Abstract: A method for detecting 1,3-dioleyl-2-palmitoyl-glycerol (OPO) in infant formula has been established. The sample is hydrolyzed with ammonia, and the fat is extracted with an organic solvent. Amino solid-phase extraction column is used for purification, and silver ion chromatography column is used for separation. Elution is performed using a mobile phase of 0.55% acetonitrile-hexane, and detection is carried out using high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). This method achieves baseline separation of OPO and 1,2-dioleyl-3-palmitoyl-glycerol (OOP), allowing for accurate quantification of OPO. The method shows good linearity in the concentration range of 25-500 μg/mL, with a correlation coefficient R2= 0.9996. The limit of detection (LOD) and quantification (LOQ) are 0.30 and 0.90 g/kg, respectively. Within the range of spiked concentrations from 1 to 96 g/kg, the average recovery rate of OPO ranges from 97.1% to 104.2%, with relative standard deviations (RSD) between 1.1% and 2.9%. The method meets the requirements for precision and accuracy, and has also passed inter-laboratory collaborative validation. An investigation analyzed 39 commercially available OPO-fortified infant formula milk powders in China, revealing that the actual OPO content ranged from28.4% to 59.7% of the labeled amount. This discrepancy is primarily due to inconsistent detection methods.

Key words: High performance liquid chromatography-evaporative light scattering detector, 1, 3-Oleic acid-2-palmitoyl triglyceride, Infant formula