Location: Natural Products Utilization ResearchTitle: A comprehensive workflow for the analysis of Bio-Macromolecular supplements: Case Study of 20 Whey protein products
|AVULA, BHARATHI - University Of Mississippi|
|PARVEEN, IFFAT - University Of Mississippi|
|ZHAO, JIANPING - University Of Mississippi|
|TECHEN, NATASCHA - University Of Mississippi|
|WANG, YAN-HONG - University Of Mississippi|
|RIAZ, MOHAMMAD - University Of Mississippi|
|BAE, JI-YEONG - Jeju National University|
|SHAMI, ANTER - University Of Mississippi|
|CHITTIBOYINA, AMAR - University Of Mississippi|
|KHAN, IKHLAS - University Of Mississippi|
|SHARP, JOSHUA - University Of Mississippi|
Submitted to: Journal of Dietary Supplement
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2020
Publication Date: 3/25/2021
Citation: Avula, B., Parveen, I., Zhao, J., Wang, M., Techen, N., Wang, Y., Riaz, M., Bae, J., Shami, A.A., Chittiboyina, A.G., Khan, I.A., Sharp, J.S. 2021. A comprehensive workflow for the analysis of Bio-Macromolecular supplements: Case Study of 20 Whey protein products. Journal of Dietary Supplement. https://doi.org/10.1080/19390211.2021.1897724.
Interpretive Summary: Dietary protein supplementation is a common practice among both athletes and people who engage in recreational or health-related exercise regimes. Twenty commercial whey protein supplements containing Supplement Facts labels procured from online retailers. A comprehensive workflow to survey the supplements for protein content and adulteration, protein quantification, small molecule analysis, and quantification of heavy metal contaminants was applied. Proton Nuclear Magnetic Resonance (NMR) profiling and principal component analysis are used to profile products, detect global outliers and broadly applicable to many types of bio-macromolecule supplements. A Bradford assay is used to determine total protein content, along with bottom-up proteomics using peak intensity profiling is used to detect protein adulterants and contaminants and relative quantification of protein composition. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) gives a good overall view of protein content and integrity, especially for supplements with relatively simple protein compositions such as milk whey. Liquid chromatography coupled to both Ultraviolet (UV) and Liquid Chromatography–Diode Array Detector-Quadrupole-Time of Flight Mass Spectrometry (LC-DAD-QToF) detection is used to identify small molecule components by chromatographic retention time and accurate mass. Finally, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is implemented for the quantification of metal composition and contaminants. The result of this workflow is a comprehensive and quantitative profile of whey protein supplement contents that can detect both small molecule and macromolecule content. This is the first time, demonstrated the utility of independent methods as an integrated orthogonal technique to assure the overall quality of complex supplements such as whey protein isolates.
Technical Abstract: The presence of proteins and other bio-macromolecules as major ingredients in nutritional supplements and herbal products is a primary factor in the marketing of many biologically derived macromolecular supplements. Workflows for the analysis of these supplements for quality assurance, adulteration, and other supply chain difficulties must include the analysis of both small molecule content and the macromolecular components with independent methods. Herewith, a comprehensive workflow for the analysis of supplements where bio-macromolecules are a major constituent is described. A thorough analysis of twenty whey protein supplements was conducted to identify protein content, protein adulteration, inorganic elemental content, as well as the macromolecular and small molecule profiles with orthogonal analytical methods. This workflow included Nuclear Magnetic Resonance (NMR) fingerprinting with Principal Component Analysis (PCA) analysis for comparative gross profiling of samples, Liquid Chromatography–Diode Array Detector-Quadrupole-Time of Flight Mass Spectrometry (LC-DAD-QToF) analysis of small molecule components, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis of inorganic elemental content, determination of protein content by a Bradford assay, Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) protein profiling and bottom-up shotgun proteomic analysis by Liquid Chromatography-Mass Spectrometry (LC-MS). Comprehensive profiles of each whey protein supplement were generated. Widespread errors in intact protein content labeling were detected, with intact protein content on average 1/5 of the claimed protein content, even though 17 out of 20 of the products listed a form of intact whey protein as the primary ingredient. In contrast, the analysis of a whey protein standard obtained from a scientific supply vendor yielded protein quantities within the manufacturer’s specifications. Added flavorings and sweeteners were found in the two products labeled ‘unflavored’. Small molecule components found in whole milk but unclaimed in whey protein were also found, including betaine potentially carried over from dairy cattle feedstock. No adulteration of protein content from other unlabeled sources was identified. The use of orthogonal, an integrated workflow allowed the detection of crucial product characteristics that would have remained unidentified using traditional workflows involving either analysis of small molecule nutritional supplements or protein analysis.