Protein Analysis: Methods, Reliability and Quality

Protein Analysis: Methods, Reliability and Quality

The protein analysis is fundamental when it comes to assessing the quality of a product. This process should be done properly in order to be able to detect and prevent frauds

In HSN, we stand up for quality and transparency

That is why we also offer detailed analyses of our proteins in our website. Moreover, we have written this article in order to explain the methods that we have used as well as other techniques.

Importance of Protein Powder

At this point, everyone should know how important proteins are when it comes to nutrition. It is becoming clearer and clearer that taking a proper supply of protein in the diet is crucial for our health.

In the world of sports, proteins are even more important due to their role in supporting the muscle adaptations to exercise

Protein for athletes

Ideally, these proteins should come from food

Nevertheless, we cannot deny that protein powder is a highly nutritional product that makes it easier for us to take this nutrient. In fact, it becomes specially useful when we have lost our appetite or when we have problems to chew (like the elderly). Or perhaps it is just a matter of comfort, since it is very easy to carry and use.

The problem comes when some supplementation companies play dirty and they pretend to sell you something that is not protein

The Importance of Protein Analysis

We have been able to witness how the commercialization of protein supplements has peaked in the last few years.

The existing competitiveness between manufacturers increases the number of products available with juicy discounts and adjusted prices

It is no surprise that this has also carried the rise of fraud which have seriously damaged the credibility of this sector. That is why, we feel like there is an obligation to show clear information from laboratory tests that show the methods that we have used in detail.

An “economic adulteration” is when someone substitutes an authentic or natural component from a product for another that is cheaper and not authentic. Above all, many companies do this just to earn more money

Why do a Protein Analysis?

  • Because it helps to establish the market value of protein products.
  • It helps to assess the quality of  the ingredients from protein.
  • Moreover, it shows the conformity of the ingredients both for the consumer and the seller.
  • Also, it determines the authenticity of the ingredients.

External Protein Analysis

Strategies to detect food fraud

There are two main ways of detecting potential frauds in the food industry1:

Adulteration Analysis

In this case, a positive result would mean that there is a specific material missing. The problem of this method is that we have to know these substances previously.

In other words, if the analysis has not been designed to detect a specific component, it will go unnoticed

Purity Analysis

This strategy is quite effective when the adulterant is high enough to detect a lack of purity from the original raw ingredient.

In fact, these adulterants will be present in high proportions in order to cheapen the manufacturing cost. This strategy tends to be the best method to detect this fraud

Foundations of Protein Analysis

Even though there are many techniques to analyze the protein content from food products, we will use this article to focus on explaining the most frequent analysis in the world of sport supplementation.

Protein information on the label

Usually, there are three types of information on the label of a protein product or supplement:

  1. Source or sources of the protein: it comes in the ingredient list.
  2. Amount of protein: see nutrient facts.
  3. Amino acid profile of the protein: Aminogram.

Objective of the analyses

The objective of laboratory analyses will be to determine the amount of protein and amino acids from a specific source.

Determining the total protein content

The most common methods will determine the total nitrogen content. Then, it will extrapolate this information to the total protein content with several conversion factors.

To put it simply, these are indirect methods

Above all, this strategy relies on the fact that proteins are the only nutrients that provide nitrogen to the diet. However, the problem is that there are other non-protein compounds that also provide nitrogen.

Consequently, they could alter the results of these test, leading to an overestimation of the protein content

Amino Spiking

This is the main reason why the methods to detect nitrogen are not reliable when it comes to fraud detection. In fact, one of the most common frauds is amino spiking, which will not appear on these analyses.

To put it simply, amino spiking consists of adding one or more cheap amino acids (amino acids contain nitrogen) to a low purity protein base.

When we calculate the total nitrogen, the result would include the sum of the nitrogen from the protein and the nitrogen from the amio acids. Therefore, it would create the false illusion that the protein content is actually high

Methods to detect total nitrogen


It is basically made up of four steps that digest the sample in order to measure the nitrogen supply (both from protein and non-protein sources).

