Abstract
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) < 0.2), recovery (% RSD < 3) and robustness. The results confirm that the method developed is suitable for OPN determination in IMF.
Highlights
Osteopontin (OPN) is a highly phosphorylated acidic glycoprotein and composed of ~300 amino acid residues [1]
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) < 0.2), recovery
Schack et al [2] reported that it is present in human milk (138 mg/L) at levels much higher than in both infant formula (~9 mg/L) and bovine milk (~18 mg/L)
Summary
Osteopontin (OPN) is a highly phosphorylated acidic glycoprotein and composed of ~300 amino acid residues [1]. Schack et al [2] reported that it is present in human milk (138 mg/L) at levels much higher than in both infant formula (~9 mg/L) and bovine milk (~18 mg/L). It is a multifunctional whey protein with various biological, physiological and pathophysiological activities [3,4]. Recently, OPN is being considered as a potential candidate protein to fortify infant milk formula (IMF), having proven benefits after clinical trials [3]. OPN is extracted commercially from bovine milk using ion-exchange chromatography with quality sufficient to add into food products [6]
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