The Hampering Effect of Heavy Water (D2O) on Oscillatory Peptidization of Selected Proteinogenic α-Amino Acids.

We present an overview of our studies on the hampering effect of heavy water (D2O) on spontaneous oscillatory peptidization of selected proteinogenic α-amino acids. The investigated set of compounds included three endogenous and two exogenous species. The experiments were carried out with use of high-performance liquid chromatography (HPLC), mass spectrometry (MS) and scanning electron microscopy (SEM). These techniques were chosen to demonstrate spontaneous oscillatory peptidization of α-amino acids in an absence of D2O (HPLC) and the hampering effect of D2O on peptidization (HPLC, MS and SEM). The HPLC analyses were carried out at 21 ± 0.5°C with each α-amino acid freshly dissolved in the binary liquid mixture of organic solvent + H2O, 70:30 (v/v) or in pure D2O for several dozen hours or several hours, respectively. The analyses with use of MS and SEM were carried out, respectively, after 7 days and 1 month of sample storage period in the darkness at 21 ± 0.5°C and for these experiments, each α-amino acid was dissolved in the liquid mixture of organic solvent + X, 70:30 (v/v), where X: H2O + D2O in volume proportions from 30:0 to 0:30. The results obtained with use of HPLC, MS and SEM point out to the strong hampering effect of D2O on the oscillations and peptidization yields, yet the dynamics of these processes significantly depends on chemical structure of a given α-amino acid.

Thin-layer chromatographic quantification of magnolol and honokiol in dietary supplements and selected biological properties of these preparations.

Two isomeric biphenyl neolignans, magnolol and honokiol, are considered as constituents responsible for the healing effect of magnolia bark, a traditional Oriental medicine. To survey the increasing number of dietary supplements that contain magnolia bark or its extract, an affordable quantitative thin-layer chromatography (TLC) - densitometry method was developed. The methanol extracts were analyzed on the silica gel plates after manual sample application using n-hexane - ethyl acetate - ethanol (16:3:1, v/v/v) as a mobile phase. For quantitation, the chromatograms were scanned in the absorbance mode at the wavelength λ = 290 nm. The limits of detection and quantitation were 90 and 280 ng/zone for magnolol and 70 and 200 ng/zone for honokiol, respectively. None of the two targeted neolignans were detected in two of the six analyzed supplements. In the other four samples, the measured amounts were between 0.95-114.69 mg g-1 for magnolol and 4.88-84.86 mg g-1 for honokiol. Moreover, separations of these two neolignans on the TLC and high-performance TLC (HPTLC) layers were compared and HPTLC was combined with antioxidant (DPPH) and antibacterial (Bacillus subtilis and Aliivibrio fischeri) assays and mass spectrometry (MS), using the elution-based interface. Both magnolol and honokiol exhibited effects in all bioactivity assays. The HPTLC-MS tests confirmed purity of neolignan zones in the extracts of dietary supplements and supported tentative identification of the alkaloid piperine and the isoflavone daidzein as additional bioactive components of the investigated dietary supplements. Using the same mobile phase in the orthogonal directions 2D-HPTLC-MS experiments proved degradation, i.e., instability of magnolol and honokiol on the silica gel adsorbent.

Development of a Novel Thin-Layer Chromatographic Method of Screening the Red Beet (Beta vulgaris L.) Pigments in Alimentary Products.

The aim of this study was to develop a thin-layer chromatographic method of qualitative analysis, aiming to confirm the presence of the red beetroot pigments in a given sample. The TLC system developed for this purpose consists of the precoated RP-18 F254s TLC plates and the acetonitrile + methanol + water + glacial acetic acid, 2:7:1:0.1 (v/v/v/v) mobile phase. With the use of this system, a striking horizontal separation of betacyanin pigments is obtained for both the red beetroot juice and the commercial betanin sample (with the left-to-right resolution distance of the two bands equal to ca. 6 mm), and a unique pattern of the two skewed chromatographic bands is observed. This striking phenomenon has been given a thorough consideration, and its tentative physicochemical justification was provided, based on analogical cases reported and extensively discussed in our earlier studies. Characteristic fingerprint obtained both for the beetroot juice and the commercial sample of betanin (resembling two slant butterfly wings) can prove very helpful for qualitative confirmation of the presence (or otherwise) of the betanin pigment in the red color juices and beverages, as it was demonstrated upon an example of elderberry juice with a confirmed fortification with the betanin pigment.

