Reagent sequence for planar chromatographic analysis of eight sweeteners in food products approved in the European Union

Chemicals and materials

Sucralose (Suc, E 955, > 99%) was provided by Innosweet, Braunschweig, Germany. Neohesperidin dihydrochalcone (Neo, E 959, ≥ 85%) was delivered from SAFC Supply Solution, Jerusalem, Israel. Saccharine sodium (Sac, E 954, > 98%) was obtained from Roth, Karlsruhe, Germany. Aspartame (Asp, E 951, p. a.) was provided by Barentz, Oberhausen, Germany. Acesulfame potassium (Ace, E 950, > 99%) and aspartame-acesulfame salt (E 962, 2:1 mixture) were provided by Fluka, Buchs, Switzerland. Sodium cyclamate (Cyc, E 952, > 99%) and HPTLC plates silica gel 60 F254 (20 cm × 10 cm) were supplied by Merck, Darmstadt, Germany. The steviol glycosides (E 960) rebaudioside A (Reb, 97.3%) and stevioside (Ste, 97.7%) were obtained from Phytolab, Vestenbergsgreuth, Germany. Organic solvents, sulfuric acid, glacial acetic acid, ninhydrin, and 2-naphthol, all analytical grade, were obtained from Sigma-Aldrich Fluka, Darmstadt, Germany. Primuline (p. a.) was purchased from Waldeck, Münster, Germany. Bidistilled water was prepared with Destamat Bi 18E, Heraeus, Hanau, Germany. Food samples were bought on the local market (Table 1).

Table 1 Overview on food samples investigated, their labeling, and contents foundStandard solution mixture

Individual standard solutions of the eight sweeteners E 950, E 951, E 952, E 954, E 955, E 959, E 960, and E 962 were prepared by dissolving 10 mg in 10 mL methanol each (1 µg/µL). A methanolic standard solution mixture was prepared that contained Ace 600, Suc 100, Sac 100, Asp 50, Ste/Reb 30, Neo 75, and Cyc 800 ng/µL.

Sample preparation

The solid samples (ID 1, 2, 7 and 8; Table 1) were crushed, and 1 g each (5 g for ID 8) were dissolved with 0.5 mL hot water in a 5-mL volumetric flask and filled up with methanol to the mark. ID 1 was further diluted with methanol 1:1. For the semi-solid yoghurt sample (ID 4), 20 g was dissolved in 10 mL methanol. Liquid samples (IDs 5 and 3) were directly used, whereby ID 6 was diluted 1:2 with methanol. If needed, centrifugation may be used at 3000 × g for 5 min.

Chromatographic method

HPTLC instrumentation (CAMAG, Muttenz Switzerland) was operated and data were processed with winCATS software, version 1.4.5.2027. The solutions were sprayed as 8-mm band (Automatic TLC Sampler ATS4) allowing 18 tracks to be applied on HPTLC plates silica gel 60 F254 (20 cm × 10 cm) with a 10-mm distance between bands, 14-mm distance from the left side and 8-mm distance from lower edge. For calibration, 1 − 20 µL of the standard solution mixture was applied on the plate (30 ng – 16 µg per band, depending on the sweetener). For quantification, sample application volumes (as in Fig. 3) were slightly adjusted to be between 2 and 20 µL/band, e.g., 10/2 µL for ID 1, 5/2 µL for ID 3, 3 µL for ID 4, 20/5 µL for ID 5, and 20 µL for ID 8. Development was performed in the twin trough chamber or Automated Developing Chamber 2 at a relative humidity of the air of about 30% with a mixture of ethyl acetate, methanol, and acetic acid 5:1:1, V/V, up to 65 mm. The plate was dried after application (1 min) and development (3 min). Plate images were recorded (DigiStore 2 Documentation System) at UV 254 nm, FLD 366 nm and white light illumination (Vis). Densitometry (TLC Scanner 3) was performed via absorbance measurement at selected wavelengths (Table 2), which were determined by recording UV spectra between 200 and 400 nm. Quantification was performed by peak height or area. For post-chromatographic derivatization, the plate was three times immersed (TLC Immersion Device III, vertical speed 4 cm/s and immersion time 1 s) according to the following reagent sequence, followed each by plate heating (TLC Plate Heater III) and image documentation or densitometry, if needed.

1.

Primuline reagent (100 mg primuline dissolved in 20 mL water and 80 mL acetone), followed by solvent evaporation and detection at 366 nm.

2.

Ninhydrin reagent (0.3 g ninhydrin dissolved in 95 mL isopropyl alcohol and 5 mL glacial acetic acid), followed by heating at 120 °C for 5 min and detection at Vis.

3.

2-Naphthol sulfuric acid reagent (1 g 2-naphthol dissolved in 90 mL ethanol and 6 mL 50% sulfuric acid added dropwise), followed by heating at 120 °C for 5 min and detection at Vis.

Table 2 Calibration performance for the eight sweeteners

The reagents stored in the refrigerator were stable for several months. For optional recording of full-scan HPTLC–ESI-MS spectra (m/z 100–1000; MSD, Agilent, Waldbronn, Germany), respective zones were directly eluted (TLC–MS Interface) into the ESI spray chamber using methanol at a flow rate of 0.2 mL/min (HP 1100 pump, Agilent). The capillary voltage for positive and negative ionization was set to be + 4 kV and − 4 kV, respectively. The nebulizer gas pressure was 20 psig, and drying gas temperature and flow rate were 300 °C and 10 L/min, respectively.

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