Thin-Layer Chromatography (TLC) in the Screening of Botanicals-Its Versatile Potential and Selected Applications.

The aim of this paper is to present a comprehensive overview of the main aims and scopes in screening of botanicals, a task of which thin-layer chromatography (TLC) is, on an everyday basis, confronted with and engaged in. Stunning omnipresence of this modest analytical technique (both in its standard format (TLC) and the high-performance one (HPTLC), either hyphenated or not) for many analysts might at a first glance appear chaotic and random, with an auxiliary rather than leading role in research, and not capable of issuing meaningful final statements. Based on these reflections, our purpose is not to present a general review paper on TLC in screening of botanicals, but a blueprint rather (illustrated with a selection of practical examples), which highlights a sovereign and important role of TLC in accomplishing the following analytical tasks: (i) solving puzzles related to chemotaxonomy of plants, (ii) screening a wide spectrum of biological properties of plants, (iii) providing quality control of herbal medicines and alimentary and cosmetic products of biological origin, and (iv) tracing psychoactive plants under forensic surveillance.

Determination of azoxystrobin and its impurity in pesticide formulations by liquid chromatography.

A method was developed for the simultaneous qualitative and quantitative determination of azoxystrobin and its relevant impurity (Z)-azoxystrobin using high performance liquid chromatography with diode array detector (HPLC-DAD) in suspension concentrate (SC) pesticide formulations, with the aim of the product quality control. Method validation was realized according to SANCO/3030/99 rev. 5. The proposed method was characterized by acceptable accuracy and precision. The repeatability expressed as ratio standard deviation (%RSD) to relative standard deviation (%RSDr) was lower than 1, whereas individual recoveries were in the range of 97-103% and 90-110% for azoxystrobin and (Z)-azoxystrobin, respectively. The limit of quantification (LOQ) for the impurity ((Z)-azoxystrobin) amounted to 0.3 µg mL-1 and was acceptable because it was lower than the maximum permitted level according to Commission Implementing Regulation (EU) No 703/2011 of 20 July 2011 for the active substance (azoxystrobin) being 25 g kg-1 of the azoxystrobin content found. The method described in this paper is simple, precise, accurate and selective as well as represents a new and reliable way of simultaneous determination of azoxystrobin and its relevant impurity in formulated products.

Application of different chromatographic techniques and chemometric analysis in authenticity testing of plant protection products containing azoxystrobin as an active substance.

Azoxystrobin (methyl(2E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4-yloxy] phenyl}-3-methoxyacrylate) is an active ingredient used to protect crops against fungal diseases. The experience of the Polish control laboratory indicates relatively frequent cases of counterfeit plant protection products (PPPs) containing this active substance. The present study aimed to use chemometric methods to model chemical fingerprints obtained by different chromatographic techniques to verify the original formulation of PPPs containing the active substance azoxystrobin. The pesticides used in the study came from different sources (including stores and warehouses), were manufactured at a different time and came from different production batches. The results obtained with the HPLC-DAD and HS-GC-MS techniques were then modeled using principal component analysis (PCA) and soft independent modeling by class analogy (SIMCA) classifier. The proposed approach has been confirmed as useful for verifying the authenticity of PPPs and can be used in the routine control testing of SC pesticides containing azoxystrobin.

The Forty-Sixth Euro Congress on Drug Synthesis and Analysis: Snapshot †.

The 46th EuroCongress on Drug Synthesis and Analysis (ECDSA-2017) was arranged within the celebration of the 65th Anniversary of the Faculty of Pharmacy at Comenius University in Bratislava, Slovakia from 5-8 September 2017 to get together specialists in medicinal chemistry, organic synthesis, pharmaceutical analysis, screening of bioactive compounds, pharmacology and drug formulations; promote the exchange of scientific results, methods and ideas; and encourage cooperation between researchers from all over the world. The topic of the conference, "Drug Synthesis and Analysis," meant that the symposium welcomed all pharmacists and/or researchers (chemists, analysts, biologists) and students interested in scientific work dealing with investigations of biologically active compounds as potential drugs. The authors of this manuscript were plenary speakers and other participants of the symposium and members of their research teams. The following summary highlights the major points/topics of the meeting.

