Article

pubs.acs.org/JAFC

Ecient Separation and Analysis of Triacylglycerols: Quantitation of

Palmitate (OPO) in Oils and Infant Formulas
Mikhail Vyssotski,*, Stephen J. Bloor, Kirill Lagutin, Herbert Wong, and D. Bradley G. Williams*,,

Callaghan Innovation, 69 Graceeld Road, P.O. Box 31310, Lower Hutt 5040, New Zealand
Graceeld Research Centre, Ferrier Research Institute, Victoria University of Wellington, 69 Graceeld Road, Lower Hutt 5010,
New Zealand

S Supporting Information
*

ABSTRACT: A high-eciency, convenient, and reliable method for the separation of structurally similar triacylglycerols is
detailed and applied in the quantitative analysis of 1,3-dioleoyl-2-palmitoylglycerol (OPO) in infant formulas and OPO oils.
OPO is an important lipid component in humanized infant formula. A fast preparative isolation of an OPO-containing fraction
from the crude complex mixture, by nonaqueous reversed phase HPLC, followed by Ag+-HPLC with detection at 205 nm
allowed ne separation and detection of the desired fraction. OPO was quantitated independently of its regioisomer 1,2-dioleoyl3-palmitoylglycerol (OOP) and isomers of stearoyl-linoleoyl-palmitoyl glycerol that might be present in infant formulas. For
samples with low OPO content, an evaporative light-scattering detector (ELSD) was more preferable than UV detection, with a
calculated LOD of 0.1 g of OPO injected and LOQ of 0.3 g. The method, which showed high reproducibility (RSD < 5%),
was suitable for both high OPO content oils and low OPO products such as unenriched infant formula. A number of possible
interference issues were considered and dealt with.
KEYWORDS: regiospecic analysis, HPLC, ELSD, triacylglycerol, fatty acid, infant formula

their use in the manufacture of humanized formula.9 Despite

the eorts of manufacturing companies to include OPO in their
formulas, Kurvinen et al.10 have found in a now-dated study
analyzing 32 commercially available infant formulas that only 4
of the milk formulas contained more OPO than OOP, and only
3 of these had an OPO/OOP ratio close to that of human milk.
Interestingly, although the 1,3-dimonounsaturated-2-saturated
pattern (to which OPO belongs) is typical for human milk, no
TGs matching this description were found in 11 infant formulas
studied by Son et al.11
To quantitate TGs in a regiospecic manner, one or more of
a number of techniques may be used, each with its own
advantages and limitations. These include methods employing a
chemical or enzymatic hydrolysis step, NMR spectroscopic
approaches, and chromatographymass spectrometry techniques.12,13 Methods that involve the partial hydrolysis of TGs
using Grignard14 or enzymatic15,16 cleavage are low throughput,
being multistep and complex. The enzymatic approach (e.g.,
using pancreatic lipase) may not be suitable for the analysis of
milk and milk substitutes that contain short-chain fatty acids
due to variable and unpredictable selectivity for fatty acid chain
length.17 There is also ongoing controversy about which TG
hydrolysis products deliver better accuracymonoacylglycerols
or diacylglycerolsdue to the possibility of isomerization of
partially acylated glycerols and the likelihood of accumulating
errors as a consequence.18 The problems associated with
existing analytical methods are summarized in the words of

INTRODUCTION
Lipid analysis is critical in several research and commercial
elds. For example, human milk substitutes are highly sought
after for bottle-fed infants to ensure optimum nutrition.
Because human milk diers in a number of parameters from
the dairy sources used in the production of infant formulas,
successful infant nutrition relies on reducing this dierence by
supplementing the dairy ingredients to more closely resemble
human milk. There is a signicant gap between human milk and
dairy products in their qualitative and quantitative composition
of neutral and polar lipid classes. A useful summary of many of
the dierences in lipid content between human and cow milk1
is supplemented by other works detailing dierences in specic
lipid classes.24 One lipid component of ongoing commercial
interest is 1,3-dioleoyl-2-palmitoylglycerol (OPO), the major
triacylglycerol (TG) species in human milk, comprising 11.8%
of the total triacylglycerols there.5 According to current views,
TGs possessing saturated fatty acids at the glycerol side
positions (also referred to as -, or sn-1 and sn-3 positions) may
have an adverse eect on a babys digestion due to the
formation of insoluble calcium salts. This is believed to reduce
the bioavailability of calcium and is a cause of constipation.6 In
contrast, TGs lacking saturated fatty acids in the sn-1 and sn-3
positions are viewed more positively and are said to increase
the bioavailability of calcium.7,8 In OPO (also known in the
industry as -palmitate),5,6 the palmitic acid residue is less
accessible to digestive tract sn-1,3-specic lipases. However, the
claim that OPO supplementation contributes to increases in
calcium absorption was recently rejected by the European Food
Safety Authority.6 Even with this nding, OPO oils and
advanced infant formulas containing OPO continue to be
produced, likely due to their benecial marketing status and
XXXX American Chemical Society

