Chromatography Tests Research Articles

Structure-Directed Screening and Analysis of Thyroid-Disrupting Chemicals Targeting Transthyretin Based on Molecular Recognition and Chromatographic Separation.

Exposure to thyroid-disrupting chemicals (TDCs) poses a great threat to human health. However, the screening and analysis of TDCs in environmental samples remain a tough work. In this study, we reported a structure-directed strategy for analyzing TDCs targeting transthyretin (TTR) based on molecular imprinting and chromatographic separation. The imprinted composites were prepared using l-thyroxine (T4) as a template and a tryptophan-like monomer screened from the amino acid library. The imprinted composites exhibited an adsorption capacity of 22.2 μmol g-1 for T4 and an imprinting factor of 2.1. Chromatographic testing was then conducted among 72 chemicals using the imprinted composites-packed column. High retention factors were observed for chemicals that were structurally similar to T4. The chromatographic results were compared with a data set of 45 chemicals with known activities toward TTR. The results suggested that chemicals can be distinguished as TTR binders and nonbinders by retention factors, with a predictive accuracy of more than 90%. Moreover, the retention factors of chemicals were highly correlated with the reported relative potencies obtained from TTR assays. Thus, screening of TTR-binding chemicals can be realized through this simple chromatographic method. The imprinted composites were applied for target analysis and nontarget analysis of TTR-binding chemicals in dust samples. Three new TTR binders were successfully identified and verified by this method. The combination of molecular imprinting and chromatography opens up a new approach for screening TDCs targeting TTR.

Evaluating the influence of paper characteristics on the efficacy of new poly(vinyl alcohol) based hydrogels for cleaning modern and ancient paper

Wet cleaning of paper artworks by using hydrogels is a recently proposed process in the cultural heritage field. In this contest, on one side, research has focused on more effective and tuned cleaning hydrogels; on the other, the cleaning performances of the proposed hydrogels have been studied on ancient paper and very little on modern paper. This lack of information is not negligible, since the two kinds of paper differ strongly in composition. The difference, in turn could affect the performance of cleaning gels. In this article, new chemical hydrogels were synthetized using an oxidized polyvinyl alcohol (PVA) bearing an aldehyde group at each chain end, as cross-linking agent, i.e. telechelic PVA. PVA hydrogels have been characterized as candidates for paper cleaning applications. The possibility of tuning the mechanical and retentive properties, simply by changing the ratio of two polymer concentrations, their stability and transparency, indeed, render them, potentially suitable in cultural heritage field. The cleaning capability of two hydrogels, with different PVA/telechelic PVA ratios, has been assessed both on ancient paper, than a modern one and the results have been compared. The efficacy of these hydrogels has been demonstrated characterizing the samples before and after the cleaning process by means of a multidisciplinary approach involving spectroscopic and chromatographic tests.

Polyvinyl alcohol based hydrogels as new tunable materials for application in the cultural heritage field

Hydrogel-based cleaning of paper artworks is an increasingly widespread process in the cultural heritage field. However, the search for tuned (compatible, highly retentive and not perishable) hydrogels is a challenging open question. In this paper, a complete characterization of chemical hydrogels based on polyvinyl alcohol (PVA) crosslinked with telechelic PVA and their remarkable performances as gels for cleaning paper artworks are reported. The rheological properties, porosity, water content of these gels were determined and analyzed as a function of the components concentration during synthesis. Due mechanical and retentive properties, the reported gels are optimum candidates for paper cleaning applications. The efficacy of these PVA-based gels has been demonstrated applying them on the surface of the sheets of several paper artworks, and characterizing the samples before and after the cleaning process by means of a multidisciplinary approach involving spectroscopic and chromatographic tests.