To do so, this nitrogen becomes ammonia, which is isolated through a distillation process. Then, it determines the total nitrogen content indirectly through a series of chemical reactions.

Its main weakness lies in its inability to differentiate between protein a non-protein content.

To solve this problem, there is a previous process known as protein precipitation. This process will isolate the protein and measure the nitrogen that comes exclusively from this source.

The Kjeldahl method is also known as “true protein”

Dumas or combustion method

This method relies in the formation of gaseous substances that result from the combustion of the sample. Consequently, it produces NOx and N2, among others. After reducing the NOx to N2, it determines the total N2.

Currently, it uses nitrogen analyzers that simplify, cheapen and make the procedure safer and faster than traditional methods such as Kjeldahl.

However, the problem is similar to that of Kjeldahl. There is no way to differentiate between protein and non-protein nitrogen, which is why it is susceptible to adulteration

Conversion factors

Like we mentioned previously, we need to determine the amount of nitrogen before assessing the protein content.

Traditionally, people have relied on factor 6.25 in order to calculate the protein from nitrogen.

Above all, this is based upon the assumption that the nitrogen content from protein is a 16%

However, it is not that simple, since the nitrogen content changes depending on the type of protein. In fact, the amino acid profile will be different.

Conversion factors

Despite their limitations, the most common factors are the ones proposed by Jones in 19312


Depending on the study and methodology, these factors can cause severe oscillations, even when we use the same type of protein.

Another big limitation is applying the factor 6.25 when we do not know the specific factor of a protein source. Consequently, this can result in over- and underestimations of the protein content.

However, we will use them as reference due to the lack of other valid methods. Moreover, the FAO considers that these factors are valid and the debate about their precision is not the topic of this article.

The importance of the aminogram

Seeing that the methods to assess the total nitrogen are quite susceptible to manipulations, we will need more precise strategies that will help us unmask possible frauds.

What is the aminogram from a protein?

The aminogram is nothing else than the list of amino acids that make up a protein.

This allows us to carry two types of protein analysis:

  1. Qualitative: due to the differentation of each one of the amino acids.
  2. Quantitative: if we add on each amino acid we can obtain the total protein content.
Moreover, it is necessary to know the aminogram of the protein or sample in order to interpret the aminogram analysis rigorously. This will help us draw a comparison between the obtained result and the value that we should have theoretically obtain.

Aminogram example

Here is a simple example for a whey protein and the amino acid glycine

According to the literature and the previous laboratory analyses, 100g of whey protein isolate should have between 1.6 – 1.8 grams of glycine.

If we analyze the aminogram and observe that 100g of product have much more glycine content than that, we will know that the protein has been adulterated. For instance, it could have been by adding more of this amino acid or blending the product with a cheaper protein.

If we had analyzed the total nitrogen with the Dumas or Kjeldahl methods, this alteration would have gone completely unnoticed

How can we analyze the aminogram from a protein?

Without any doubt, one of the best techniques could be the HPLC (High Performance Liquid Chromatography).

With the HPLC method, we will obtain something that looks like this graphic:


Before the analysis, the sample is previously treated, so that it will be able to detect each amino acid individually

The basis of this technique is that each amino acid has different physicochemical properties. Changing the conditions of the analysis will allow us to detect these amino acids.

Each peak corresponds to an amino acid and the area under the curve of each peak helps us determine its concentration

Interpretation of the analyses by

If you have visited our website in order to purchase some of our products, you have probably seen this section:

Product information

External Laboratory

The reason why is that we have analyzed samples of many of our products and we wanted to share the results with you. The aim is no other than to show that we do not cheat, since transparency is one of our core values.

ENAC Accreditation

We have not done these analyses, in fact, they have been carried out by an independent laboratory. Moreover, this laboratory has the ENAC accreditation (Accreditation National Entity). Actually, you can find their seal in the documents from the analyses.


The ENAC literally defines the accreditation as: “an international tool used to generate confidence in the correct performance of a very specific type of activity called Conformity Assessment Activities. These include testing, calibration, inspection, certification and verification, among others.”