Received: April 13, 2015

Revised: June 9, 2015
Accepted: June 13, 2015

DOI: 10.1021/acs.jafc.5b01835
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prepared from 1-monopalmitoylglycerol and a mixture of stearic and

linoleic acids (1.00:1.03 mol/mol) using the same method. Similarly, a
mixture of tristearin, triolein, 1-stearoyl-2,3-dioleoyl-, 1,3-dioleoyl-2stearoyl-, 1,2-distearoyl-3-oleoyl-, and 1,3-distearoyl-2-oleoylglycerols
was prepared from glycerol and a mixture of stearic and oleic acids
(1.00:1.00 mol/mol). Synthetic samples were puried by column
chromatography on Merck silica gel 70230 mesh 100 grade 10184
(Sigma-Aldrich, St. Louis, MO, USA) with the consecutive use of
hexane, hexane/dimethyl ether (99:1, by v/v), and hexane/diethyl
ether (95:5, by v/v) solvent systems and controlled by TLC using
Merck HPTLC silica gel 60 10 10 cm (Darmstadt, Germany) in
hexane/diethyl ether/formic acid (80:20:2, v/v/v) with detection by
iodine vapor. The composition of the samples was assessed by GCFID. The regiospecic distribution of the saturated, monounsaturated,
and diunsaturated fatty acids in triacylglycerols was assessed using
NMR spectroscopy,27 with the spectrum acquisition parameters
modied as presented under NMR Spectroscopy below. DCC was
from Acros Organics (Fisher Scientic, Pittsburgh, PA, USA). The
same approach was used to synthesize a mixture of OOO, OOE, OEO,
OEE, EOE, and EEE starting from oleic and elaidic acids (1.00:1.04
mol/mol) and glycerol. HPLC solvents (acetone, acetonitrile, nhexane) were Merck LiChrosolv solvents for liquid chromatography
(Sigma-Aldrich).
Extraction of TGs from Infant Formula. A 2 g sample of the
infant formula powder (sample 4) was extracted using a modication
of the method of Svennerholm and Fredman28 to ensure that polar
lipids (required for other assays, not related to this work) were also
extracted. The original method was modied as follows: 2 g of powder
was combined with 6 mL of water and mixed with 16 mL of methanol.
This solution was then extracted sequentially with chloroform (8 mL)
and 1:1 chloroform/methanol (16 mL) and twice with 2:1
chloroform/methanol (12 mL) with the use of ultrasonication (5
min/extraction). The centrifuged lipid extracts from each extraction
were combined and transferred to a separating funnel and washed with
water (23 mL) and then with 10% aqueous KCl (3 mL). The lower
(organic, chloroform-rich) layer was dried and constituted the crude
lipid extract. A portion (40 mg) of the crude lipid extract was dissolved
in CHCl3 (2 mL) and puried by passage through a Phenomenex
Strata 500 mg silica SPE column (Torrance, CA, USA). Elution with
chloroform (4 mL) produced a TG-rich fraction, which was dried
under a stream of nitrogen gas and used directly for RP-HPLC.
GC-FID. Fatty acid methyl esters (FAMEs) were prepared from
samples 13 by direct transmethylation without prior lipid extraction.
Sample 4 was extracted as described above and converted into
FAMEs.29 GC analysis of the FAMEs was performed on a Trace GC
Ultra gas chromatograph (Thermo Fisher Scientic, Waltham, MA,
USA) equipped with a ame ionization detector (FID) and TraceGold
TG-WaxMS capillary column of dimensions 30 m 0.25 mm i.d., 0.25
m (Thermo Fisher Scientic, Waltham, MA, USA). Helium (99.99%)
was used as carrier gas (Air Products, Allentown, PA, USA), and a split
ratio of 100:1 was maintained. Injector and detector temperatures
were both set at 280 C, and the oven was held at 190 C for 50 min.
Individual peaks of FAME were identied by comparison with
standards of FAME and by equivalent chain length values.30
GC-FID analysis of acylglycerols was performed on the same gas
chromatograph equipped with a CP-TAP CB UniMetal capillary
column of dimensions 25 m 0.25 mm i.d., 0.1 m, supplied by
Agilent (Santa Clara, CA, USA). Samples were injected in splitless
mode. Helium (99.99%) was used as carrier gas (Air Products).
Injector and detector temperatures were set to 350 and 370 C,
respectively. The oven temperature was raised from 60 to 200 C at a
rate of 120 C/min, then increased to 320 C at a rate of 20 C/min,
followed by a rise to 360 C at a rate of 1 C/min, and was maintained
at that temperature for 6 min.
NMR Spectroscopy. In a typical experiment 2045 mg of a
sample was dissolved in 0.75 mL of CDCl3. 13C NMR spectra were
recorded at 125.7 MHz with WALTZ16 proton composite pulse
decoupling on a Bruker Avance III NMR spectrometer (Bruker,
Billerica, MA, USA) equipped with an automatic tuning 5 mm
multinuclear probe at 303 K. The pulse program ZGIG, which was

