The Institute of Food and Beverage Innovation of the Zurich University of Applied Sciences in Wädenswil deals with delicious, healthy, safe and sustainable food. Besides the education of food technology engineers, practical work with the food industry is fundamental to its strength in innovation. The research group, Food Chemistry analyzes food ingredients using different analytical techniques like HPLC-MS, FT-IR and HPTLC-MS. One of the research objectives is the determination of secondary plant metabolites like alkaloids and polyphenols in food. Monitoring the changes through the entire value chain is of primary importance. In this study, determination of secondary metabolites was performed on fresh cocoa beans and diverse intermediate products of the chocolate production process.

Polyphenols are not only known for their astringency but also for their great antioxidant potential. They are found in large amounts in cocoa and its derived product chocolate. The characterization and determination of high molecular oligomeric proanthocyanidins (PA) are of great interest because of their higher antioxidant activity compared to low molecular monomeric flavan-3-ols [1]. The changes of the PA profile are also essential for the organoleptic evaluation, which shows that monomeric PAs are more responsible for the bitter taste in contrast to oligomeric PAs, which are responsible for the astringency. Also of great interest are the two alkaloids caffeine and theobromine, not only because of their bitter taste but also for their stimulating and “prime pumping” effect. Anthocyanins can be used as general indicator for determining the degree of cocoa fermentation.

In the following application, HPTLC was used to determine oligomeric PAs, alkaloids and anthocyanins through the entire value chain – from fresh cocoa beans, through roasted cocoa, cocoa mass and up to molded chocolate bars. The established method has been proven as a suitable tool for comprehensive compound analysis in laboratories with high sample throughput. Besides the acquisition of the characteristic HPTLC fingerprint for the specific manufacturing step, the visual information can be used to build up an imaging database.

HPTLC plates silica 60 F254 (Merck), 20 x 10 cm

One single batch of 10 kg fresh cocoa beans was studied across different processing steps [2]. Changes in the PA profile were recorded on a lab-scale model and may not be fully in line with real-life crop or industrial scale processing. Nevertheless, one batch was processed and aliquots of about 50 g were sampled from each processing step. Samples were collected from (1) raw fresh cocoa beans, (2) fermented, dried cocoa beans, (3) roasted cocoa mass, (4) 1 h conched cocoa mass, and (5) molded chocolate bars.

1 g of the fine grinded and defatted cocoa powder was extracted three times with 3 mL acetone – water 1:1. The combined supernatant was diluted 1:10 with acetone – water 1:1.

Anthocyanin standard solution (0.01 mg/mL in methanol) with cyanidin-3-O-arabinoside (cn-ara) und cyanidin-3-O-glucoside (cn-glc); alkaloid standard solution (0.2 mg/mL in acetone – water 1:1) with caffeine and theobromine; PA standard solution (0.1 mg/mL in methanol) with (-)-epicatechin (EC), proanthocyanidins B2 (PA B2) and C1 (PA C1) as well as cinnamtannin A2 (Cinn A2)

Bandwise with Automatic TLC Sampler (ATS 4), 15 tracks, band length 8 mm, distance from left edge 20 mm, distance from the lower edge 8 mm, application volume between 5 and 10 μL for standard solutions and 2 and 10 μL for the sample solutions

In the Automatic Developing Chamber (ADC 2) with chamber saturation (with filter paper) for 20 min and after conditioning at 33% relative humidity for 10 min using a saturated solution of magnesium chloride, development with ethyl formate – formic acid – water – toluene 30:4:3:1.5 to the migration distance of 70 mm (from the lower edge), drying for 5 min

The plate was heated at 100 °C and immersed with Chromatogram Immersion Device (immersion speed 5 cm/s, immersion time 0 s) in Fast Blue Salt B reagent (140 mg Fast Blue Salt B in 140 mL methanol, 10 mL water, and 50 mL dichloromethane), followed by 30 s drying in a cold air stream. Alternatively, the Derivatizer can be used.

TLC Visualizer under UV 254 nm and white light, also after derivatization

TLC Scanner 4 and visionCATS, absorption measurement at 280 nm for alkaloids and 510 nm for anthocyanins and derivatized PA, slit dimension 5.00 x 0.20 mm, scanning speed 50 mm/s, evaluation via peak area, polynomial regression, spectrum recording from 190 to 600 nm

Elution of target zones was done after derivatization with Fast Blue Salt B with the oval elution head (4 x 2 mm) of the TLC-MS Interface. Hereby, acetone – water 1:1 was used as elution solvent at a flow rate of 0.1 mL/min using a HPLC pump. The recording of mass spectra was performed in the positive ionization mode.

