Determination of Lactose in dairy products using TLC-MS


Today food intolerances play a big role in our society. For example a lot of people are lactose intolerant, usually some more in Asian countries than in Europe or North America. So far, analysis of lactose in foodstuff, especially in dairy products is important for food industries. Foodstuff with a lactose content less than 10 mg/100 g could be labeled as “lactose-free”. Traditionally, analysis is done by use of photometric measurements in single cuvettes, or could also be done in a 96-well plate by use of an enzymatic test kit. Less time consuming and complicated is the use of HPTLC (High performance thin layer chromatography). As shown in this application sheet, milk, yoghurt and an instant sauce are applied onto the TLC plate without a complex sample preparation. Only centrifugation and dilution is necessary. Lactose can be quantified by scanning the TLC plate and subsequently a MS (mass spectrometry) measurement is possible by extracting the analyte directly from the TLC plate with a TLC-MS interface.

Results and Discussion

As it is not possible to detect lactose directly under VIS- or UV-light, the plate has to be derivatized with Aniline-di-phenylamine-phosphoric acid reagent before scanning. Figure 1a and 1b show the derivatized plate under white light and at 366 nm.

For quantification a lactose standard with 1.0 mg/mL was used and volumina between 0.1 μL and 4.0 μL were applied on the plate. Details about sample preparation and chromato-graphic data are shown in the tables on next page. Lactose content of the samples is calculated by means of a calibration curve (poly-nominal). Lactose content of the samples is shown in table 1.

Derivatized plate under white light

Figure 1a: Derivatized plate under white light.



Derivatized plate at 366 nm

Figure 1b: Derivatized plate at 366 nm.


The mass spectrum in figure 2 shows three plain peaks, two of lactose with adducts and one of an in-source fragment.


M = 342.3 g/mo


By the use of HPTLC the difference of lactose content in dairy products could be shown. HPTLC is a straightforward and economic method to analyze samples with complex sample matrix, such as milk or yoghurt. By coupling TLC to MS it is possible to identify the different compounds of a sample.

Determined Lactose values

Organic fresh milk 4.68 g/ 100 g
UHT milk 4.36 g/ 100 g
Lactose free UHT milk < 100 mg/ 100 g
Instant sauce 1.64 g/ 100 g
yoghurt 5.49 g/ 100 g


Chromatographic data

Track Compound Conc. [mg/mL] Application
volume [µL]
hRf Detected mass m/z
1, 11 Lactose standard 1


0.1 0.35 365.2 406.1
2, 12 Lactose standard 0.5
3, 13 Lactose standard 1.0
4, 14 Lactose standard 2.0
5, 15 Lactose standard 4.0
6, 16 organic fresh milk 0.4
7, 17 UHT milk   0.4
8, 18 lactose free UHT milk   2.0
9, 19 instant sauce   0.3
10, 20 yoghurt   0.3

Application data

Plate HPTLC Silica gel 60 F254 MS-grade, 20x10 cm
Sample preparation


Milk: 2 x 5 min centrifuged, afterwards 1mL diluted in 50 mL water
Yoghurt: 1 g diluted in 10 mL water, afterwards 1 x centrifuged
Instant sauce: sauce cooked as directed, 1 g sauce diluted in 10 mL water, afterwards 1 x 5 min centrifuged
Sample application ATS 4 sample applicator (Camag), 6 mm bandwise
Application volume 0.1 - 4 µL
Mobile phase Acetonitrile / Water 3/1 (v/v) + 0.1% Trifluoroacetic acid
Migration distance 5 cm
Migration time 12 min
Extraction equipment “TLC-MS Interface” from Camag
Extraction solvent Acetonitrile / Water 95/5 (v/v) + 0.1% Formic acid
Extraction flow 0.2 mL/min
Documentation equipment TLC Visualizer (Camag)
Wavelength scan at white light
Staining Aniline-diphenylamine-phosphoric acid reagent heated 10 min at 120°C
MS equipment single-quadrupole mass spectrometer expression CMS (Advion)
MS detection ESI (+) mode (m/z 50 - 1200)

Ordering information

Description Product No.
HPTLC Silica gel 60 F254 MS-grade, 20x10 cm 1.00934
Acetonitrile hypergrade for LC-MS LiChrosolv® 1.00029
Trifluoroacetic acid for spectroscopy Uvasol® 1.08262
Diphenylamine for synthesis 8.20528
Aniline for analysis EMSURE® 1.01261
Ortho-Phosphoric acid  for analysis EMSURE® 1.00573