world-leading contributors to the development of methods for

positional analysis of glycerolipids: the assays of stereo- and
regioisomers are more demanding than conventional lipid
analyses...All of the methods are subject to improvement.19
Attempts to solve these problems include a comprehensive
procedure employing an o-line two-dimensional HPLC-MS
technique.2023 Its advantage is an ability to identify and
quantitate a number of TG regioisomers (e.g., the data for 11
TG species were presented in one study).20 However, with MSbased detection other issues arise: some TG species in the
sample present linear calibration plots, whereas others possess
nonlinear plots, complicating the data processing.24 Here, it was
concluded that linearity should not be assumed as nonlinear
calibration plots may be obscured by scatter in the data or by
plots where there is only slight curvature. Additional care and
processing of the data are thus required to ensure the highest
condence in the results. Indeed, dierent MS detectors are
known to give dierent ratios of isomeric TGs.23 For example,
when ionizing OOP, the OO/OP ratio of fragment ions varies
from 51 with a Thermo Orbitrap to 64 with a Bruker Ion trap,
whereas ionization of OPO with the same instruments provides
OO/OP ratios of 16 and 21, respectively. Similarly, ionization
of SOO provides OO/SO ratios ranging from 50 for a Bruker
Ion trap to 70 for a Bruker QqTOF instrument, whereas OSO
provides OO/SO ratios of 17 and 22, respectively, for the same
instruments. Another recently reported study uses o-line twodimensional chromatography by applying nonaqueous reversed
phase (RP)-HPLC followed by Ag+-HPLC of the selected
fractions using evaporative light scattering detection (ELSD).25
When two-dimensional chromatography is not used, separation
of isomers and potential contaminants remains problematic,
and the resulting chromatograms can be complex and
unresolved;22,23 among others, the separation of the OPO/
POO pair has not been solved.
The goal of this work was to establish a convenient and
reliable method by which to separate structurally similar TGs,
with the targeted quantitation of OPO. A range of techniques
was investigated with a view to comparing the advantages and
limitations of each. This paper details eorts to provide a robust
separation and analytical method that provides data with a high
level of condence and for which a range of potential
contaminants were considered.

MATERIALS AND METHODS

Chemicals and Materials. Oleic, linoleic, elaidic, and stearic acids

and glycerol were purchased from Sigma-Aldrich (USA). 1Monopalmitoylglycerol, 1,3-dipalmitoylglycerol, 1,2-dipalmitoylglycerol, 1,3-dioleoyl 2-palmitoylglycerol (OPO), rac 1,2-dioleoyl-3palmitoylglycerol (OOP), 1,2-dipalmitoyl-3-oleoylglycerol (PPO),
1,3-dipalmitoyl-2-oleoylglycerol (POP), tripalmitin (PPP), triheptadecanoin (HHH), triolein (OOO), trielaidin (EEE), trilaurin, trimyristin,
tristearin, triarachidin, tribehenin, tripalmitolein, tripetroselinin,
trielaidin, tri-11-eicosenoin, trierucin, trilinolein, and trilinolenin
were purchased from Sigma-Aldrich (St. Louis, MO, USA). The
triglyceride mix TG2-TG10 was from Supelco (Sigma-Aldrich, St.
Louis, MO, USA). Commercial samples for analysis included sample 1,
a dietary ingredient comprising dairy milk solids blended with OPOcontaining oil; sample 2, OPO oil; sample 3, OPO oil (samples, 1, 2
and 3 were kind donations of industrial food grade products from a
New Zealand company); and sample 4, infant formula.
POP and PPO were synthesized from 1,3-dipalmitoylglycerol and
1,2-dipalmitoylglycerol, respectively, and oleic acid, with dicyclohexylcarbodiimide (DCC) as the coupling agent.26 A mixture of 1palmitoyl-2,3-distearoyl-, 1-stearoyl-2-linoleoyl-3-palmitoyl-, 1-linoleoyl-2-stearoyl-3-palmitoyl-, and 1,2-dilinoleoyl-3-palmitoylglycerols was
B