Clinical Profile of Adult Patients Suffering from Scrub Typhus in Kasturba Hospital Wardha, Central India: A Study of 125 Patients

Background: Scrub Typhus is among the commonest causes of Acute Undifferentiated Fever during the monsoon in central India but there is a gap in knowledge regarding the data of the clinical profile in tropical rural area. Objective: To study the clinical profile of adult patients admitted with scrub typhus during the monsoon in rural central India. Material and methods: A Hospital based retrospective study was carried out in the 125 patients admitted to Medicine department of MGIMS during the monsoon period of June to September 2017. The History, clinical features, Lab profile, Treatment, complications and outcomes were studied closely. The data was retrieved from the Hospital Information System. The Outcome was studied in association with the severity of presentation, complications and treatment given. Diagnosis depended on recognizing the syndrome of non-specific symptoms of fever and cough, while diagnosis was confirmed using the IgM Scrub Typhus Rapid Chromatographic Test, which was considered as a Gold Standard in our study. Statistical analysis was done by descriptive and inferential statistics using chi-square test and software SPSS 22.0 version, EPI Info version 7 and Graph Pad Prism 6.0 version. Results: The clinical illness was characterized by Fever with chills (60.8%) and Cough (68.8%). The Disease was mostly acute (97%) presenting with a history of less than 10 days. The most characteristic physical sign was fever which was present in almost 60 percent of cases and the traditionally mentioned rash called Eschar was found in only 38 percent of patients. ARDS, nephropathy, Liver failure and Multi organ dysfunction occurred in 16%, 20%, 20% and 13.6% respectively. Though Scrub typhus occurs secondary to a chigger bite, only 6 patients out of 125 gave the actual history of insect bite. 9 patients out of 125 (7.2%) succumbed to death. Early treatment with Doxycycline shortened the duration of illness. No relapses occurred in those patients who received Doxycycline or Azithromycin for at least 7 days. Conclusion: Serious complications can occur if Scrub typhus is not recognized promptly. Early institution of Doxycycline can save lives in the patients of acute undifferentiated fever pending investigations. General physicians should be Scrub typhus minded to suspect and accurately treat the patients of fever.

Current Approaches to Valerian Tincture Standardisation in Terms of Assay

The quality control of the «Valerian rhizome and roots» herbal substance is carried out using high performance liquid chromatography (HPLC) according to the State Pharmacopoeia of the Russian Federation, XIV edition. The quantitative analysis of the active ingredients in valerian tincture is performed using a non-specific and non-selective spectrophotometric method. Therefore, it is important to introduce in Russia a more modern test procedure for quantitative determination of active ingredients in valerian tincture.The aim of the study was to develop a selective and sensitive HPLC procedure for quantitative determination of the total content of sesquiterpenic acids, expressed as valerenic acid, for the purpose of valerian tincture standardisation.Materials and methods: valerian tincture samples produced by seven Russian manufacturers were used as test samples, and valerenic acid was used as the reference standard. The quantitative analysis of the active ingredients was performed by two methods: spectrophotometry at 512 nm following the reaction of valerenic acid ethylester with hydroxyalamine and ferric chloride, and by HPLC using a Nucleosil C18 column, 125×4.6 mm, 5 µm particle size, in gradient elution mode, with detection at 220 nm.Results: the spectrophotometric technique was shown to be insufficiently specific. The authors of the study validated the chromatographic test procedure, established system suitability criteria, and compared the results obtained by the two test procedures. They also determined a tentative standard of the total content of sesquiterpenic acids, expressed as valerenic acid, obtained by HPLC.Conclusions: the HPLC assay developed for quantitative determination of active ingredients in valerian tincture is more specific as compared to the spectrophotometric technique, as the sum of the peaks of valerenic and acetoxyvalerenic acids and the results for the reference standard are taken into account during calculations. The new test procedure is in line with the cross-cutting standardisation principle and can be recommended for inclusion into the draft monograph «Valerian tincture».

Genetic association of MBL-2 gene polymorphisms with Filarial chyluria.

