Sensitive Analysis of the Lactose Content of Lactose‑Free Labelled Products Using HPAEC-PAD


The Application Notebook

The Application NotebookThe Application Notebook-12-01-2015
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The market for lactose-free products is growing rapidly and constantly and Europe is a worldwide leader in the lactose‑free market. Between 2012 and 2016 the sales of lactose-free products are expected to increase by 75%.

Mareike Margraf and Kate Monks, Knauer

The market for lactose-free products is growing rapidly and constantly and Europe is a worldwide leader in the lactose‑free market. Between 2012 and 2016 the sales of lactose-free products are expected to increase by 75% (1,2). Studies have stated that customers who are lactose intolerant, or believe they are, will pay a big premium for the right product (1). From these statements it becomes obvious that a huge demand for lactose-free products exists in the industry. A high performance liquid chromatography (HPLC) method that easily reaches the required limits of detection (LOD) by using high performance anion-exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) on a Knauer AZURA® analytical HPLC system coupled to the DECADE II electrochemical detector is presented. Besides lactose, sucrose and glucose were also analyzed to prove the method´s ability to differentiate several sugars. 

Generally, lactose intolerance is the inability to digest lactose caused by the deficiency in the enzyme lactase, which hydrolyzes lactose into glucose and galactose. More than 65% of the world´s population loses the ability to completely digest lactose after infancy in what is called primary or late onset lactose intolerance (3). Reduction in lactase activity is rarely total but decreases to 

10–30% of the initial level between the ages 5 and 20 (4). In addition, secondary and developmental lactose intolerance can occur. It can be stated that lactose intolerance is an important subject worldwide. 

Although in European countries like Sweden and Finland lactose tolerance levels of 74% and 82% are widespread, the market for lactose-free products is growing and the regulations are getting more strict (5).

In many European countries, the limit of lactose in lactose‑free labelled products has recently been decreased from 100 to 10 mg/100 g product(6). This makes an HPLC method with low detection limits inevitable for the quality control of these products. Special methods and systems are needed because classical determination of sugars in food products is not sensitive enough  in this special case. The lactose content of food products can generally be determined in several ways. Validated methods do exist for enzymatic essays, polarimetry, gravimetry, differential pH, and HPLC.

Today, HPLC is the method of choice when sugar contents in dairy products have to be analyzed because it is a highly specific method with the ability to differentiate other sugars. HPLC on an ion exclusion column coupled to RI detection is the typical method used. However, in the special case of lactose analysis in lactose‑free products, this method is nowhere near sensitive enough. Therefore, special methods have to be applied to reach the wanted low detection limit of 10 mg/100 g sample.

Experimental Sample Preparation

Samples from different non-dairy food products were extracted using various extraction protocols, filtered, and injected into the HPLC system.

Experimental Preparation of Standard Solution

Standards of lactose, sucrose, and glucose were weighed in and dissolved in deionized water. They were afterwards diluted in deionized water to reach low concentrations down to less than 100 µg/L lactose. Food labelled as lactose free must contain less than 10 mg lactose in 100 g food product. When sample prep is taken into consideration, the target LOD becomes < 10 mg/L.

Method Parameters

The HPLC analysis was performed using a Knauer AZURA Analytical HPLC system consisting of an isocratic high pressure pump P 6.1L in the metal-free ceramic edition, an autosampler 3950, and the DECADE II electrochemical detector. The mobile phase was continuously sparged with helium to keep it inert. A schematic drawing of the system can be seen in Figure 1. The applied anion exchange column is stored in the tempered section of the DECADE II detector.

Figure 1: System for the sensitive analysis of lactose.

The system was flushed and allowed to equilibrate overnight because the applied method is very sensitive to any changes. A sufficient equilibration time is recommended especially when the system is running this method for the first time.

Method Parameters

Column: 250 × 4.6 mm, 7-µm RCX-10 PEEK hardware

Column order no.: 25EE158HML

Eluent A: 30 mM NaOH continuously sparged with helium

Gradient: Isocratic 

Flow rate: 2 mL/min

Injection volume: 50 µL

Temperature: 30 °C (column and flow cell)

System pressure: approximately 870 bar

Detection: ECD (electrochemical detection)

E cell: E1, E2, E3: 0.05, 0.75, -0.80 volts 

ts, t1, t2, t3: 0.06, 0.5, 0.13, 0.12 s

I-cell: 300–500 nA


Figure 2 shows an overlay of four chromatograms measured with the described method. It is obvious that a separation of the three applied sugars is possible. In addition, really low lactose concentrations could still be detected.

Figure 2: Chromatograms of a standard solution at different concentrations: Blue: 1090.0 µg/L, green: 218.0 µg/L, violet: 109.0 µg/L, red: 21.8 µg/L.

To figure out the limit of detection and the limit of quantification of the method, standard dilutions from around 1000 down to 21 µg/L were injected. Using the resulting peak heights, the parameters could be calculated. Figure 3 shows the calibration curve and the method performance parameters.

Figure 3: Calibration curve for lactose concentrations in the range of 21.8 µg/L to 1090.0 µg/L and results for method performance.

The following analysis of four samples from typical German food proved that most of them were lactose-free and therefore allowed to use this label, even with the new lactose limits given by the EU. Figures 4 to 7 show the chromatograms of the sugar standard (blue) overlaid with the samples (red) from Hähnchenspieß (chicken skewers), Leberkäse (meatloaf), Paprikalyoner (sausage), and Nürnberger (sausage). Only in one case could a significantly high lactose peak be found. Using the presented method, 3 out of 4 samples could be declared as lactose‑free; one sample is not allowed to be called a lactose-free product.

Figure 4: Chromatogram of “Hähnchenspieß” chicken skewers (red) with an overlay of the sugar standard (blue).

Figure 5: Chromatogram of “Paprikalyoner” sausage (red) with an overlay of the sugar standard (blue).

Figure 6: Chromatogram of “Leberkäse” meatloaf (red) with an overlay of the sugar standard (blue).

Figure 7: Chromatogram of “Nürnberger” sausage (red) with an overlay of the sugar standard (blue).

In addition the chromatograms show that few disturbing peaks were detected. This is caused by the specialized detection method that is sensitive to sugar analysis and does not show many of the samples impurities.

Using the calibration, the lactose contents in the samples were determined. Table 1 shows the results.

Three out of four samples can be declared as lactose-free according to the definition.

If there is a need in the future to detect even lower concentrations, this method allows for optimization by less dilution of the samples. This becomes possible by the very specific detection method where nearly no interfering matrix peaks are seen.


The presented method of HPAEC-PAD on a Knauer HPLC system was well-suited to determine low limits of sugars in food products. The detection principle was quite specific for sugars and only showed very little interference by matrix peaks. Using the AZURA analytical system combined with the DECADE II electrochemical detector, the analysis of lactose in lactose-free labelled products can be performed in a robust and reproducible manner. This system reaches the detection limits defined by the EU and can therefore be used in food control.


  1. New Nutrition Business: Lactose-free dairy Opportunities, strategies and key case studies (2012).
  2. B. Pribila et al., Journal of the American Dietetic Association 100(5), 
  3. 524–528 (2000).
  4. The Royal College of Physicians and The British Nutrition Foundation, Food intolerance and food aversion. Report number: 18 (1984).
  5. Vuorisalo et al., Perspect. Biol. Med. 55(2), 163–74 (2012).
  6. EFSA Journal 8(9), 1777 (2010)
  7. Leo M.L. Nollet et al., Handbook of Dairy Foods Analysis (Taylor and Francis Group LLC, USA, 2010).
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