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Osteopontin (OPN) is a multifunctional whey protein which has recently received much attention for possibly applications in fortifying infant milk formula (IMF) with its bioactivity. However, to date, there is no established high-performance liquid chromatography (HPLC) method to quantify this protein in milk or IMF. In this study, a rapid, simple, isocratic and reliable reversed-phase HPLC method was developed and validated to quantify the OPN in IMF. A C18 column (4.6 × 150 mm × 5 micron) was employed with 20% of 0.1% trifluoroacetic acid (TFA) and 80% of 60% acetonitrile in 0.1% TFA for 10 min detected at 214 nm. The flow rate was 0.3 mL/min with an injection volume of 10 µL. The column temperature was 40 °C, and the peak appeared after 4 min. The validation was based on the system suitability, linearity (r2 = 0.999), limit of detection (LOD) (0.14 mg/L), limit of quantitation (LOQ) (0.41 mg/L), precision (% relative standard deviation (RSD) &lt; 0.2), recovery (% RSD &lt; 3) and robustness. The results confirm that the method developed is suitable for OPN determination in IMF.

A Reversed-Phase HPLC Method for Determination of Osteopontin in Infant Formula