Antioxidant Activity of Selected Thyme (Thymus L.) Species and Study of the Equivalence of Different Measuring Methodologies.

This study presents the results of comparative evaluation of the antioxidant activity of the phenolic fraction exhaustively extracted with aqueous methanol from 18 different thyme (Thymus L.) specimens and species. This evaluation is made with use of the same free radical source (DPPH• radical), three different free radical scavenging models (gallic acid, ascorbic acid, and Trolox), and three different measuring techniques (the dot blot test, UV-Vis spectrophotometry, and electron paramagnetic resonance spectroscopy, EPR). A comparison of the equivalence of these three different measuring techniques (performed with use of hierarchical clustering with Euclidean distance as a similarity measure and Ward's linkage) is particularly important in view of the fact that different laboratories use different antioxidant activity measuring techniques, which makes any interlaboratory comparison hardly possible. The results obtained confirm a semiquantitative equivalence among the three compared methodologies, and a proposal is made of a simple and cost-effective dot blot test that uses the DPPH• radical and provides differentiation of antioxidant activity of herbal matter comparable with the results of the UV-Vis spectrophotometry and EPR.

A Comparison of Antibacterial Activity of Selected Thyme (Thymus) Species by Means of the Dot Blot Test with Direct Bioautographic Detection.

Bioautography carried out with the aid of thin-layer chromatographic adsorbents can be used to assess antibacterial activity in samples of different origin. It can either be used as a simple and cost-effective detection method applied to a developed chromatogram, or to the dot blot test performed on a chromatographic plate, where total antibacterial activity of a sample is scrutinized. It was an aim of this study to compare antibacterial activity of 18 thyme (Thymus) specimens and species (originating from the same gardening plot and harvested in the same period of time) by means of a dot blot test with direct bioautography. A two-step extraction of herbal material was applied, and at step two the polar fraction of secondary metabolites was obtained under the earlier optimized extraction conditions [methanol-water (27+73, v/v), 130°C]. This fraction was then tested for its antibacterial activity against Bacillus subtilis bacteria. It was established that all investigated extracts exhibited antibacterial activity, yet distinct differences were perceived in the size of the bacterial growth inhibition zones among the compared thyme species. Based on the results obtained, T. citriodorus "golden dwarf" (sample No. 5) and T. marschallianus (sample No. 6) were selected as promising targets for further investigations and possible inclusion in a herbal pharmacopeia, which is an essential scientific novelty of this study.

Determination of selected protoporphyrins in parma ham with use of 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin as a surrogate standard in the recovery study.

A high-performance liquid chromatographic method for the determination of hemin, protoporphyrin IX (PPIX), and zinc(II)protoporphyrin IX (Zn(II)PPIX) in Parma ham was developed. The detection was done by means of a universal DAD-detector, whereby quantification of the three naturally occurring protoporphyrins was carried out at lambda = 414 nm, i.e., very close to the respective maxima of their Soret bands. The extraction thereof from the meat matrix was done by a mixture of acetone and chloroacetic acid (100 mL + 0.2 g). Usage of 5,10,15,20-tetra(4-hydroxyphenyl)porphyrin (THPP) as a surrogate standard and its detection fixed at lambda = 444 nm, allowed to obtain accurate (ca. 96%) recovery results. Established concentrations of hemin, Zn(II)PPIX, and PPIX in the Parma ham samples were 15.97, 19.96 and 1.52 µg g(-1), respectively.

A Modified Device for Pressurized Planar Electrochromatography and Preliminary Results with On-Line Sample Application.

