l-Arabinose (Ara), d-cellobiose (Cel), l-fucose (Fuc), d-fructose (Fru), d-galactose (Gal), N-acetyl-d-galactosamine (GalNAc), d-glucose (Glc), N-acetyl-d-glucosamine (GlcNAc), d-mannose (Man), d-melibiose monohydrate (Mel), d-raffinose pentahydrate (Raf), l-rhamnose (Rha), d-ribose (Rib), l-sorbose (Sor), d-sucrose (Suc), and d-trehalose (Tre) (all saccharides ≥ 98%), acetonitrile (HPLC grade), 2-propyl acetate (> 99%), methanol (HPLC grade), natural product reagent A (≥ 98%), sodium dihydrogenphosphate monohydrate (> 98%), and sodium chloride (> 99.5%) were delivered by Carl Roth (Karlsruhe, Germany). d-galacturonic acid (GalU), d-glucosamine hydrochloride (GlcN), sodium d-glucose-6-phosphate (GlcP), d-lactose monohydrate (Lac), d-melezitose monohydrate (Mlz), d-sorbitol (Sol), stachyose monohydrate (Sta), sucralose (Sul) (all saccharides ≥ 98%), ethylamine (aqueous solution 70%), and α-amylase from human saliva (Type IX-A, lyophilized powder, 1,000–3,000 units/mg protein) were obtained from Sigma-Aldrich (Taufkirchen, Germany). Purified water was prepared by a Destamat Bi 18E (Heraeus, Hanau, Germany). HPTLC plates silica gel 60 were from Merck (Darmstadt, Germany). Wheat starch, wheat flour, spring honey, summer honey, blossom honey, and the lactose-free milk samples were purchased from a local supermarket.

Saccharide stock solutions were individually prepared in water (10 mg/mL, stored at 4 °C for maximal one week) and freshly diluted with water to 1 mg/mL to obtain individual working standard solutions. Standard mixes for quantifications (0.1 mg/mL) were prepared by pipetting 100 µL of the respective working standard solutions, filled up to 1000 µL with water.

For the digestion of starches, 100 mg of soluble starch, wheat starch, and wheat flour were weighed into 25-mL falcon tubes and suspended in 10 mL of buffer (20 mM sodium dihydrogen phosphate with 6.7 mM sodium chloride, pH 6.7). The falcon tubes were placed in a boiling water bath for 15 min to inactivate enzymes. A total of 1 mL of each freshly vortexed suspension was pipetted into a 2-mL Eppendorf tube, followed by the addition of 1 U α-amylase; except for native samples (0 min). Incubation was carried out at ambient temperature for 1, 3, 5, 7, and 9 min using a Vortex Genie 2 with multi-tube holder (Scientific Industries, Bohemia, NY, USA) at speed 10. After incubation, α-amylase was deactivated in a boiling water bath for 15 min. The cooled samples were made up to a total volume of 2 mL with water, vortexed, and centrifuged at 17,000g (Heraeus Pico 17, Thermo Fisher Scientific, Schwerte, Germany).

Milk samples (100 µL) were pipetted into a 10-mL volumetric flask and filled up with methanol. After vortexing, the flask was allowed to stand for 10 min to settle down the proteins, whereafter the supernatant was filtrated through a 0.45-μm membrane filter (cellulose acetate) into an HPTLC vial.

Honey samples (1 g) were weighed into 25-mL falcon tubes, followed by the addition of 10 mL of water. After 15 min on a vortex with multi-tube holder at speed 10, the sample solutions were filtrated through a 0.45-μm membrane filter (cellulose acetate) into a 2-mL Eppendorf tube. For the qualitative and quantitative analyses, the honey solutions were diluted 1:25 and 1:250, respectively, with water in an HPTLC vial.

HPTLC instrumentation (CAMAG, Muttenz, Switzerland) consisted of Automatic TLC Sampler (ATS 4), Automatic development chamber (ADC 2), Derivatizer, TLC Visualizer 2, TLC Scanner 4, and Plate Heater III. The instruments were controlled by visionCATS software version 3.2 SP2. HPTLC silica gel 60 plates were prewashed with methanol and dried for 10 min in an oven at 110 °C. Samples and standard solutions were applied as 6‑mm bands with a track distance of 7 mm, distance from the lower edge 8 mm, and left edge minimal distance of 12 mm with the following settings: water as sample solvent type, standard (qualitative analyses) or quantification (quantitative analyses) as filling/rinsing quality, and 25 µL syringe volume. During the method development, standard solutions (1 mg/mL) were applied at 0.2 µL. For quantifications, standard solutions (0.1 mg/μL) were applied at 0.2, 0.5, 0.8, 1.4, and 2.0 μL. Starch samples were applied at 6 µL, milk samples at 1.0, 3.0, and 5.0 µL, and honey samples at 0.5 μL and 1.5 μL (1:25 and 1:250 dilutions).

After plate preconditioning for 20 min in a 20 cm × 10 cm twin-trough chamber (using filter paper and 10 mL mobile phase in the opposite trough), the plate was developed with 10 mL 2-propyl acetate–methanol–water (7:3.5:1.5, V/V) containing 1 mg/mL natural product reagent A up to 60 mm, taking 12 min, and thereafter dried inside a fume hood for 15 min. A second development was performed analogously. Alternatively, development was performed in the ADC 2 with chamber saturation (using filter paper) for 5 min and plate preconditioning for 15 min with 20 mL and 10 mL mobile phase, respectively, followed by plate drying for 10 min. Relative humidity of the surrounding air during experiments was < 50%.

Derivatization was carried out with 1 mL ethylamine solution (15% in water, Derivatizer, yellow nozzle, level 2), followed by plate heating at 150 °C for 15 min. Chromatograms were captured under 366 nm followed by fluorescence measurements (366 nm > 400 nm, measurement slit 5 mm × 0.2 mm). An optional second derivatization was performed with 2 mL aniline diphenylamine orthophosphoric acid reagent (yellow nozzle, level 5), followed by plate heating at 110 °C for 10 min. Chromatograms were documented at white light illumination (transmittance mode).

Limit of detection (LOD) and limit of quantification (LOQ) were determined for glucose-6-phosphate, maltose, lactose, fructose, and rhamnose. The standard solutions (0.1 mg/mL) were applied with 0.2, 0.5, 0.8, 1.4, and 2.0 µL (20–200 ng), followed by development, derivatization, and fluorescence measurement. The calculations followed the Deutsches Institut für Normung (DIN) method [17], the International Council for Harmonisation (ICH) guidelines [18], and the United States Pharmacopeia (USP) procedure [19] (available in visionCATS).