Waters : Determination of Lactose in Lactose-Free and Lactose-Reduced Products

It is estimated that 65% of the world's population is lactose intolerant - a digestive disorder with characteristic symptoms that occur when foods that contain lactose are consumed - with variable frequency within different regions of the world and among different ethnicities.

As such, the lactose-free product market is projected to reach $18.4 billion in 2025, and the increasing demand for reduced or no sugar products combined with the growing awareness among global consumers around lactose intolerance are expected to drive this growth.

With regulations and specific requirements in manufacturing, it's critical to routinely test samples for quality assurance, ingredient & allergen levels and to meet product specifications and label claims. Waters LC-MS technologies enable food and beverage laboratories to accurately measure low levels of lactose in dairy products, ensuring that manufacturers can meet product specifications and label claims.

About lactose intolerance and lactose-free products

Lactose is a disaccharide (sugar) found in dairy products like cow's milk, cheese, and yogurt. Lactose intolerance is typically a result of the body not producing enough of the lactase enzyme. Lactase is produced in the small intestine and is responsible for breaking lactose down into glucose and galactose (monosaccharides).

A common approach to removing lactose from dairy products is the addition of lactase during processing. The addition of lactase results in lactose being converted to glucose and galactose which are absorbed into the small intestine, stopping the symptoms of lactose intolerance occurring.

Lactose regulations vary globally

Regulations around the values and labeling of lactose-free or lactose-reduced products are not uniform - they vary around the world, with two examples below.

European Union (EU)regulation 1169/2011, on the provision of food information to consumers includes lactose on the list of substances or products which cause allergies or intolerances. Currently, in the EU there are no harmonized rules on the use of claims to show the reduction or absence of lactose in foods. EU Member States have adopted their own limit values with Finland, for example, applying the Nordic limit values of:

• Lactose-free foods - lactose content less than 10 mg/100 g or 100 mL
• Low lactose foods - lactose content less than 1 g/100 g or 100 mL

The Brazilian Health Regulatory Agency, ANVINSA, have released two recent resolutions which regulate food stuffs for special needs diets (RDC 135/2017) and sets the requirements for the mandatory declaration of the presence of lactose on food labels, for products containing over 100 milligrams of lactose per 100 grams/millilitres of product (RDC 136/2017).

Use of liquid chromatography to test for lactose

With regulation around the world, it's critical for manufacturers to test and monitor products regularly. Liquid chromatography (LC) is a widely used technique for the determination of sugars in foods and beverages. Alongside speed of analysis and sensitive detection, the ability to separate and accurately quantify several monosaccharides and disaccharides in the same analysis are key advantages of using LC for the analysis of sugars.

There are several different approaches to sugar analysis by LC, they involve the use of different column stationary phase technologies to retain and separate common sugars, they include:

Use of HILIC for sugar analysis

HILIC is used as an alternative to traditional reversed-phase chromatography for the retention of very polar analytes. HILIC columns can include silica, hybrid, diol, and amide as well as several other polar stationary phases. Initial HILIC mobile phase compositions are typically a combination of high organic solvent (80%) typically acetonitrile, with lower amounts of aqueous (20%). The higher the percentage ratio of aqueous in the mobile phase the greater the elution strength. Buffers and additives are commonly used for HILIC to improve peak shapes and separations, methods can also either be isocratic or a gradient.

HILIC provides excellent separation efficiency for sugars with an analysis time typically shorter than other chromatographic techniques. The BEH Amide stationary phase is well suited to the analysis of sugars, with low salt retention being one key advantage of the Amide ligand. The stationary phase also provides excellent high pH stability allowing the use of mobile phase conditions which avoids the loss of reducing sugars and collapses anomers to a single peak. The application note Quantification of Mono and Disaccharides in Foods provides an example HILIC separation using the XBridge BEH Amide XP column. In this application note, the challenging separation between glucose and galactose is demonstrated as well as evaluating other possible co-elution's between sugar isomers.

Detection of lactose

HPLC (High Performance Liquid Chromatography) coupled to a UV-Vis detector is a common LC set-up in routine food testing laboratories involved in ensuring products are meeting specifications and label claims for ingredients. However, sugars such as lactose do not contain strong chromophores, which means alternative detectors are required for analysis. These detectors can include refractive index (RI), evaporative light scattering (ELS), and mass spectrometry (MS). When considering which detector best fits your laboratory needs, allowing you to confirm the levels of lactose in food and beverage products, some factors to consider include:

• Sample complexity
• Sensitivity requirements
• Elution method (isocratic vs gradient)
• Laboratory infrastructure (gas supply and extraction)
• Ease of implementation and use

Determination of lactose in lactose-free products by LC-MS

The need to be able to detect low levels of lactose in lactose-free products, such as the 10 mg/100 g shown above, means that a selective and sensitive detector is required. Whilst RI detection is commonly used for the determination of sugars, this detector may be less suited to the analysis of lactose in lactose-free dairy products and may not meet the required method sensitivity and selectivity.

Single quadrupole mass spectrometry using selective ion recording (SIR) provides greater selectivity for this analysis compared to RI, reducing the impact of co-elution with other sample components. Along with lower detection limits, mass detectors such as the ACQUITY QDa are compatible with gradient chromatography methods and provide greater peak confirmation through the capture of mass spectral data.

The application note Determination of Low Level Lactose in Dairy Products Using UHPLC-MS, co-authored by Waters Corporation and the Eurofins Nutrition Analysis Center, Des Moines, demonstrates the use of an LC-MS method for the determination of lactose in dairy products, including lactose-reduced and lactose-free products.

Protecting brands with accurate and reliable food and beverage testing workflows

Waters' instrument and service solutions enable food and beverage testing laboratories to create efficient testing methods, supporting manufacturers in ensuring:

• Products meet nutritional value and specifications for added sugars
• Lactose-free products meet regulations around levels and label claims