Determination of Lactose in Dairy Products Using TLC-MS

Introduction

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.

Figure 1a. Derivatized plate under white light.

Figure 1b. Derivatized plate at 366 nm.

The mass spectrum in figures 2 and 3 shows three plain peaks, two of lactose with adducts and one of an in-source fragment.

Figure 2. Lactose structure.

Figure 3. Plain peaks two of lactose.

Conclusion

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.

Table 1.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

Table 2.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

Table 3.Application data

Chromatography
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
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
Detection
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)

Figure 4. Lactose product.