol (OOS); 1,2-stearoyl-3-oleoyl-sn-glycerol
(SSO); 1-palmitoyl-2-stearoyl-3-oleoyl-sn-glycerol (PSO); 1,2-oleoyl-3-linoleoyl-sn-glycerol (OOL); 1,2-linoleoyl-3-
oleoyl-sn-glycerol (LLO); 1,2-oleoyl-3-linolenoyl-sn-glycerol (OOLn); 1,3-linoleoyl-2-oleoyl-sn-glycerol
Molecules 2020, 25, 3881 10 of 11

(LOL); tristearoyl-glycerol (SSS); tripalmitoyl-glycerol (PPP); 1,2-linoleoyl-3-steareroyl-sn-glycerol (LLS);

High Temperature Gas Chromatography- Flame Ionization Detector (HTGC-FID).

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Sample Availability: Samples of the compounds are not available from the authors.

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