To sum up, it is a way to ensure that the certified entity meets a series of requirements. These tend to be regulated by international regulations such as the ISO, UNE or EN. In other words, they need to meet global quality standards

Example of an HSN Protein Analysis: 100% Whey Protein Isolate

Let’s see what information can we find in the analyses available in To do so, we will use the analysis of the whey protein isolate.

Protein content

First of all, we are going to see the total protein content according to the analyses.

WPI Analysis

If we start at the top, we will see the product description or reference (blue) with its corresponding lot number (green).


The method used for this product has been the Dumas method (yellow) which we have explained previously. Therefore, they have determined the total nitrogen content to multiply it for the factors 6.25 or 6.38 and obtain the protein value.

Dry matter

Finally, we can see that the protein value has been calculated in dry matter after multiplying it for 6.38 (red).

As we have seen before, this is the conversion factor used for milk proteins


According to this analysis the protein value is of a 90.22%. Although we need to take into account that this value is an average. Therefore, the percentage may vary.

This variation is what is known as uncertainty

In this case, the uncertainty for this value is of a ±3,791. In other words, this means that the results may vary between an 86.43% and a 94.01%

Comparison between the nutrient facts

This information corresponds to the amount of protein indicated in our label: 93g per 100g of product, a 93%.

Nutrient Facts

What about the Aminogram?

But after reading the article, you may be thinking that the Dumas method is not useful when it comes to detecting frauds. In fact, that 935 could be the result of any type of adulteration.

Do not worry! That is why we have also provided the amino acid analysis from this protein:

Aminogram from WPI

Once again, we can find the reference and lot number of the product (blue and green respectively).

HPLC Analysis

If we focus on the analysis, the methodology corresponds to the ISO 13903:2005 international regulation or HPLC.

As we have previously seen, it is easy to detect frauds with this technique.

The first test verifies that the previous protein value is real. The sum of all the amino acids should provide a similar result to the protein grams per 100g of product obtained with the Dumas method.

Then, if we combine all these numbers (which also have a level of uncertainty), the result is 94.99 grams of amino acids per 100g of product.

Therefore, the first test is satisfactory and it equals the values mentioned previously

The second test is to see if these amino acid values are similar to the ones obtained for the whey protein.

This is an example of an aminogram from a study3:

Comparative aminogram

Keeping in mind the inter-laboratory variations, we can see that the results are quite similar in general.

Consequently, we can confirm that our sample has not been adulterated in any way

Comparing the aminogram and label

Finally, the last thing left to do is comparing the aminogram from the external laboratory with the one from our label


After all, we can see that the results are practically identical for all the amino acids

Evidently, there are small variations, since the analysis from our suppliers and the external laboratory may vary, Therefore, the label reflects the typical values of the product.

In other words, the typical values can be slightly different from the values of each lot


There are many other analyses available, all of them with their strengths and weaknesses. But we have chose the ones that seem to be more frequent. In HSNstore, we have a compromise with all our customers and we want to offer the best quality and transparency about our products.

I hope that this article will help you interpret the analyses we have published in our website. Above all, our aim is to help you choose a good protein supplement


  1. Moore JC, DeVries JW, Lipp M, Griffiths JC, Abernethy DR. Total protein methods and their potential utility to reduce the risk of food protein adulteration. Compr Rev Food Sci Food Saf. 2010;9(4):330-357. doi:10.1111/j.1541-4337.2010.00114.x
  2. Mariotti F, Tomé D, Mirand PP. Converting nitrogen into protein – Beyond 6.25 and Jones’ factors. Crit Rev Food Sci Nutr. 2008;48(2):177-184. doi:10.1080/10408390701279749
  3. Sinha R, Radha C, Prakash J, Kaul P. Whey protein hydrolysate: Functional properties, nutritional quality and utilization in beverage formulation. Food Chem. 2007;101(4):1484- 1491. doi:10.1016/j.foodchem.2006.04.021

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About Carlos Sánchez
Carlos Sánchez
Carlos Sánchez has a degree in Human Nutrition and Dietetics, and therefore all his actions are rigorously backed by science.
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