Vulnerability of anthocyanins to the components of a thin-layer chromatographic system and comprehensive screening of anthocyanes in alimentary products.

The aim of this study was to revisit the TLC authentication of alimentary products concept based on analysis of anthocyanes with the foodstuffs of plant origin. To this effect, we used two anthocyanins (cyanin and keracyanin) and two anthocyanidins (pelargonidin and delphinidin) as phytochemical standards. The first step was to develop a novel method making use of the RP-18 F254s stationary phase (which ensures mixed-mode retention mechanism with the localized adsorption on the non-bonded silanols) and acetic acid as the mobile phase component. Importantly, similar TLC systems are currently used for the analysis of anthocyanes. Individual steps of our method development enabled a deeper insight in vulnerability of anthocyanins to external conditions resulting in hydrolysis thereof. In this study, it was impossible to fully separate the products of hydrolytic degradation of the test anthocyanins in a single development run and it was only triple development which ensured distinct and symmetrically shaped chromatographic spots, further scrutinized with use of mass spectrometry. The identity of the hydrolytically split fractions was additionally studied with use of the p-aminobenzoic acid (PABA) test. To obtain calibration curves, triple development was employed for cyanin, keracyanin, and pelargonidin, while delphinidin was developed in one development run. The respective LOD and LOQ values were: for spot (i) derived from the cyanin standard, 0.005 and 0.016 µg spot-1; for spot (ii) derived from the cyanin standard, 0.006 and 0.017 µg spot-1; for spot (i) derived from the keracyanin standard, 0.092 and 0.274 µg spot-1; for spot (ii) derived from the keracyanin standard, 0.035 and 0.104 µg spot-1; for the pelargonidin standard, 0.013 and 0.040 µg spot-1; and for the delphinidin standard, 0.036 and 0.108 µg spot-1. The developed method was used to identify and quantify cyanin, keracyanin, pelargonidin and delphinidin in selected alimentary products (syrups, juices and herbal infusions), keeping in mind that the obtained numerical results were of semi-quantitative nature only.

Novel thin-layer chromatographic method of screening the anthocyanes containing alimentary products and precautions taken at the method development step.

The purpose of this study was to develop a novel and cost-effective thin-layer chromatographic method (TLC) using cellulose powder as stationary phase for authentication of the selected fruit-based alimentary products and targeting anthocyanes as the authenticity markers. Our method outperformed the HPTLC method earlier developed by another research team using silica gel as stationary phase. It was demonstrated that due to a limited chemical stability of anthocyanes, employing them as authenticity markers is burdened with a non-negligible uncertainty risk. Hydrolytic split of the glycosides into the aglycone and carbohydrate moieties can lead to a confusing multiplication of chromatographic bands and therefore it is advisable to use for the authentication purposes a limited set of well selected and stable enough anthocyane markers. Cyanin chloride, keracyanin chloride, pelargonidin chloride and delphinidin chloride were selected as the external standards and for the development of the calibration curves. The TLC-obtained LOD and LOQ values were 0.025 and 0.075µgspot-1 for cyanin, 0.055 and 0.166µgspot-1 for keracyanin, 0.047 and 0.140µgspot-1 for pelargonidin, and 0.171 and 0.513µgspot-1 for delphinidin, respectively. The analogous HPTLC-obtained LOD and LOQ values were 0.107 and 0.321µgspot-1 for cyanin, 0.189 and 0.566µgspot-1 for keracyanin, and 0.161 and 0.484µgspot-1 for pelargonidin, respectively. Delphinidin was not detectable with use of the HPTLC method. Consequently, quantification of anthocyanes in the alimentary products carried out with use of TLC allowed identification of more target compounds and in a higher number of alimentary products than it was possible with use of HPTLC, apparently due to the LOD levels by one magnitude order lower for TLC than HPTLC.