DOI: 10.1021/acs.jafc.5b01835
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Journal of Agricultural and Food Chemistry

supplied with Bruker TopSpin2.6 spectrometer software, was used for

suppression of nuclear Overhauser eects via inverse gate proton
decoupling. Acquisition parameters were as follows: 90 13C observe
pulse, 11.0 s; spectral width, 2011 Hz (16 ppm) centered on the
carbonyl region at 171 ppm; 32768 real and imaginary FID data points
collected during the 8.146 s FID data acquisition time, followed by a
22 s recovery delay time to ensure a pulse repetition time of >5 times
the longest expected longitudinal relaxation time T1. A total of 800
scans were collected, requiring almost 7 h per spectrum.
Because this part of the study involved quantitation, some
comments on digital handling at the receiver stage are warranted.
Although analog to digital converter oversampling and the standard
Bruker sharp digital lter were used in conjunction with a 125 kHz
analog lter, routine on the y linear prediction (as implemented by
the Bruker baseopt parameter) was not used to correct the initial
FID data points to align them to the true time zero of the FID near the
center of the excitation observe pulse. The FID was zero lled once to
32768 real data points, and a 0.06 Hz exponential weighting function
was applied before Fourier transformation.
RP-HPLC. Semipreparative nonaqueous RP-HPLC was performed
using a Gilson HPLC system (Middleton, MI, USA) comprising two
Gilson 306 pumps and a Gilson 811C dynamic mixer. The mobile
phase was 70:30 acetone/acetonitrile run under isocratic conditions at
a ow rate of 4 mL/min for a duration of 72 min. The column, a
Phenomenex Prodigy 5 m 100A 250 10 mm ODS column, was
operated at room temperature. Detection was at 210 nm using a
Gilson UVvis 156 detector. Because the methods employed isocratic
elution, no further column conditioning was necessary between
samples. Nevertheless, 80 mL of solvent was allowed to ow through
the column between runs, at 4 mL/min. Samples of the oil were
prepared by dissolving approximately 100 mg of the oil in 1 mL of the
mobile phase. Three injections of 300 L were made for each sample,
and the collected fractions were pooled for subsequent analysis. For
the infant formula, 3040 mg of the extracted TG sample was
dissolved in 300 L of acetone and injected in one dose. Fractions
were evaporated using a rotary evaporator and transferred to a vial
using hexane. The hexane was evaporated under a stream of nitrogen,
the sample weighed, and the solution made up with an accurately
measured volume of hexane or isooctane.
Ag+-HPLC. Analytical silver ion HPLC was performed on a Waters
H-class Acquity UHPLC system (Milford, MA, USA) with UV at 205
nm (PDA) and ELSD detectors in series, using Agilent ChromSpher 5
Lipids columns (Santa Clara, CA, USA).31,32 The Waters Acquity ELS
detector settings were as follows: detector gain, 500; gas pressure
(compressed air), 240 kPa; nebulizer set to cooling; and drift tube
temperature, 60 C. Three columns (250 4.6 mm) were connected
in series and maintained at 25 C. The mobile phase was 0.5%
acetonitrile in n-hexane (sonicated to ensure complete mixing), and
the ow rate was 1.0 mL/min. Samples were prepared at 10 mg/mL in
hexane, and injection volumes of 25 L were used for the high OPO
samples and UV detection. Lower concentrations were employed
when the ELSD was used for detection (approximately 2 mg/mL).
For high OPO samples, the method involving preparative
nonaqueous RP-HPLC to generate an OPO-enriched fraction with
analysis by Ag+-HPLC with UV detection at 205 nm was the most
eective. For the low OPO samples (<1% OPO), a weighed amount of
the extracted TG fraction was used for the nonaqueous RP-HPLC, and
an amount of 40 mg proved useful. There was no need to weigh the
recovered fraction, which was dried of solvent and made up in 500 L
of n-hexane for Ag+-HPLC using ELSD as the detector. A standard
curve could be prepared using OPO at 0.2 mg/mL in n-hexane.
Injections of between 1 and 10 L were sucient to prepare a
standard curve, using the syringe on board the HPLC apparatus.
Similar volumes were found appropriate for injection of samples in
both the high OPO and low OPO procedures. For the work described
here the UV-based standard curve for OPO was linear for the range
from 3 to 16 g injected. The ELSD standard curve conformed to a
power curve in the range 0.21.2 g of OPO injected.33

