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

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.

Analytical Ultracentrifugation-Calibrated Anion-Exchange Chromatography for Sensitive and Intact Determination of Osteopontin in Infant Formula and Dairy Products

“…method for intact OPN analysis was presented in a recent paper. 21 However, it showed several critical flaws: (1) It used one of the above Sigma materials as its reference standard, which might lead to inaccurate quantification. (2) It failed to show chromatographic baseline separation between the OPN and other interferences from the infant formula matrix and also did not present any other method specificity or selectivity tests to prove the purity of the targeted OPN peak.…”

“…If those reagent-grade OPN materials are used with an assumption of 100% purity, the result will be overestimated. A rapid and easy HPLC-UV method for intact OPN analysis was presented in a recent paper . However, it showed several critical flaws: (1) It used one of the above Sigma materials as its reference standard, which might lead to inaccurate quantification.…”

Isocratic Reverse-Phase HPLC Method for Determination of Aspirin, Paracetamol, and Naproxen

“…In combination with mass spectrometry, chromatography has been an essential part of proteomic analysis. [8][9][10][11][12][13]. High-performance liquid chromatography (HPLC) has been adapted to analyze multi-component drugs by adjusting the affinity between the surfactant of column beads and the drug molecules according to size and electric charges.…”