Contact: Dr. Vasilisa Pedan, Zurich University of Applied Sciences, Institute of Food and Beverage Innovation, 8820 Wädenswil, Switzerland, vasilisa.pedan[at]zhaw.ch

Dr. Gawande, Assistant Professor in the Quality Assurance Techniques at Sinhgad Institute of Pharmacy, Pune, Maharashtra, India, is involved in the development and validation of chromatographic methods for stability of pharmaceutical drugs, both standalone and fixed dose combinations.

Stability is the capacity of a drug product to remain within specification for a given time. It is a prime requirement to ensure its identity, strength, quality and purity. Forced degradation studies are an integral part of drug development programs. For detecting the number and types of degradation products that are formed under various conditions, different chromatographic techniques in conjunction with UV and MS are used.

HPTLC is especially beneficial for stability testing due to its disposable stationary phase, as well as in the case of forced degradation, when appreciable amounts of acidic, alkaline and peroxide reagents are used. Furthermore, HPTLC permits the analysis of a large number of samples in a short time. There are also multiple derivatization and detection options that can help to characterize degradation products. Apart from this, simplicity and low cost of analysis add to the benefits.

10 mg of cefixime trihydrate (CEFI) or azithromycin dihydrate (AZI) dissolved in 10 mL methanol

1. Hydrolytic degradation: 1 mL of drug standard solution was mixed with 1 mL water or 0.5 N HCl or 0.5 N NaOH, kept for 1 h at RT and neutralized with acid or base
2. Oxidative degradation: 1 mL of drug standard solution was mixed with 1 mL of H2O2 (3 and 30 %) and kept for 1 h at RT
3. Thermal degradation: The drug powder placed in a sealed glass ampoule was heated at 100 °C and 200 °C for 1 h and 2 h in a hot oven
4. Photolytic degradation (according to ICH guidance Q1B, option 2): A thin layer of solid drug powder was exposed with fluorescent cold white light (1.25 million lux h) and UV light (200 Whm-2) in a photo stability chamber

All degradation samples were dissolved (solid samples) or diluted with methanol to a final concentration of 100 μg/mL.

HPTLC plates silica gel 60 F254, aluminium backed (Merck), 20 x 10 cm, prewashed by developing first with methanol and then with the mobile phase followed by drying for 15 min with cold air

Bandwise application with Linomat 5 or Automatic TLC Sampler (ATS 4), 15 tracks, band length 8 mm, distance from left edge 20 mm, distance from lower edge 8 mm, application volumes 10.0 μL for sample solutions and 2.0 and 5.0 μL for standard solutions (CEFI resp. AZI); for preparative isolation: band length 180 mm and application volume 200.0 μL

In the Twin Trough Chamber 20 x 10 cm with chamber saturation (with filter paper) for 20 min, development with ethyl acetate – methanol – acetone – toluene – ammonia 2:10:14:1:1 to the migration distance of 80 mm (from the lower edge), drying for 15 min with cold air

For detection of AZI, the plate was immersed with the Chromatogram Immersion Device into sulfuric acid reagent (1:4 in ethanol; immersion speed 3 cm/s, immersion time 6 s), dried for 30 s with cold air, and heated at 100 °C for 5 min using the TLC Plate Heater.

TLC Visualizer under UV 254 nm, UV 366 nm, and white light

TLC Scanner 3 and winCATS, absorption measurement at 235 nm for CEFI and 530 nm for AZI, slit dimension 6.00 x 0.45 mm, scanning speed 20 mm/s, spectra recording from 190 to 550 nm

The target zones were marked with a pencil, and plates were cut carefully to separate different bands. Individual bands were cut and sonicated with methanol for extraction of degradation products. Methanol fractions were concentrated and evaporated to obtain solid residues which were analysed by Q-TOF and Ion Trap mass spectrometry in the positive ionization mode

Contact: Dr. Vandana Gawande, Department of Pharmaceutical Chemistry, STES’s Sinhgad Institute of Pharmacy, Narhe, Pune – 411041, Maharashtra, India, gawandevandana848[at]gmail.com