Article

RESULTS AND DISCUSSION

C NMR Spectroscopy. The use of 13C NMR spectroscopy of carboxyl carbons is accurate and convenient to
determine the regiospecic distribution of saturated, cis-9 and
cis-11 monounsaturated and polyunsaturated fatty acids in
TGs.27 Although this method was unable to provide data
sucient to detail the regiospecic distribution of individual
fatty acids in complex mixtures, it was helpful to determine the
ratio of saturated fatty acids in the -positions (sn-1,3) of
triacylglycerols to those in the -position (sn-2). The optimal
conditions for recording the spectra and securing details on the
allocation of the signals to link those with the identity of the
responsible compound were facilitated by the use of TG
standards, both as discrete individual components and in
mixtures of molecules. Dairy-derived samples (sample 1)
aorded a distinctive set of signals for the carboxyl carbon
atoms of the saturated fatty acids, in both the sn-1,3 and sn-2
positions. This was attributed to the presence of short-chain
saturated fatty acids in the dairy TGs (Figure 1).
13

Figure 1. 13C NMR spectra: (A) sample 1 (dietary ingredient); (B)

OOP; (C) OPO standard; (D) mixture of tricaproin (CCC) with
tristearin (SSS), with peak allocations. Note that the commercially
obtained reference sample of OOP in fact contained 19.7% of an
impurity, identied as OPO, with a saturated acid in the -position.

If it is benecial to have saturated fatty acids (any) in the

middle of the TG molecule and unsaturated acids (any) at the
side carbons of a glycerol moiety, then 13C NMR spectroscopy
is seemingly the best option to provide the relevant data for
such indiscriminate requirements. For example, using the data
from a previous study,34 one may calculate that in human milk
triacylglycerols, 47.1 mol % of colostrum-derived saturated fatty
acids are located in the sn-2 position (72.0 2.6% of total fatty
acids in the sn-2 position are saturated), ignoring the identity of
the fatty acids concerned. In transitional milk 45.9 mol % of the
saturated fatty acids are located in the sn-2 position (77.1
8.8% of sn-2 fatty acids are saturated), and in mature milk 45.5
mol % of the saturated fatty acids are located in the sn-2
position (79.1 1.3% of sn-2 fatty acids are saturated). There is
evidently very little variation in the level of saturated fatty acids
in the sn-2 position between colostrum, transitional milk, and
mature milk. Our data on saturated fatty acids in the sn-2
C

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position of the samples investigated in the present study, given
in mole percent of total saturated fatty acids present in the
sample, are as follows: sample 1, 34.9 mol % (46.0% of sn-2
fatty acids are saturated); sample 2, 52.9 mol % (72.6% of sn-2
fatty acids are saturated); and sample 3, 52.4 mol % (80.3% of
sn-2 fatty acids are saturated).
Given the data presented above, it would seem reasonable to
use total sn-2 saturates as one of the parameters when infant
formulas are compared to human milk. Although limited, the
method is appealing and may provide sucient information
when dealing with infant constipation issues. Specically, the
work of Quinlan et al.,7 published before OPO-enriched
formulas started to gain popularity, leads to the identication of
some interesting trends. Using data from that study it was
possible to calculate that the amount of insoluble calcium salts
of saturated fatty acids in the stools of formula-fed infants was
substantially higher than that in breast milk-fed infants.
Between formula-fed and breast milk-fed infants, the dierence
was approximately 36 times higher for lauric, 33 times for
myristic, 24 times for palmitic, and 8 times for stearic acid,
excreted in the form of calcium salts. These elevated levels of
insoluble soaps, seemingly produced from triacylglycerols with
saturated fatty acids in sn-1 and/or sn-3 positions, were linked
to an increased incidence of hard stools and constipation in
infants. An advantage of the use of the 13C NMR spectroscopic
method was that the results were produced as mole percent,
making it easy to estimate the potential loss of calcium due to
the formation of calcium soaps from non-sn-2-linked fatty acids
in dietary TGs.
A downside of the method was in its low throughput,
because the time required for recording a spectrum is 67 h
using a sample size of 2030 mg. It is possible that the use of
larger samples with dierent NMR probes may reduce the time
required for analysis while retaining the accuracy of the
method. A recent publication shows that optimization of the
parameters reduces the recording time even in the case of
complex mixtures of triacylglycerols from sh oils.35
Other signals in 13C NMR spectra, for example, those of CH2 carbon atoms or of olenic carbons, may also be helpful in
providing information about the regiospecic distribution of
fatty acids in triacylglycerols.36 An alternative HSQC-TOCSY
NMR method37 was employed for the analysis of palmitoyland oleoyl-containing standards of triacylglycerols, but cannot
resolve the present analytical problem.
GC-FID and GC-MS analysis. In contrast to 13C NMR
spectroscopy, GC-based analysis of TGs provides information
on molecular species composition but no data on the
regiospecic distribution of fatty acids. Due to the diculties
faced in volatilizing TGs, a high-temperature methylsilicone
phase presented an obvious choice. This allows the separation
of the compounds according to the number of carbon atoms in
the molecule. An improved option would be medium-polarity
phenyl-methylpolysiloxane phases that allow separation according to the number of the double bonds within a group of TGs
with the same carbon number.38
The target compound, OPO, is a triacylglycerol with 52
carbon atoms in the fatty acid moieties containing two double
bonds, TG52:2 in shorthand notation. The column employed
in the present study allowed the baseline separation of TG52:0,
TG52:1, TG52:3, and TG52:4 (see Figure 2). Despite this
ability to separate isomers, OPO remained indistinguishable
from its regioisomer, OOP, when this column was used (Figure
2). Incomplete separation of OPO/OOP from stearoyl-linoleyl-

