Identification and quantification of cannabinol as a biomarker for local hemp retting in an ancient sedimentary record by HPTLC-ESI-MS

Cannabis products have been used in various fields of everyday life for many centuries, and applications in folk medicine and textile production have been well-known for many centuries. For traditional textile production, hemp fibers were extracted from the stems by water retting in stagnant or slow-moving waters. During this procedure, parts of the plant material‚ among them phytocannabinoids‚ are released into the water. Cannabinol (CBN) is an important degradation product of the predominant phytocannabinoids found in Cannabis species. Thus, it is an excellent indicator for present as well as ancient hemp water retting. In this study, we developed and validated a simple and fast method for the determination of CBN in sediment samples using high-performance thin-layer chromatography (HPTLC) combined with electrospray ionization mass spectrometry (ESI-MS), thereby testing different extraction and cleanup procedures‚ as well as various sorbents and solvents for planar chromatography. This method shows a satisfactory overall analytical performance with an average recovery rate of 73%. Our protocol enabled qualitative and quantitative analyses of CBN in samples of a bottom sediment core‚ having been obtained from a small lake in Northern India, where intense local retting of hemp was suggested in the past. The analyses showed a maximum CBN content in pollen zone 4 covering a depth range of 262–209 cm, dating from approximately 480 BCE to 1050 CE. These findings correlate with existing records of Cannabis-type pollen. Thus, the method we propose is a helpful tool to track ancient hemp retting activities

Performance of Electropun Polyacrylonitrile Nanofibrous Phases, Shown for the Separation of Water-Soluble Food Dyes via UTLC-Vis-ESI-MS

Research in the miniaturization of planar chromatography led to various approaches in manufacturing ultrathin-layer chromatography (UTLC) layers of reduced thickness (<50 µm) along with smaller instrumentation, as targeted in Office Chromatography. This novel concept merges 3D print & media technologies with miniaturized planar chromatography to realize an all-in-one instrument, in which all steps of UTLC are automated and integrated in the same tiny device. In this context, the development of electrospun polyacrylonitrile (PAN) nanofiber phases was investigated as well as its performance. A nanofibrous stationary phase with fiber diameters of 150–225 nm and a thickness of ca. 25 µm was manufactured. Mixtures of water-soluble food dyes were printed on it using a modified office printer, and successfully separated to illustrate the capabilities of such UTLC media. The separation took 8 min for 30 mm and was faster (up to a factor of 2) than on particulate layers. The mean hRF values ranging from 25 to 90 for the five food dyes were well spread over the migration distance, with an overall reproducibility of 7% (mean %RSD over 5 different plates for 5 dyes). The individual mean plate numbers over 5 plates ranged between 8286 and 22,885 (mean of 11,722 over all 5 dyes). The single mean resolutions RS were between 1.7 and 6.5 (for the 5 food dyes over 5 plates), with highly satisfying reproducibilities (0.3 as mean deviation of RS). Using videodensitometry, different amounts separated in parallel led to reliable linear calibrations for each dye (sdv of 3.1–9.1% for peak heights and 2.4–9.3% for peak areas). Coupling to mass spectrometry via an elution head-based interface was successfully demonstrated for such ultrathin layers, showing several advantages such as a reduced cleaning process and a minimum zone distance. All these results underline the potential of electrospun nanofibrous phases to succeed as affordable stationary phase for quantitative UTLC

