“Nitrogen Spiking” of Protein-Based Nutritional Products


How does it happen?

Kjeldahl/Dumas and other protein tests are not selective – they determine nitrogen – not protein. A protein factor is used to convert nitrogen to protein. Anything containing nitrogen is measured as if it were protein. Free amino acids, creatine, melamine, urea, etc are examples.

why has it been happening for so long?

Brands/Manufacturer’s have little motivation to change the status quo. Tremendous pressure exists on testing labs to produce a “passing” result over an “accurate” result. A more selective quantitative protein test will produce equal or lower protein concentrations – not higher – than Kjeldahl/Dumas. This is low cost already – the Kjeldahl/Dumas test at Dyad Labs is only $35/sample. So, why would brands/manufacturer’s pay more for a test that will give them a “lower” result? Another question to consider is how great is the safety concern? Regulatory agencies rarely get involved.

testing challenges

Developing an alternative protein quantitation method is challenging for many reasons. Proteins are complex and varied. Protein standards are not readily available, and developing a selective, accurate, and precise test is not easy. However, the Kjeldahl/Dumas methods are already accurate and precise. Protein sources in samples typically have many different types of specific proteins. For example: milk is ~80% casein (4 major proteins) and ~20% whey (5 major proteins). Another challenge is that relative amounts of specific proteins changes between samples.

dyad labs observations

Dyad Labs is an independent contract lab. We have hundreds of clients. We analyze a lot of different samples. Rarely do we get requests to do additional testing beyond just protein. This includes raw materials. When we do, it is typically for melamine only. There has been an increase in products that contain both protein and amino acids.

protein & amino acid products

For products that have both protein and amino acids, separate tests are needed to determine amino acid and nitrogen concentrations; amino acids are subtracted from nitrogen (i.e. Kjeldahl/Dumas) to obtain protein amounts. Protein-bound amino acids require hydrolysis, which release the amino acids. A few Hydrolysis challenges are that there is lots of variability in the peptide bond cleavage, and not all amino acids have the same stability in acid.

So how do we distinguish from amino acids that may have come from protein breakdown vs those that were free?

what is being done?

Dyad Labs has developed a qualitative method for protein analysis. This method confirms identity of whey, casein, pea, soy, rice. Protein is digested by trypsin into peptides. Three unique peptides for each protein source is monitored by LC/MS/MS. This method has AOAC First Action Status: AOAC 2017.11 Pea, Soy, and Rice Proteins, and AOAC 2017.12 Milk Protein (Whey and Casein). No other labs have volunteered yet to be a part of the reproducibility study; Please contact Spencer Carter or AOAC to become involved.

USP Dietary protein expert panel

Purpose: Develop, validate, and recommend new specifications, analytical tests, and related scientific documents to USP for dietary proteins.

Outcomes: Support the creation of new and modernized FCC monographs, FCC Identity Standards, general tests and assays, as well as USP reference materials.

Duration proposed: Dec 2017-June 2022.

USP Panel considerations

A few questions to consider in regards to the USP panel are: What matrices are applicable to whey protein concentrate and whey protein isolate? Should method(s) be considered as Identity and/or Assay (i.e. Qualitative/Quantitative test) in a monogragh, Identity Standard or as a standalone general test? What is the performance data: specificity, LOD/LOQ, sensitivity, precision, accuracy, etc.? We also need to consider the availability of physical standards and instrumentation, as well as costs and challenges.

USP Panel Methods being Considered

  • LC/MS/MS Peptide Mapping (ID & Assay, in progress)
  • Capillary Gel Electrophoresis / SDS-PAGE (ID, in progress)
  • Intact LC-MS (ID, in progress)
  • Amino Acid Profile (ID & Assay, to be tested)
  • Kjeldahl related methods (total nitrogen & non-protein nitrogen for ID & Assay, to be discussed and tested)