Figure 2. GC separation of sample 1 triacylglycerols in the area of

interest according to the number of carbon atoms and unsaturation in
the fatty acid residues. Only the peaks with retention times identical to
those of the reference TG samples used are labeled.

palmitoyl (SLP) glycerols present in both human milk and

some infant formulas10 was also seen (marked with an arrow in
Figure 2).
There are a number of questions yet to be addressed in the
GC analysis of TGs, one being the applicability of dierent
injectors to various types of oils.39 An observation made in one
study39 is that, at least for some oils, a common split/splitless
injector may produce results as good as more sophisticated
programmed temperature injectors. The study also shows the
importance of determining and using the response factors for
individual TGs. For example, the dierence in relative response
between TG50:1 (POP) and TG54:2 (SOO) varied by almost
50%. In our experiments the dierence in relative response
between POP and OPO was approximately 15%. If average
common values were used instead of specic values for
response factors, the error can be signicant, leading to
unreliable data. Attempts in the present study to add a MS
dimension by using a Shimadzu GCMS-QP2010-FID instrument were unsuccessful due to the upper temperature limit for
the heated GC-MS interface being too low (350 C) for a
reliable transfer of the TGs of interest and were thus not
pursued.
HPLC. Initial attempts to analyze TG mixtures by Ag+HPLC alone, even with two or three Ag+ columns in line,
yielded poor results. The sample being injected was thus
simplied by adding a semipreparative RP-HPLC step and
collecting the group of TGs that would contain the target OPO
(Figure 3). Obviously, if other TGs are also of interest for
regiospecic analysis, the relevant fractions may be collected
during the same run. This TG52:2 fraction (outlined area in

Figure 3. RP-HPLC trace of sample 3. Detection was by UV at 210

nm. The fraction collected for Ag+-HPLC assay is identied.
D

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Figure 3) produced by RP-HPLC could, apart from OPO,
theoretically contain TGs 48:0, 50:1, 52:2, 53:4, 56:4, 58:5,
60:6, 62:7, 64:8, 66:9, etc., because these species demonstrate
chromatographically similar behavior under the conditions
employed. Of these, the following have been reported in
analyses of human milk and infant formulas: 48:0 (PPP), 50:1
(PPO, POP, SMO and its isomers), TG52:2 (OOP, OPO,
SLP), and 54:3 (OOO, SOL, SLnS).10
Ag+-HPLC of an OPO/OOP mixture demonstrated that
these isomers are clearly separable using this type of
chromatography (Figure 4, trace A). Saturated, monounsatu-

Figure 5. Separation of homologous unsaturated TGs by Ag+-HPLC:

SSO and PPO, SOO and OOP, and OSO and OPO. Detection was by
ELSD.