Lymphatic filariasis has become a significant public health issue in North India. The association of polymorphisms in MBL2 gene with filarial chyluria (FC) is evaluated in the North Indian patients for the first time. Hence, a tertiary care hospital based case-control study was conducted in north India where FC is endemic. Therefore, 186 confirmed patients of FC as cases and 210 age-, sex- and residence-matched subjects as controls were enrolled for the study. Filarial etiology was confirmed using diethylcarbamazine (DEC)-provocation test, immune chromatographic test and IgG/IgM antibody test. MBL2 gene polymorphisms at codon 54 and -221 promoter region were genotyped by PCR followed by RFLP. Wild-type, heterozygous and homozygous mutant frequencies of MBL2 genotype at the codon 54 were 57.5%, 32.8% and 9.7% in the case group and 62.9%, 30.5% and 6.7%, in controls, respectively. The same at the -221 position were 51.1%, 44.1% and 4.8% in FC patients and 44.3%, 40.0% and 15.7% in controls, respectively. Thus, results no significant association between MBL2 polymorphism at codon 54 and FC. However, polymorphism at the -221 promoter region is linked with FC with a significant odd-ratio of 0.27 (confidence interval at 95% was 0.12-0.59; p<0.001). This preliminary finding is intriguing for further confirmation using a larger study with more patients.

Laboratory investigation of the effect of GO-ZnO nanocomposite on drilling fluid properties and its potential on H2S removal in oil reservoirs

Drilling fluid loss is one of the basic problems in the drilling industry, which includes 10 to 20 percent of the cost of a drilling operation. It can cause some complications, such as creation of the water in oil emulsion. Furthermore, the presence of toxic hydrogen sulfide (H2S) gas in the excavation formations and the risk of its transfer to the surface, is a hazard to the health of employees on drilling rigs and causes corrosion of surface and subsurface equipment. In this study, the effect of adding Graphene Oxide-Zinc Oxide (GO-ZnO) nanocomposite on the drilling fluid properties and H2S removal were investigated. First, the GO-ZnO nanocomposite was synthesized. After XRD, SEM and EDS characterization tests and assuring the formation of the nanocomposite, drilling fluid samples were prepared by adding different concentrations of GO-ZnO nanocomposite to the base fluid (used in a real reservoir). Then the rheology, filtration, and gas chromatography tests were performed on various fluid samples. The results indicated desirable increases in the rheological properties such as plastic viscosity (5%–28%), yield point (25%–42%) and gel strength (25%–33%) and a significant decrease in the filtration rate. Besides, the results of the chromatographic tests indicated the ability of GO-ZnO nanocomposite to entirely remove the H2S in 15 min.

Deteksi Antibodi dan Antigen Cacing Filaria dan Indeks Entomologi Vektor Potensial Filariasis di Kota Langsa Provinsi Aceh

Humans can be infected with filariasis through mosquito bites with filaria worms in infective larva stage (L3). This study was conducted to detect the antibody, antigen, and the entomology index of potential mosquito vectors of filariasis in Langsa City. This research was conducted in Matang Seulimeng Village and Sungai Paoh Firdaus in Langsa City on November 2016 with 600 respondents. To determine the level of exposure to the infection source, a finger blood survey was conducted with a quick check using the rapid diagnostic test (RDT). RDT used is Brugia test to measure antibodies and ICT (Immuno Chromatographic Test) to measure filaria worm antigens. The existence of potential vectors is measured by entomology index, which are mosquito density, relative abundance, frequency of mosquitoes caught and species dominance. As the result, there were no respondent have been found positive for antibodies or filarial worms antigens in their blood. For the vector data, the potential mosquito vector of filariasis in Langsa City is Culex quinquifasciatus (23%) with peak biting activity at 01.00 02.00 pm.

KARAKTERISASI SENYAWA FENOLIK DARI BIJI BUAH RAMBUTAN (Nephelium lappaceum L.)

