How is Protein Tested in Whey Supplements?

by Christine Hronec

Standard procedures for cGMP include validation of the macronutrients declared on the supplement facts label. According Title 21 of the Code of Federal Regulations (21 CFR 101.9(g)), the FDA allows a variance between analytically measured values and label values due to the nature of testing variability.

FDA analyzes food samples that have been randomly collected from lots to determine compliance with labeling regulations. The agency defines a food lot as a collection of the same size, type and style of the food that is designated by a common container code or marking, or that constitutes a day’s production. The sample for nutrient analysis shall consist of a composite of 12 subsamples (consumer units), taken 1 from each of 12 randomly chosen shipping cases. FDA will then analyze the nutrient content of this 1 composite test sample.

The agency generally analyzes composites by appropriate methods found in the most recent edition of Official Methods of Analysis of AOAC International (AOAC International, Gaithersburg, MD, 16th edition, 1995, and yearly revisions/updates) (see below for additional information on selection of methods). The ratio between the nutrient level derived by analytical testing and the label value is calculated to determine whether the nutrient in question is in compliance with applicable regulations. The ratio is defined as:
(Laboratory value / label value) x 100 = %

Class II nutrients are vitamins, minerals, protein, total carbohydrate, dietary fiber, other carbohydrate, polyunsaturated and monounsaturated fat, or potassium that occur naturally in a food product. Class II nutrients must be present at 80% or more of the value declared on the label. As an example: If vitamin C is a naturally occurring nutrient in a product, and the product declares 10% DV vitamin C (i.e., 6 mg/serving) on its label, then laboratory analysis must find at least 80% of the label value (80% of 6 mg or 4.8 mg vitamin C/serving) for the product to be in compliance.

Protein content is analytically measured by the Kjeldahl Method. This is based on the principle that protein has a unique chemical composition compared to carbohydrates and fat due to the presence of nitrogen as the backbone fingerprint atom naturally found in amino acids. An analytical lab will chemically manipulate a sample to isolate the nitrogen content and then use that measured value to calculate protein content by then multiplying the percent nitrogen by a factor of 6.38 for milk and milk products. The factor used will vary according to the type of protein being measured (i.e. vegetable proteins use a factor of 6.25).

This method involves the following chemical processes: Sample is digested in H2SO4, using CuSO4*5H2O as a catalyst with K2SO4 as a boiling point elevator to release nitrogen from protein and collect and retain the nitrogen content in the form of an ammonium salt. Concentrate NaOH is added to released NH3, which is distilled, collected in H3BO3 solution, and then titrated. While this is the most accurate approach, it can easily be seen that this is an intricate multi-stage test method that will have inherent repeatability and reproducibility values. Due to the complex nature of this test, the FDA has deemed protein to be a class II ingredient allowing for a 20% variance between the measured value and analytically tested value.

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