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.

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.

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.

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.

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.

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.

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.

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.

Piperazinyl fragment improves anticancer activity of Triapine.

A new class of TSCs containing piperazine (piperazinylogs) of Triapine, was designed to fulfill the di-substitution pattern at the TSCs N4 position, which is a crucial prerequisite for the high activity of the previously obtained TSC compounds-DpC and Dp44mT. We tested the important physicochemical characteristics of the novel compounds L1-L12. The studied ligands are neutral at physiological pH, which allows them to permeate cell membranes and bind cellular Fe pools more readily than less lipid-soluble ligands, e.g. DFO. The selectivity and anti-cancer activity of the novel TSCs were examined in a variety of cancer cell types. In general, the novel compounds demonstrated the greatest promise as anti-cancer agents with both a potent and selective anti-proliferative activity. We investigated the mechanism of action more deeply, and revealed that studied compounds inhibit the cell cycle (G1/S phase). Additionally we detected apoptosis, which is dependent on cell line's specific genetic profile. Accordingly, structure-activity relationship studies suggest that the combination of the piperazine ring with Triapine allows potent and selective anticancer chelators that warrant further in vivo examination to be identified. Significantly, this study proved the importance of the di-substitution pattern of the amine N4 function.

The newest cathinone derivatives as designer drugs: an analytical and toxicological review.

PURPOSE: Currently, among new psychoactive substances, cathinone derivatives constitute the biggest group, which are mainly classified into N-alkylated, 3,4-methylenedioxy-N-alkylated, N-pyrrolidinyl, and 3,4-methylenedioxy-N-pyrrolidinyl derivatives. These derivatives are actively being subjected to minor modifications at the alkyl chains or the aromatic ring to create new synthetic cathinones with the goal of circumventing laws. In this review, the new synthetic cathinones that have appeared on the illegal drug market during the period 2014-2017 are highlighted, and their characterization by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry is presented. METHODS: Various key words were used to conduct an extensive literature search across a number of databases, specifically for synthetic cathinones that emerged between 2014 and 2017. RESULTS: More than 30 new cathinone derivatives were discovered. The preexisting parental compounds for the new derivatives are also referenced, and their mass spectral data are compiled in a table to facilitate their identification by forensic toxicologists. CONCLUSIONS: To our knowledge, this is the most current review presenting new synthetic cathinones. Political authorities should take measures to implement and enforce generic scheduling (comprehensive system) laws to control the diversely modified synthetic cathinones. Supplementing the existing databases with new findings can greatly facilitate the efforts of forensic toxicologists.

Antibacterial potential of the Cistus incanus L. phenolics as studied with use of thin-layer chromatography combined with direct bioautography and in situ hydrolysis.

The main aim of this study was to detect and identify antibacterial components of fraction I derived from eleven commercial C. incanus herbal teas. Fraction I obtained by a well-established phytochemical protocol of a multi-step extraction was expected to contain flavonoid aglycons alone. Antibacterial profile of fraction I was demonstrated by means of thin-layer chromatography - direct bioautography (TLC-DB) using a Gram positive B. subtilis and a Gram negative A. fischeri strain. Six chromatographic zones of fraction I exhibited a well pronounced antibacterial potential. In qualitative terms, a good agreement was observed among chromatographic fingerprints and the corresponding bioautograms of the eleven samples. The compounds isolated from the six zones were analyzed by HPLC- diode array detector (DAD)-electrospray ionization (ESI)-MS. High numerical m/z values valid for certain constituents of these isolates suggested that some selected antibacterial components are, unexpectedly, flavonoid glycosides. In order to confirm this suggestion, three independent HPTLC methods (multi-development on amino phase and two two-dimensional developments on silica gel phase) were devised to in situ hydrolyze flavonoid glycosides and then separate and visualize the liberated glucose and some other building blocks of the zones' components. Additionally, the sensitivity of glucose detection with p-aminobenzoic acid reagent was enhanced by paraffin. In that way, the presence of the kaempferol glycosides (and not only the aglycones alone) in fraction I was confirmed. Beside kaempferol, p-coumaric acid as a building block unit was shown by HPLC-DAD-MS analysis of the hydrolyzed isolates. Results proved apigenin, kaempferide and acylated kaempferol glycosides (cis- and trans-tiliroside and their conjugates with p-coumaric acid) to be antibacterial components of fraction I. Because isomers of the coumaric acid conjugated tiliroside were detected only in fraction I and not in the crude C. incanus extract, they are regarded as artifacts produced through fractionation.