Pressurized planar electrochromatography (PPEC) is a separating technique in which an electric field is applied to force the mobile phase movement through a porous media (electroosmotic effect). High separation efficiency, fast separations and changes in separation selectivity in comparison to liquid chromatography, especially thin layer chromatography (planar chromatography, TLC), are features of this technique. Constructional methodological challenges to PPEC are obstacles to its development and application in laboratory practice. In this article, an attempt to overcome the challenges related to device construction and sample application/injection is described. The introduced device enables both prewetting of the adsorbent layer and electrochromatogram development with a single PPEC device. It also enables simultaneous application/injection of six samples on a chromatographic plate in a stream of the mobile phase (on-line application/injection). In addition, the PPEC chamber was equipped with a thermostat. The device is characterized by an impressive throughput in comparison to the other planar technique, TLC/HPTLC. Although the developed device still needs improvement, it is, in our opinion, a considerable step toward possible automation of this planar separation technique.

Susceptibility of Staphylococcus aureus clinical isolates to propolis extract alone or in combination with antimicrobial drugs.

The objective of this study was to assess in vitro the antimicrobial activity of ethanolic extract of Polish propolis (EEPP) against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates. The combined effect of EEPP and 10 selected antistaphylococcal drugs on S. aureus clinical cultures was also investigated. EEPP composition was analyzed by a High Performance Liquid Chromatography (HPLC) method. The flavonoid compounds identified in Polish Propolis included flavones, flavonones, flavonolols, flavonols and phenolic acids. EEPP displayed varying effectiveness against twelve S. aureus strains, with minimal inhibitory concentration (MIC) within the range from 0.39 to 0.78 mg/mL, determined by broth microdilution method. The average MIC was 0.54 ± 0.22 mg/mL, while calculated MIC50 and MIC90 were 0.39 mg/mL and 0.78 mg/mL, respectively. The minimum bactericidal concentration (MBC) of the EEPP ranged from 0.78 to 3.13 mg/mL. The in vitro combined effect of EEPP and 10 antibacterial drugs was investigated using disk diffusion method-based assay. Addition of EEPP to cefoxitin (FOX), clindamycin (DA), tetracycline (TE), tobramycin (TOB), linezolid (LIN), trimethoprim+sulfamethoxazole (SXT), penicillin (P), erythromycin (E) regimen, yielded stronger, cumulative antimicrobial effect, against all tested S. aureus strains than EEPP and chemotherapeutics alone. In the case of ciprofloxacin (CIP) and chloramphenicol (C) no synergism with EEPP was observed.

Synthesis and applications of [60]fullerene nanoconjugate with 5-aminolevulinic acid and its glycoconjugate as drug delivery vehicles.

The 5-aminolevulinic acid (5-ALA) prodrug is widely used in clinical applications, primarily for skin cancer treatments and to visualize brain tumors in neurosurgery. Unfortunately, its applications are limited by unfavorable pharmacological properties, especially low lipophilicity; therefore, efficient nanovehicles are needed. For this purpose, we synthesized and characterized two novel water-soluble fullerene nanomaterials containing 5-ALA and d-glucuronic acid components. Their physicochemical properties were investigated using NMR, XPS, ESI mass spectrometry, as well as TEM and SEM techniques. In addition, HPLC and fluorescence measurements were performed to evaluate the biological activity of the fullerene nanomaterials in 5-ALA delivery and photodynamic therapy (PDT); additional detection of selected mRNA targets was carried out using the qRT-PCR methodology. The cellular response to the [60]fullerene conjugates resulted in increased levels of ABCG2 and PEPT-1 genes, as determined by qRT-PCR analysis. Therefore, we designed a combination PDT approach based on two fullerene materials, C60-ALA and C60-ALA-GA, along with the ABCG2 inhibitor Ko143.

Oligomerization oscillations of L-lactic acid in solution.

We employ high-performance liquid chromatography with diode array, evaporative light scattering, and mass spectrometric detection to monitor the oligomerization of L-lactic acid in pure acetonitrile and in 70% aqueous ethanol. The production of higher oligomers appears to proceed in an oscillatory fashion. A model is presented that involves the formation of aggregates (micelles), which catalyze the oligomerization.

Binary HPLC-diode array detector and HPLC-evaporative light-scattering detector fingerprints of methanol extracts from the selected sage (Salvia) species.