HPTLC assay of nicotine and cotinine in biological samples

This study presents the development of a simple high-performance thin layer chromatography (HPTLC) method for the determination of nicotine and its metabolite cotinine in human plasma and urine. The following mobile phases: methanol: ammonia (100:1.5, v:v), chloroform: acetone: ammonia (48.75: 48.75: 2.5, v:v:v), methanol: chloroform: ammonia (48.75: 48.75: 0.5, v:v:v) and glass plates precoated with silicagel 60 F254 (20x20) as a stationary phase were used. Densitometric scanning was performed at 263 nm. Two different extraction procedures have been applied: liquid-liquid extraction using dichloromethane at alkaline pH and solid-phase extraction using C18 cartridges. Preliminary tests in order to establish the system of solvents for development, as well as the range of linearity, were conducted. The best separation of nicotine and cotinine was obtained by using methanol: chloroform: ammonia (48.75: 48.75: 0.5, v:v:v) as the mobile phase. The liquid-liquid extraction technique led to better results than solid phase extraction. The regression curves were linear (with a corresponding correlation coefficient higher than 0.99) in the quantities range of 200 ng–1000 ng/spot for both nicotine and cotinine. The UV spectra confirm the identification of nicotine and cotinine both in the standards and in the extracts after liquid-liquid extraction. The proposed method can be applied for the simultaneous evaluation of nicotine and cotinine in biological samples at toxic/lethal levels. Thus, the method may be applicable in lethal nicotine intoxication cases in forensic toxicological analysis

Analysis of food supplement with unusual raspberry ketone content

In recent years food supplement market increased constantly, including slimming products and against obesity. The case of rasberry ketone (RK) is here reported. HPTLC and HPLC-DAD analyses on a marketed product containing raspberry juice evidenced an abnormal quantity of RK, not in accordance with the juice natural content. The reported data confirm the need of adequate controls on marketed food supplements and the necessity of a complete adherence between labelling and real constitution of the product. Practical Applications: Determining the natural origin and assuring the consumers' safety for raspberry-based food supplement

Overpressured layer chromatography: from the pressurized ultramicro chamber to BioArena system

The pressurized ultramicro (UM) chamber as a closed adsorbent layer chamber enables the use of a special chromatoplate and a pump to increase and optimize the mobile phase flow velocity through an optional development distance in an adsorbent layer. This chamber is the basic instrument of overpressured-layer chromatography (OPLC), which is a separation technique that combines the advantages of conventional TLC/HPTLC with those of HPLC. The versions of OPLC instrument, the character and achievement of off-line and on-line OPLC systems in analytical and preparative use are described. The development of BioArena as a complex bioautographic system means an exploitation of the unique advantages of planar-layer system for detection, isolation and identification of new antimicrobials, antineoplastics, biopesticides and other biologically active substances as well as for studying fundamental biochemical reactions and mechanisms

Preventive medicine center and health care for students of medicine and health professions at the Sapienza University of Rome: a research protocol

This project aims to develop a Center of Preventive Medicine and Health Care for the students of Medicine and Health profession at Sapienza University of Rome. At the beginning of the university career students, both residents and nonresident s, have to face several difficulties such as: starting smoking or the increase in cigarette consumption ; the independent management of their own health (especially for non residents consequently to the distance of the family doctor) ; unhealthy diet; tuberculosis (TB) biological risk during their university training. These aspects , especially if present at the same time, act as a source stress and adversely affect the quality of life and the academic performance. Specific aims of the project will be: implementing an ambulatory of Preventive Medicine; implementing a virtual ambulatory of general medicine; creating a website on the problems mentioned above. Data collected will be computerized to keep an electronic health record (HER) and to use the information for the purposes of scientific research. The Centre will act in close relationship with the Central Administration, with the Headmasters of the Medical Faculties, and in close collaboration with the Center of Occupational Medicine of Sapienza University

Seagrass Posidonia oceanica (L.) Delile as a marine biomarker: A metabolomic and toxicological analysis