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.

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.

Chromatographic Enantioseparations in Achiral Environments: Myth or Truth?

Direct chromatographic enantioseparations are among the most important practical tasks of chromatography. The accepted rules and concepts of stereochemistry anticipated one type of chromatographic systems applicable only to such enantioseparations consisting either of chiral stationary phase, or chiral selector present in mobile phase. In such a model of chromatographic system, both racemic and non-racemic mixtures could be enantioseparated. Over the years, however, reports on successful chiral enantioseparations in non-chiral chromatographic systems have cumulated as well. To adapt the initial model to novel experimental evidence, an assumption had to be added stating that these were only non-racemic mixtures (and not racemic ones), which could be enantioseparated in achiral chromatographic systems, by granting them an inevitable chirality from the enantiomeric excess of a more abundant enantiomer. There still exists an overlooked portion of experimental evidence for successful enantioseparations of racemic mixtures in the non-chiral chromatographic systems, which cannot be explained by the accepted model. Facing this incompatibility between the model and practical results, we reflect on how to reconcile these two by questioning the possibility of pinpointing a true racemate with the help of our imperfect analytical tools.

Identification and physicochemical characterization of 4-fluorobutyrfentanyl (1-((4-fluorophenyl)(1-phenethylpiperidin-4-yl)amino)butan-1-one, 4-FBF) in seized materials and post-mortem biological samples.

During the last decade, a substantial growth in new psychoactive substances (NPS) has been recorded. Within this group, a considerably fast-growing sub-group is represented by the opioids, which are based on modifications of the fentanyl structure. In this study, identification and analytical characterization of a new fentanyl derivative, 4-fluorobutyrfentanyl (1-((4-fluorophenyl)(1-phenethylpiperidin-4-yl)amino)butan-1-one, 4-FBF), is described. Apart from the seized powder, 4-FBF was also identified in the e-cigarette liquid secured in Case 1. The concentration of the compound in the liquid was 35 mg/mL. The main component of the liquid was glycerol, and nicotine was also present. This substance was detected in seized material that originated from the illegal drug market in Poland. To the best of the authors' knowledge, this report presents the first two analytically confirmed cases of fatal intoxication associated with 4-FBF. Case 1 was a 26-year-old male drug user who was found dead at home. Case 2 was a 25-year-old female, occasional user of NPS and drugs, who was also found dead at home. The concentrations of 4-FBF in blood were 91 and 112 ng/mL and in urine 200 and 414 ng/mL. The concentrations of 4-FBF in liver and kidney were 902 and 411 ng/g, and 136 and 197 ng/g, for Case 1 and Case 2, respectively. Copyright © 2016 John Wiley & Sons, Ltd.

Spontaneous Pulsation of Peptide Microstructures in an Abiotic Liquid System.

We report observations of pulsating peptide formation and depeptidization in 70% aqueous acetonitrile solutions of l-Pro-l-Phe and l-Cys, resulting in the oscillatory appearance and disappearance of solid masses of microfibers and microspheres, respectively. We monitor the concentration changes of the monomeric amino acids by high-performance liquid chromatography. The concentration of all amino acid solutions used is 1.0 mg mL(-1), due to solubility limitations in 70% aqueous acetonitrile. The nonlinear concentration changes of l-Pro, l-Phe and l-Cys, and the amounts of the main peptidization products observed within our monitoring periods (for l-Pro-l-Phe, 250 h, and for l-Cys, 70 h) are typically from several to 20% of the original monomer concentrations. We follow the formation and decay of the insoluble peptides by turbidimetry. We also investigate the materials formed using scanning electron microscopy and mass spectrometry. We carry out numerical simulations on a simple model that reflects the main features of spontaneous pulsation of peptide fiber or sphere formation in this abiotic liquid system.