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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
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
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
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.
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
ASSOCIATED CONTENT
S Supporting Information
*
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
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 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
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
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.
REFERENCES
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX
Article
DOI: 10.1021/acs.jafc.5b01835
J. Agric. Food Chem. XXXX, XXX, XXXXXX