incurred. Additionally, the danger that polyunsaturated TGs

with higher retention times will accumulate in the columns and
interfere with subsequent assays made the RP-HPLC/Ag+HPLC option more attractive. Moreover, due to the low
loading capacity of analytical Ag+-HPLC columns, the RPHPLC step was benecial, because it allowed an increase in the
relative content of all TG52:2 components, thus increasing
the sensitivity and accuracy of the method. An extra advantage
of employing the two-step HPLC is that there was no need to
prepare high-purity triacylglycerols for injection, because
contaminants that might be present in oils or lipid extracts
(e.g., phospholipids, sterol esters, etc.) were either rejected at
the RP-HPLC step or did not t into the OPO window in Ag+HPLC. Also, potential overlap with OSO was avoided by the
two-step method.
Two more possibilities exist in which the OPO fraction may
be contaminated with triacylglycerols of similar mobility during
Ag+ chromatography. The rst was that dairy fats are known to
contain trans fatty acids,41 which demonstrate higher mobility
than their cis isomers in Ag+ chromatography.42 It was
consequently possible that triacylglycerols, which contain
shorter chain fatty acids, and trans fatty acids may have
retention times similar to that of OPO. Approximations have
been made from analyses of trielaidin (EEE) versus OOO
behavior, and the eect of chain-shortening on the behavior of
saturated TGs has been investigated.40 These observations
suggest that, to compensate for an increase in mobility due to
one trans double bond present, the triacylglycerol in question
must have a lower number of carbon atoms. It would therefore
be rejected at the RP-HPLC step in the present method and
cause no problems during Ag+ chromatography. In the second,
it was possible that entities with the same or even higher
number of carbon atoms and higher levels of unsaturation than
OPO and containing a trans fatty acid (e.g., a TG54:3) may be
a source of contamination in the TG52:2 fraction from the
RP-HPLC step. It was anticipated that the chromatographic
behavior of one or more of EEE, EEO, EOE, EOO, OEO, and
OOO may overlap with OPO. To address this possibility a
mixture of TG54:3 containing oleic and elaidic acids was
prepared and subjected to Ag+-HPLC. Pleasingly, OPO was
clearly separated from its nearest neighbor EEO (Figure 6).
A reversed-order approach (compared to ours) two-dimensional for analysis of TGs has been reported. Online Ag+-HPLC
and RP-HPLC with detection by UV, ELSD, and PI-APCI-MS
was employed.43 Both UV and ELSD methods of detection
performed poorly (probably due to a low loading capacity of

Figure 4. Ag+-HPLC separation of OPO from the other TGs that may
be present in the RP-HPLC TG52:2 fraction: (A) OOP/OPO
mixture; (B) SLP/LSP mixture; (C) sample 2 TG52:2 fraction; (D)
sample 2 TG52:2 fraction + SLP/LSP mixture.

rated, and triunsaturated TGs are not expected to overlap with

OPO and/or OOP in Ag+-HPLC. It was still important to
exclude the possibility of OPO/OOP overlapping with other
diunsaturated TGs that might be present in the sample, namely,
SLP and its regioisomers. Accordingly, a mixture of SLP
isomers was prepared, and it was conrmed that those are
clearly separated from OPO in Ag+-HPLC under the conditions
used (Figure 4, trace B). Under these conditions, OPO and
OOP were also distinct from POP and PPO (Figure 4, trace
C). Although the complete separation of LSP and OOP was
not achieved (Figure 4, trace D), LSP is not reported among
the TGs found in human milk and its substitutes10 and is
therefore unlikely to interfere if OOP content is to be
determined. Conrmation of the peak identity was obtained
by collection of the putative OPO peak and subsequent FAME
analysis thereof by GC after hydrolysis and conversion of the
free fatty acids into their methyl esters. Additionally, a sample
spiked with authentic OPO showed coelution of the OPO
peaks. Sample-derived OOP was also found to coelute with
authentic OOP. Adlof40 reported that silver ion HPLC also
allows the separation of saturated TGs possessing shorter
homologues due to their higher retention times. In the present
experiments with three Ag+-HPLC columns connected in
series, good separation of monounsaturated homologues (SSO
and PPO) was achieved, whereas diunsaturated TGs (SOO,
OOP, OSO, and OPO) demonstrated good but incomplete
separation (Figure 5).
Although it was tempting to try to improve the separation by
increasing the number of columns (e.g., four in line) in the
hope that the RP-HPLC fractionation step might be omitted, a
signicant increase in the time required for analysis would be
E

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Journal of Agricultural and Food Chemistry

found to be reproducible when using three individual lipid

samples (extracted from the infant formula as described above).
For n = 13, the average content of OPO in the lipid was found
to be 5.77 0.24 mg/g of lipid, with RSD = 4.1%. The median
was 5.72 mg/g and the range, 0.90 mg/g. For ELSD, the LOD
was calculated as 0.1 g injected weight of OPO, whereas the
LOQ was calculated as 0.3 g injected weight of OPO.44
In summary, the results demonstrate that 13C NMR
spectroscopy was useful to provide high-level rough data
relating to the level of saturated fatty acids in the sn-2 position
of TGs, but lacked the power (with the protocols employed) to
provide structural information on those saturated fatty acids.
The combination of RP-HPLC with UV detection at 210 nm,
followed by Ag+-HPLC of the selected OPO-containing
fraction with either UV detection or ELSD, presented a reliable
and robust method by which quantitative data for OPO content
could be generated from complex matrices. Usefully, OPO was
well separated from all other likely diunsaturated and/or trans
triunsaturated TGs, thereby avoiding signals from non-OPO
TGs that would give falsely elevated readings for OPO. The UV
and ELSD detection methods provide complementary ranges
for detection as indicated by their respective calibration curves.
The method presented allowed good limits of detection and of
quantitation. Because the combination of techniques draws
together a set of procedures that are designed to specically
deal with complex matrices, the applicability of the method is
likely to be wide-ranging and, thus, readily transferrable to
other complex matrices and foodstus. It is expected, however,
that some ne-tuning of one or both of the HPLC methods
would be required, depending on the analytes of interest.

