Worldwide, milk and dairy products are vital sources of nutrition for humans [1]. Beside nutrients, minerals, proteins, and fat, lactose is a major component of dairy products, serving as an energy source. To efficiently metabolize lactose, the enzyme lactase is indispensable [2]. However, globally nearly 70% of the population is lactose intolerant, i.e., they have difficulty digesting lactose [3, 4]. Lactose malabsorption leads to numerous gastrointestinal and extra-intestinal symptoms and other complaints with varying extents. While some lactose intolerant people are sensitive to any amounts, others can consume a small quantity without ill effects. A specific cutoff value and regulations for lactose-free product labels and production are still lacking— therefore it is necessary to accurately analyze and label foods using sensitive standard techniques. Ion chromatography with pulsed amperometric detection (IC-PAD) enables the determination of very low lactose contents. Validation according to AOAC requirements shows the high sensitivity and reliability of this method as a routine analysis. 

Lactose determination was performed for a broad selection of sample matrices comprising infant formulas and follow-up baby food (AN-P-088), certified references (e.g., the low lactose milk reference muva ML-2312), as well as 15 commercially available low lactose and lactose-free products including yogurt, butter, cream, cottage cheese, milk drinks, milk chocolate, and supplements.

Solids (e.g., cheese and milk chocolate) were chopped, while the powders and liquid materials were homogenized. Following this pretreatment, the samples were weighed directly into suitable containers (0.1–5 g, 50 mL polypropylene tubes). The sample weight (W_S in g) was recorded to the nearest 0.001 g for later calculations. An aqueous extract was prepared by adding ultrapure water (UPW) to a total volume of 50 mL (W_UPW in kg). Afterwards, the vials were capped and mixed vigorously with a vortex mixer for approximately 20 seconds. To improve the solubility of certain samples (e.g., cream cheese or chocolate), vials were heated up in a 70 °C tempered water bath for 10 minutes.

Carrez precipitation is a standard method to remove proteins and larger molecules in order to protect the analytical system. Following this common practice, the reagents were added and the final weight noted (W_UPWc in kg). After thorough mixing, the samples were centrifuged (5000×g) for 10 minutes and decanted. The covered vials were placed directly into the autosampler. Increased column protection can be achieved by an additional ultrafiltration step. 

Alternatively, automated sample preparation using Inline Dialysis with the Low Volume dialysis cell is recommended. For this, samples were prepared as described previously regarding aqueous extracts, shaken well, and covered before placing them directly on the autosampler rack. When using dialysis, no Carrez precipitation is necessary prior to analysis, saving time and chemicals. Using the Low Volume dialysis cell requires only 5 mL of sample. 

The quantity of lactose in the aqueous sample extracts was determined by ion chromatography (IC) with a Metrosep Carb 2 - 250/4.0 separation column using an isocratic hydroxide eluent (400 mmol/L NaOH) and pulsed amperometric detection (PAD) with the sweep waveform (AN-P-088, WP-077). A long electrode lifetime with minimal maintenance requirements is possible by using the Metrohm Thin-Layer amperometric cell (Au working and Pd reference electrode). The sweep mode combined with the less turbulent flow in the Thin-Layer cell results in a smooth baseline with low noise – a necessary precondition to analyze very low concentrations, such as in low lactose products.

Flow schemes for the direct analysis of lactose with mandatory sample preparation such as Carrez precipitation are shown in Figure 1.

Sample stability is improved using the 889 IC Sample Center – cool. Automated sample preparation by Inline Dialysis can be added to any existing configuration setup. More details are available in Metrohm literature for Metrohm Inline Sample Preparation as well as in AN-P-088.

Any liquid handling for sample transport or sample path cleaning can also be automated using Metrohm’s most flexible tool for liquid handling – the Dosino.