Value assignment technology for 1，3-oleic-2-palmitic triglyceride purity reference material

doi:10.3724/SP.J.1123.2025.10031

Abstract

Abstract:

1，3-Oleic-2-palmitic triglyceride （OPO） has received widespread attention as a nutritional fortifier allowed to be added to infant formulas， and there is an urgent need to develop high-purity certified reference material （CRM） to meet the metrological traceability requirements of relevant tests. Value assignment methods for pure materials are a key technical challenge in the development of high-purity CRMs. The commonly used mass balance （MB） method indirectly determines the purity of the material by subtracting impurities， which involves the accurate quantification of structurally similar impurities， moisture， volatile solvents， and non-volatile impurities. Among these steps， the separation and analysis of structurally similar impurities of the main component are the key to MB method. Owing to the lack of ultraviolet/fluorescence chromophores， OPO cannot be effectively detected by liquid chromatography with terminal absorption （190–210 nm）； this technique lacks sufficient sensitivity for low-level related impurities. Therefore， with a universal detector， this study established a high performance liquid chromatography-charged aerosol detector （HPLC-CAD） method for the analysis of structural analogue impurities in OPO， and applied the mass balance method to certify the purity of the reference material. On the other hand， based on quantitative nuclear magnetic resonance （qNMR） technology， a qNMR purity assignment method for OPO purity was established by optimizing key parameters such as NMR internal standards and quantitative peaks. In the mass balance method， an HPLC-CAD was selected as the core technique for quantifying trace structurally similar impurities of OPO. The chromatographic conditions were as follows： Nova Pak C8 column （150 mm×3.9 mm， 4 μm）， acetonitrile-isopropanol （90∶10， volume ratio） at a flow rate of 1.0 mL/min， sample dissolved in n-hexane-isopropanol （1∶1， volume ratio） at a mass concentration of 2.0 mg/mL with an injection volume of 5 μL. Under these conditions， four structurally related trace impurities were baseline-separated and accurately quantified. Method validation showed that in the mass concentration range of 0.001-0.025 mg/mL， good linear relationship was obtained （R²=0.999 1）. The limits of detection （LOD） and quantification （LOQ） were 0.000 5 mg/mL and 0.001 mg/mL， respectively， meeting the requirements for quantitative analysis. Six replicate measurements gave an average total impurity content of 1.23% （standard deviation 0.03%）， corresponding to an OPO main component content of 98.77%. In addition， Karl Fischer coulometric titration was used to determine moisture content， headspace gas chromatography-flame ionization detection （HS-GC-FID） was used to analyze residual volatile organic solvents （VOC）， and thermogravimetric analysis was used to determine non-volatile impurities. The purity of OPO was calculated as 98.51% by subtracting related impurities， moisture， VOC and non-volatile residues. In qNMR， direct and accurate quantification of the main component was achieved by optimizing key parameters： deuterated chloroform was selected as the solvent； ethyl paraben was used as the internal standard； selected chemical shifts of 2.33 （OPO） and 6.88 （ethyl paraben） as quantitative peaks. Accurately weigh approximately 10.0 mg of OPO and 3.0 mg of the internal standard in a brown bottle， dissolved in 0.50 mL of deuterated chloroform， and transferred to a 5 mm NMR tube. After six parallel measurements， the average purity of OPO was calculated to be 98.82%， with a standard deviation of 0.21%. Additionally， the homogeneity and stability of the candidate high-purity OPO reference material were evaluated. The uncertainties introduced by value assignment， inhomogeneity， and instability were evaluated. The results showed that the certified purity value of OPO was 98.7%， with a relative expanded uncertainty of 0.7% （k=2，where k is the coverage factor corresponding to a confidence probability of approximately 95% under the normal distribution）. The successful development of this certified reference material provides a traceability standard for relevant detections and fills the gap in OPO purity reference material.

Key words: 1, 3-oleic-2-palmitic triglyceride （OPO）, purity reference materials, value assignment, high performance liquid chromatography-charged aerosol detector （HPLC-CAD）, quantitative nuclear magnetic resonance （qNMR）

CLC Number:

O658

WANG Xinyu, WANG Ying, XU Mengqian, ZHOU Xia, CHU Hongtao, ZHANG Qinghe, LI Xiuqin. Value assignment technology for 1，3-oleic-2-palmitic triglyceride purity reference material[J]. Chinese Journal of Chromatography, 2026, 44(7): 785-794.