Figure 6. Ag+-HPLC separation of OPO from isomeric TG54:3 with

various numbers of trans double bonds.

the column used for the separation in the rst dimension (a

cation exchanger coated with silver nitrate).
As ELSD detectors are in common use in many analytical
laboratories, the application of either UV or ELSD for the
detection of a range of molecules is now routine. In this work a
UV detector and ELSD in series were used for optimum
reliability of the data. It was clear that UV detection was only
useful for the high OPO samples. The limit of detection with
UV at 205 nm was approximately 1.5 g of OPO injected (at
this level the ELSD peak was above scale). Using ELSD, the
limit of detection was an order of magnitude lower at around
0.2 g of OPO injected. Lipid samples with low levels of OPO
such as the infant formula used in this work would be dicult
to analyze using UV detection. Whereas ELSD was particularly
useful for low OPO samples, the range of detection was
narrower and the detector quickly moved o scale with samples
containing higher levels of OPO; this would require sample
dilution to obtain accurate data. For this reason, the use of an
UV detector for those samples where the OPO level is high
remained the method of choice. The UV standard curve for
OPO was found to be linear for the range from 3 to 16 g
injected, whereas the ELSD standard curve conformed to a
power curve in the range of 0.21.0 g of OPO injected. The
nondestructive nature of UV detection was also useful for
collection of fractions from Ag+-HPLC for any subsequent
analysis (e.g., GC-FID of FAMEs). The combined results
produced by dierent approaches to the analysis of OPOcontaining mixtures are presented in Table 1.
Calibration and Statistical Data. For ELSD, good
linearity (R2 = 0.999 over nine discrete calibration curves set
up over a period of 3 days using freshly prepared samples) was
shown between 0.2 and 1.0 g injected when using a log curve
of the response. This also demonstrated the good repeatability
of the method. For UV detection, good linearity (R2 = 0.999)
was obtained between 3 and 16 g injected. The method was

ASSOCIATED CONTENT

S Supporting Information
*

Abbreviations used, response factors, calculations of saturated

fatty acids in the sn-2 position, calibration data, linearity data,
reproducibility results, determination of limit of detection. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jafc.5b01835.

AUTHOR INFORMATION

Corresponding Authors

*(M.V.) Phone: +644 931 3394. Fax: +644 931 3055. E-mail:
mikhail.vyssotski@callaghaninnovation.govt.nz.
*(D.B.G.W.) Phone: +644 463 0065. Fax: +644 931 3055. Email: bradley.williams@vuw.ac.nz.
Notes

The authors declare no competing nancial interest.

Table 1. Analysis of OPO Oils, Dietary Ingredient, and Infant Formula by Dierent Techniques
RP-HPLC/GCa RP-HPLC/GCa,b

GCa,c

GCa,c

OPO+OOP

TG52:2

TG52

saturated FA in sn-2, of total

saturated acids

saturated FA in sn-2, of
total FA in sn-2

Pe

area %

area %

wt %

wt %

mol %

mol %

wt %

11.9
28.7
24.3
naf

9.9
27.6
23.8
na

8.3
20.5
20.6
0.08

14.8
33.2
29.2
2.8

34.9
52.9
52.4
33.9

46.0
72.6
80.3
81.6

18.4
37.6
46.7
31.9

method:

RP-HPLC/Ag+-HPLC

analyte:

OPO

OPO+OOP
+SLP

expressed
as:

wt %

sample
sample
sample
sample

9.2
20.4
20.8
0.07

1
2
3
4

GCd

13

C NMR

GC-FID of triacylglycerols. bSLP calculated using fatty acids data (GC). cInternal standards: HHH, POP. Relative response factor determined for
OPO. dGC-FID of fatty acid methyl esters. eP = palmitic acid. fna, not assessed.
F

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ACKNOWLEDGMENTS
We are grateful to Callaghan Innovation for funding and to
colleagues from Callaghan Innovation as follows: David Kyle
for identifying the OPO assay problem, Rosemary Webby for
skilled extraction of the infant formula, Andrew MacKenzie for
reviewing the manuscript, and Donal Krouse for assistance with
the statistics calculations.

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