This study is focused on an important family of the sage (Salvia) species, with Salvia officinalis L. having a long-established position in European traditional medicine. Binary fingerprints (chromatographic profiles) of six different sage species were compared using HPLC coupled with two different detectors: the diode-array detector and the evaporative light-scattering detector. Advantages of using binary fingerprinting over single-detector fingerprinting are demonstrated and discussed, with selected examples. Experimental data are provided for a comparison of the chemical composition of sage samples originating from two harvesting seasons (2007 and 2008). A number of phytochemical standards (i.e., certain phenolic acids, flavonoids, and coumarin) were used that allowed identification and semiquantitative estimation of these particular compounds in the analyzed methanol extracts.

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.

Preliminary evaluation of application of a 3-dimensional network structure of siloxanes Dergall preparation on chick embryo development and microbiological status of eggshells.

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P < 0.05) in the group sprayed with a 6% concentration of the solution. This phenomenon was caused by embryo mortality in the first week of incubation. At the concentration of 0.6%, Dergall exhibited strong antibacterial properties against bacteria such as Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella flexneri, and Salmonella typhimurium. For Streptococcus pyogenes, the highest antibacterial activity of Dergall was reported in the concentrations of 100 and 50%. For Pseudomonas aeruginosa, no antibacterial activity of Dergall was generally observed, but in vivo testing showed a strong decrease of all gram-negative bacteria growth. Moreover, a prolonged antimicrobial effect lasting until 3 D after disinfection was observed, which makes Dergall a safe and efficient disinfectant.

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.

Effect of long-term cadmium and copper intoxication on the efficiency of ampullate silk glands in false black widow Steatoda grossa (Theridiidae) spiders.

The aim of the study was to compare cellular effects of xenobiotic cadmium and biogenic copper in ampullate silk glands of false black widow Steatoda grossa spider after long-term exposure via ingestion under laboratory conditions. Both the level of selected detoxification parameters (glutathione S-transferase, catalase, and the level of total antioxidant capacity) and degree of genotoxic changes (comet assay) were determined in the silk glands. Additionally the contents of selected amino acids (L-Ala, L-Pro, L-His, L-Phe, DL-Ile, and DL-Asn) in the hunting webs produced by spiders of this species were assessed. The ability of S. grossa females to accumulate cadmium was higher than that for copper. Long-term exposure of spiders to copper did not change the level of detoxification parameters, and the level of DNA damage in the cells of ampullate silk glands was also low. Cadmium had a stronger prooxidative and genotoxic effect than copper in the cells of the analyzed silk glands. However, regardless of the type of metal used, no significant changes in the level of amino acids in silk were found. The obtained results confirmed the effectiveness of metal neutralization mechanisms in the body of the studied spider species, which results in the protection of the function of ampullate silk glands.

Fatal case of poisoning with a new cathinone derivative: α-propylaminopentiophenone (N-PP).

PURPOSE: Similar to synthetic cannabinoids, synthetic cathinone derivatives are the most popular compounds among novel psychoactive substances. Along with a growing number of new cathinones, the number of consumers wishing to enrich their experience with these compounds is also growing, and the same can be said about the growing numbers of poisonings. The reason for overdosing is a lack of consumer awareness regarding composition of the product, with which they experiment, and even more, regarding concentration of psychoactive substances contained in the taken product. In this paper, we report a case of the purposeful intake of a high dose of powder containing a novel cathinone derivative, α-propylaminopentiophenone, which resulted in the deadly poisoning of a woman. METHODS: Aiming to identify this psychoactive substance causing the fatality, the postmortem specimens collected from the autopsy was analyzed by means of high-performance liquid chromatography coupled with mass spectrometry, and the analysis of a powder material found with the victim was additionally analyzed by means of gas chromatography with mass spectrometric detection. RESULTS: In the course of analysis performed on the specimens originating from autopsy (blood, eyeball fluid, liver, kidney and brain), high concentrations of α-propylaminopentiophenone were established, which was responsible for the death of a young woman. The same psychoactive compound was also identified in the powder material. CONCLUSIONS: To the best of the authors' knowledge, this is the first case reported in the literature on fatal poisoning with α-propyloaminopentiophenone.

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.