Milk is an excellent source of protein, fat, carbohydrate, minerals, and vitamins. Therefore, it is a popular choice for people of all age groups.


With growing demand, it has been found that milk is being adulterated for economic gain. Due to inadequate monitoring and lack of proper law enforcement, this situation is worse in developing and undeveloped countries, particularly when milk is produced locally and sold directly to individuals. Studies have shown that, occasionally, 100% of liquid milk has been diluted in some way. 

How is Milk Adulterated?

The adulteration of milk is an act of intentionally reducing its quality, offering it for sale either by admixture, substitution of inferior substances or by the removal of some valuable ingredient. Adding water to liquid milk is the simplest means to increase the volume.

The most common approach to measuring and controlling milk quality is to determine its protein and fat content. This has resulted in both components being substituted with nitrogenous compounds (melamine, urea, whey) and vegetable oil to improving apparent protein and fat content. In recent years, the use of melamine has become an important issue and attracted worldwide attention because of its harmful nature, which has resulted in several food safety incidents. As vegetable oil cannot be dissolved in liquid milk detergents are added as an emulsifying agent. 

To extend the shelf life of milk by inhibiting bacterial growth other substances such as formaldehyde, hydrogen peroxide, benzoic acid and salicylic acid have also been introduced. These compounds can cause serious adverse health effects.

Besides chemical adulterants, milk from different species is also assessed in order to avoid allergenic effects in children as a result of admixing of bovine milk with milk from goats or sheep.

To address concerns relating to health, safety and to prohibit mislabeling in milk, Codex set a standard for milk and milk products in 2003. It only allows milk to be a mammary secretion from milking animals without addition or subtraction. More recently, the Food and Safety Standard Authority of India (FSSAI) and Hong Kong Centre for Food Safety have also regulated adulterants in milk.

Analytical Techniques for Milk Adulteration 

Over the last decade, many analytical methods have been proposed for rapid screening and confirmation of milk adulterants. Chromatographic techniques such as high performance liquid chromatography with UV, or fluorescence detection (HPLC/UV, HPLC/FLD) and gas chromatography with flame ionization (GC/FID) are commonly used for checking food preservatives and fat analysis. Recently, LC and GC coupled with mass spectrometry (MS) have become state of the art for highly sensitive and selective determination. Spectroscopic techniques including near infrared (NIR) and mid infrared (MIR) are powerful and rapid tools which enable simultaneous detection of multiple adulterants in milk with high sample throughput needing little or no sample preparation. However, the latter technique suffers from a lack of standardization and no regulatory recognition.

To determine milk fraud from different animal species, various techniques such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE), and matrix-assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF) have been studied, based on specific proteins, DNA and peptides. The more ‘classical’ DNA based analysis based on PCR is able to detect the presence of cow, sheep or goat milk, but only when it is specifically looked for and when treatment of the sample has not lead to breakdown of the DNA protein into smaller fragments that can prevent detection with this technique. In this case, NGS should be considered because not only is it quick, accurate and relatively cheap, the true advantage is that all animal DNA will automatically be detected in one screen, usually referred to as ‘untargeted’. Therefore, you will know if a milk sample is 100% of a specific animal species or if it is a mixture of milk with different animal species origin so for example cow’s milk in sheep milk.

It should always be borne in mind that some traditional methods, such as the analysis of freezing point depression (FPD) remains a quick and reliable way of detecting even very small amounts of added water. 

The main target markers of adulterants and the analytical methods which are widely used to analyze milk are summarized in Table 1.

Table 1 Overview of main milk adulterants, target markers, and their analytical methods

Adulterants Analytical techniques Target markers
Water NIR
Thermistor cryoscope
Freezing point
Melamine LC-MS/MS
Urea NIR, MIR Urea
Whey NIR, MIR Whey
Cow’s milk ELISA 
Whole bovine 
Bovine 12S rRNA 
Bovine para-κ-casein 
Protein components
Animal species
Detergents Rapid paper chromatography Methylene blue dye-detergent complex
Hydrogen peroxide
Batch injection analysis (BIA) with amperometric detector H2O2
Formaldehyde HPLC/UV Formaldehyde derived with 2,4-dinitrophenylhydrazine
Salicylic acid HPLC/FLD Salicylic acid
Benzoic acid HPLC/UV Benzoic acid

Future Prospects

In future, the emphasis will be on analytical techniques in the ongoing development of rapid methods for automated direct sample analysis. The “Electronic nose” is an example of new technology that is cheap, quick and easy. It has been widely used in dairy industry to check milk aging, shelf-life prediction, off-flavors and the geographic origin of milk. However, modification of the sensor for more specific toxic substances is still in the early stages. Nuclear magnetic resonance spectroscopy (NMR), a fairly traditional tool in the lab, is also proposed as a rapid method which can analyze a large number of adulterants in milk with highly reliable results. Nevertheless, its use is confined to a few food testing laboratories due to its high cost and the requirement of specialist or experienced staff to operate and maintain it.

For more information, please contact:

Amornpun Dajsiripun
Global Competence Support Centre Food Specialist 
t: +66 2683 0541 ext 2423