Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins

Fabian Ostertag; Christian M. Schmidt; Sonja Berensmeier; Jörg Hinrichs

doi:10.1016/j.foodchem.2020.128176

Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins

Fabian Ostertag, Christian M. Schmidt, Sonja Berensmeier, Jörg Hinrichs

Chair of Bioseparation Engineering

Hohenheim University

Research output: Contribution to journal › Article › peer-review

41 Scopus citations

Abstract

Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% − 103%) and recovery (96% − 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.

Original language	English
Article number	128176
Journal	Food Chemistry
Volume	342
DOIs	https://doi.org/10.1016/j.foodchem.2020.128176
State	Published - 16 Apr 2021

Keywords

Column screening
HPLC-DAD
Lactoferrin
Method validation
Whey protein

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title = "Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins",

abstract = "Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% − 103%) and recovery (96% − 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.",

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author = "Fabian Ostertag and Schmidt, {Christian M.} and Sonja Berensmeier and J{\"o}rg Hinrichs",

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AU - Schmidt, Christian M.

AU - Berensmeier, Sonja

AU - Hinrichs, Jörg

PY - 2021/4/16

Y1 - 2021/4/16

N2 - Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% − 103%) and recovery (96% − 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.

AB - Whey represents a valuable protein source for human nutrition. Whey composition varies with respect to process characteristics during milk processing. For efficient exploitation of this dairy side stream, reliable analytical methods are essential. The aim of this study was to develop and validate an RP-HPLC-DAD method for the simultaneous quantification of the minor (lactoferrin, lactoperoxidase, bovine serum albumin) and major (α-lactalbumin, β-lactoglobulin) whey proteins. Seven RP-columns were compared and the composition of the mobile phase was optimized to achieve baseline separation. In validation experiments the limits of detection (LOD < 8 mg/L) and quantification (LOQ < 24 mg/L) were determined. Validity was proofed by precision (>96%), accuracy (95% − 103%) and recovery (96% − 102%) measurements. Peak homogeneity was confirmed by SDS-PAGE. The individual working ranges were adjusted to the estimated protein concentrations in whey, allowing direct analysis without sample preparation at a method runtime of 23 min.

KW - Column screening

KW - HPLC-DAD

KW - Lactoferrin

KW - Method validation

KW - Whey protein

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VL - 342

JO - Food Chemistry

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ER -

Development and validation of an RP-HPLC DAD method for the simultaneous quantification of minor and major whey proteins

Abstract

Keywords

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