A Comprehensive Workflow for the Analysis of Bio-Macromolecular Supplements: Case Study of 20 Whey Protein Products

Abstract

The presence of bio-macromolecules as major ingredients is a primary factor in marketing many biologically derived macromolecular supplements. Workflows for analyzing these supplements for quality assurance, adulteration, and other supply-chain difficulties must include a qualitative assessment of small-molecule and macromolecular components; however, no such integrated protocol has been reported for these bio-macromolecular supplements. Twenty whey protein supplements were analyzed using an integrated workflow to identify protein content, protein adulteration, inorganic elemental content, and macromolecular and small-molecule profiles. Orthogonal analytical methods were employed, including NMR profiling, LC-DAD-QToF analysis of small-molecule components, ICP-MS analysis of inorganic elements, determination of total protein content by a Bradford assay, SDS-PAGE protein profiling, and bottom-up shotgun proteomic analysis using LC-MS-MS. All 20 supplements showed a reduced protein content compared to the claimed content but no evidence of adulteration with protein from an unclaimed source. Many supplements included unlabeled small-molecule additives (but nontoxic) and significant deviations in metal content, highlighting the importance of both macromolecular and small-molecule analysis in the comprehensive profiling of macromolecular supplements. An orthogonal, 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.

Keywords:

• whey protein
• dietary supplements
• LC-MS
• SDS-PAGE
• ICP-MS
• NMR

Declaration of interest

The authors report no conflict of interest. The authors are responsible for the content and writing of the article.

Additional information

Funding

This research is supported in part by Science-Based Authentication of Botanical Ingredients, funded by the Center for Food Safety and Applied Nutrition, US Food and Drug Administration grant number 5U01FD004246, and Discovery & Development of Natural Products for Pharmaceutical & Agrochemical Applications, funded by the United States Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No. 58-6060-6-015.

Notes on contributors

Bharathi Avula

Bharathi Avula, is a principal research scientist at the National Center for Natural Products Research, University of Mississippi. Her research interests are the development and validation of analytical methods for a wide range of botanicals and dietary supplements using HPLC/UPLC, HPTLC, CE, GC-MS, LC-MS, GC-FID, and ICP-MS techniques; chemical fingerprinting and standardization of marker or active compounds in botanicals; and analysis of drugs and their metabolites in biological samples (blood, saliva, and urine).

Iffat Parveen

Iffat Parveen, is a research scientist at the National Center for Natural Products Research, University of Mississippi. Her research interests are identification and authentication of botanicals using different molecular markers and the DNA barcoding approach.

Jianping Zhao

Jianping Zhao, is a senior research scientist at the National Center for Natural Products Research, University of Mississippi. His research interests are the isolation and structural identification of constituents in natural products; metabolomics techniques to validate, authenticate, and characterize the properties of botanicals/herbs that are traditionally used in alternative, complementary, and integrative health-care systems; and quality assessment of natural products that are used as dietary supplements, functional foods, nutraceuticals, and drugs.

Mei Wang

Mei Wang, is a research scientist at the National Center for Natural Products Research, University of Mississippi. Her research interests are the application of analytical tools in evaluating the quality and safety of botanicals and dietary supplements; establishment of fingerprint profiles and standardization of chemical markers in medicinal plant products; and development of novel methods for the analysis of drugs and their metabolites.

Natascha Techen

Natascha Techen, is a senior research scientist at the National Center for Natural Products Research, University of Mississippi. Her research interest is the development of molecular (DNA) markers that can help with the identification of medicinal plants of raw drugs or present in dietary supplements.

Yan-Hong Wang

Yan-Hong Wang, is a principal research scientist at the National Center for Natural Products Research, University of Mississippi. His expertise focuses on the application of mass spectrometry–based analytical tools to characterize active or marker compounds in medicinal plants and developing and validating methods for evaluation of the quality and safety of dietary supplements and herbal medicines. His interests include isolation, characterization, and analysis of natural products, clinical pharmacokinetics of candidate drug molecules, and characterization of metabolites in drug discovery.

Mohammad Riaz

Mohammad Riaz, was an MS student at the Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi and currently working as an Associate Scientist II at AbbVie. His research interest is the method of development of biomacromolecules using LC-MS, focusing on implementing mass spectrometry for the structural and compositional analysis of proteins.

Ji-Yeong Bae

Ji-Yeong Bae, is an assistant professor at the College of Pharmacy, Jeju National University, and was a senior R & D chemist at the National Center for Natural Products Research, University of Mississippi. Her research interests are the development of analytical methods for the quality control of medicinal plants and dietary supplements, and chemical fingerprinting of useful materials from natural resources.

Anter A. Shami

Anter Shami, is an associate R & D chemist at the Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi. His research interest is an application of mass spectrometry for the analysis of macromolecules.

Amar G. Chittiboyina

Amar G. Chittiboyina, is an assistant director and principal research scientist at the National Center for Natural Products Research, University of Mississippi. His research interests are many aspects of natural and biologically active compounds; biogenesis of secondary metabolites; implementation and data integration with orthogonal methods for the analysis of phytochemicals; development of new synthetic methodologies; synthesis and structure activity relationships; development of non-animal alternative methods in toxicology; and application of computer-aided drug design techniques for the safety and efficacy of natural products.

Ikhlas A. Khan

Ikhlas A. Khan, is a director at the National Center for Natural Products Research and a professor of pharmacognosy major at the Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi. His research interests are applications of analytical tools in evaluation of quality and safety of dietary supplements; microbial transformation; fingerprinting and standardization of marker compounds in medicinal plant products; optimization of large-scale extraction; and isolation of natural products of commercial value.

Joshua S. Sharp

Joshua S. Sharp, is an associate professor at the Department of BioMolecular Sciences, School of Pharmacy, University of Mississippi. His expertise focuses on the development and application of new mass spectrometry–based technologies for studying the structure-function relationships of proteins and carbohydrates of biomedical interest.