Figures/Tables 9

Fig. 1 Secondary mass spectrum of 1，3-dioleoyl-2-palmitoyl glycerol （OPO） standard substance candidate reference materials in electron-activated dissociation （EAD） cleavage mode

Fig. 2 NMR spectrum of OPO standard substance candidate reference materials in deuterated chloroform

Fig. 3 Liquid chromatograms of OPO under different detectors and chromatographic columns with isocratic elutiona. HPLC-ELSD Ag+ column， 0.6% acetonitrile in n-hexane， 1.8 mL/min； b. HPLC-ELSD-BEH C18 column， acetonitrile-isopropanol-n-hexane （80∶10∶10， volume ratio）， 0.3 mL/min； c. HPLC-CAD-Nova-Pak C8 column， H2O-acetonitrile-isopropanol （10∶45∶45， volume ratio）， 1.0 mL/min.

Fig. 4 Liquid chromatograms of OPO under different mobile phase conditionsCondition Ⅰ： isocratic elution， water-acetonitrile-isopropanol （10∶45∶45， volume ratio）， 30 min； condition Ⅱ： isocratic elution， acetonitrile-isopropanol （90∶10， volume ratio）， 30 min； condition Ⅲ： A=acetonitrile， B=isopropanol； 10%B initial， ramp to 30%B in 10 min （hold 3 min）， drop to 10%B at 13-14 min， re-equilibrate 4 min.Peaks Nos. 1-4： impurities 1-4.

Fig. 5 Standard curves of OPOa. standard curve of OPO within the range of 0.001-1.0 mg/mL； b. standard curve of OPO within the range of 0.001-0.025 mg/mL； c. double logarithmic standard curve of OPO within the range of 0.001 0-1.0 mg/mL.

Table 1 Results of content determination of structurally related impurities in OPO samples

Sample No.	Sample content/（mg/mL）	Mass concentrations of impurities/（mg/mL）					Total impurity content/%	OPO content/%
Sample No.	Sample content/（mg/mL）	1	2	3	4	Total	Total impurity content/%	OPO content/%
1	1.9686	0.00034	0.0191	0.00035	0.0046	0.0244	1.24	98.76
2	2.0062	0.00034	0.0190	0.00039	0.0049	0.0246	1.23	98.77
3	2.0721	0.00038	0.0205	0.00046	0.0038	0.0251	1.21	98.79
4	1.9892	0.00040	0.0193	0.00043	0.0038	0.0239	1.20	98.80
5	2.0204	0.00033	0.0198	0.00043	0.0040	0.0246	1.22	98.78
6	1.9878	0.00043	0.0204	0.00045	0.0043	0.0256	1.29	98.71
Average Standard deviation （SD）						0.0247	1.23	98.77
Average Standard deviation （SD）						0.0006	0.0003	0.0003

Fig. 6 NMR spectrum of ethyl nipagin in deuterated chloroform

Fig. 7 NMR spectrum of the mixture of ethyl nipagin and OPO in deuterated chloroform

Table 2 Results of NMR determination of the mass fraction of the purity standards of OPO

Sample	$I x I s t d$	$n s t d n x$	$M x M s t d$		$m s t d m x$	$P N M R$
1	1.4587	0.3333	5.175		0.3956	99.02
2	1.4367	0.3333	5.175		0.4004	98.71
3	1.4266	0.3333	5.175		0.4027	98.58
4	1.4327	0.3333	5.175		0.4021	98.86
5	1.4079	0.3333	5.175		0.4083	98.64
6	1.4313	0.3333	5.175		0.4035	99.10
Average				98.82
SD				0.21

Table 2 Results of NMR determination of the mass fraction of the purity standards of OPO

Sample	$I x I s t d$	$n s t d n x$	$M x M s t d$		$m s t d m x$	$P N M R$
1	1.4587	0.3333	5.175		0.3956	99.02
2	1.4367	0.3333	5.175		0.4004	98.71
3	1.4266	0.3333	5.175		0.4027	98.58
4	1.4327	0.3333	5.175		0.4021	98.86
5	1.4079	0.3333	5.175		0.4083	98.64
6	1.4313	0.3333	5.175		0.4035	99.10
Average				98.82
SD				0.21

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