A human-made environmental disaster due to the shipwrecked of Costa Concordia cruise vessel on the Tuscan Island of Giglio (Italy) coast and the possible pollutants release has been feared, so requiring the activation of removal operations and the monitoring of the marine environment. In the present study, the seagrass Posidonia oceanica (L.) Delile was used as a bioindicator for the impact of the Costa Concordia accident on the marine and coastal habitat. Different P. oceanica samples were collected in the shipwrecked site under different light conditions. Using high-performance thin-layer chromatography, metabolic analysis of the samples was carried out in order to highlight possible changes in the secondary metabolism due to the permanent shading and the presence of pollutant traces. Moreover, sample mutagenicity, as a consequence of the possible absorption of environmental toxicants leaked by the wreck, was assessed by the Ames test. The results highlighted the permanence of the Concordia-induced alteration in the plant secondary metabolites. However, absorption of chemical pollutants and carcinogens was not reported; this point was confirmed by the lack of mutagenic effects found for the samples tested. Our results clearly evidence that the environmental impact of Costa Concordia wreck and removal operations on P. oceanica was mainly due to the lack of light in the marine habitat. Present methodological approach, which combines metabolomic and genetic ecotoxicological analysis, could represent a suitable strategy to evaluate the impact of human disasters on the ecosystem and to monitor the environmental changes

Analysis of Nebivolol hydrochloride and Valsartan in Pharmaceutical Dosage Form by High Performance Thin Layer Chromatographic Method

A simple, accurate and precise high performance thin layer chromatographic method has been developed for the estimation of Valsartan and Nebivolol hydrochloride simultaneously from a tablet dosage form. The method employed silica gel 60 F254 pre-coated plates as stationary phase and a mixture of Ethyl acetate: Methanol: Ammonia (6.5:2.5:0.5 %v/v/v) as mobile phase. Densitometric scanning was performed at 280 nm using a Camag TLC scanner 3. Beer&#x2019;s law was obeyed in the concentration range of 800ng/spot-2400ng/spot for Nebivolol hydrochloride and 200ng/spot-1000ng/spot for Valsartan. The Retention factor for Nebivolol hydrochloride is 0.75 &#xb1; 0.04 and is 0.27 &#xb1; 0.01 for Valsartan . The method was validated as per ICH Guidelines, proving its utility in estimation of Valsartan and Nebivolol hydrochloride in combined dosage form

A New Phytochemical Screening Programme used for Crops grown with Organic and Conventional Methods

A broad screening programme, covering the most general phytochemical groups of compounds, was developed on the basis of Thin Layer Chromatography (TLC). A total of 46 TLC systems, comprising 26 derivatization reagents, 3 stationary phases, and 4 mobile phases, were included. The TLC systems were classified according to the groups of phytochemical compounds detected: Alcohols and phenolic compounds; Carbohydrates; N-containing compounds; Organic acids and lipids; P-containing compounds; S-containing compounds, and Terpenoids. Furthermore, one group of TLC systems detected compounds from several of the mentioned groups. The screening programme was applied in the screening of potatoes (S. tuberosum L.), peas (P. sativum L.), kale (B. oleracea L.), carrots (D. carota L.), and apples (M. domestica Borkh.), cultivated with combinations of organic and conventional methods for plant protection and nutrient supply, for phytochemical differences (biomarkers). Distinctive phytochemical differences were found between the differently cultivated samples of these crops. In peas and carrots only one biomarker was found. In peas the biomarker was related to the soil conditions, while the biomarker in carrots was related to the use of pesticides. In potato, two biomarkers related to the use of pesticides were found. Three biomarkers were found in kale. Two of these could be related to the use of pesticide, while the last was related to either fertiliser or soil conditions. Several biomarkers were found apples, but a relation to the cultivation methods was not clear. Three of the biomarkers in apples could be related to either the use of pesticides or fertiliser, while no conclusions could be drawn from the other biomarkers found. The results of the screening programme form the basis for a potential development of a kit to detect whether crops are organically- or conventionally cultivated. Furthermore, the results from this part and other parts of the project "Organic food and health – a multigenerational animal experiment" provide basis for the selection of which secondary compounds to quantify by specific chemical analysis, isolate, and/or structure elucidation

Recent developments in the rapid analysis of plants and tracking their bioactive constituents

Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input