Rambutan seeds are one of the waste produced from rambutan that is potential to be used in the food and pharmaceutical fields because it is rich in nutrients and secondary metabolites. This research was carried out to characterize phenolic compounds from the ethyl acetate fraction by using chromatographic methods, phytochemical tests, and 1H-NMR analysis. The isolate was obtained as white powder with 6.2 mg and gave positive sign for phenolic by phytochemical test. 1H-NMR spectrum data (CD3OD, 500 MHz) showed the chemical shift characteristic of coumarin compounds at δH (ppm): 7.82 (1H, d, J= 8.3 Hz), 7.39 (1H, d, J= 7.55 Hz), 6.73 (1H, d, J= 8.05 Hz), and 5.61 (1H, d, J= 7.55 Hz), an anomeric proton at δH 4.8-5 ppm, the characteristics of the methyl group of L-rhamnose sugar at δH 1.29 ppm and other sugar protons at δH 3-4 ppm. Based on the 1H-NMR analysis and comparison with literature, a phenolic compound obtained from rambutan (Nephelium lappaceum L.) seeds is 7-O-β-L-rhamnose-8-hydroxycoumarin or 8-O-β-L-rhamnose-7-hydroxycoumarin.

Comprehensive HPTLC fingerprinting as a tool for a simplified analysis of purity of ginkgo products

Ethnopharmacological relevanceHerbal medicinal products based on ginkgo leaf refined dry extract (GBE) are an European development from the Eastern Asia traditionally used species Ginkgo biloba L. Nowadays, ginkgo products have increased the presence in the market, mainly as dietary supplements. Its adulteration with rutin and quercetin or herbal extracts rich in these compounds is a common practice. Tests featuring assays and detection of adulterants need to be performed on top of other existent methods (e.g. identification test). This may increase the costs of evaluating the quality of ginkgo products. Aim of the studyTo prove that comprehensive HPTLC fingerprinting can provide information beyond identification of ginkgo products, avoiding additional chromatographic tests for detection of adulterations. Materials and methodsThe information contained in the fingerprint obtained by HPTLC analysis of flavonoids was used for identification and for detection of adulterants, as well as to verify the limits of rutin and quercetin, which are normally determined by HPLC and used for detection of adulterants. For this purpose, peak profiles were generated from HPTLC chromatogram images. USP-HPLC methods were used for quantification of total flavonoids and testing the limits of rutin and quercetin. HPLC data were used to support the validity of the HPTLC method. An additional reversed phase HPTLC method was developed as a possible confirmatory method for the quercetin limit test. ResultsThe proposed HPTLC method uses a particular sequence of detections, resulting in a number of images, which are later interpreted in a certain order. It is able to identify ginkgo products, to detect adulterants (rutin, quercetin, sophora fruit and flower bud, and buckwheat), and, using peak profiles generated from the chromatogram images prior to and after derivatisation, to evaluate the limits of rutin and quercetin. Forty-eight out of fifty-nine ginkgo dietary supplements analysed contained one or more adulterants. Furthermore, results of the HPTLC and HPLC limit tests for rutin and quercetin were in agreement in 98% of the cases. Finally, a decision tree showing the sequence of interpretation of the fingerprints obtained with the different detections after a single HPTLC analysis is included to help the analyst to evaluate whether samples have the correct identity and whether they contain or not adulterants. ConclusionA single HPTLC analysis is able to provide information on identity and purity of the products. This simplifies the analytical workflow and reduces the number of analyses prescribed in the USP powdered ginkgo extract monograph.

Antibiotic-affinity chromatographic test strip for quantitative analysis and antibiotic resistance testing of Staphylococcus aureus

Bacterial analysis and antibiotic resistance testing (ART) are of great significance in clinical diagnosis and therapy of bacterial infectious diseases. In this work, a portable antibiotic-affinity chromatographic test strip has been developed for rapid analysis of Staphylococcus aureus (S. aureus) and further applied for ART of this pathogen. Porcine IgG was immobilized on a nitrocellulose membrane for capturing S. aureus based on the selective binding capability of the Fc fragment of IgG toward protein A on the surface of the target bacteria. Fluorescent microspheres modified with teicoplanin (TEI) were applied as signal substances to trace S. aureus utilizing the hydrogen bond conjugation between this antibiotic and Gram-positive bacteria. S. aureus can be analyzed within the concentration range from 1.4 × 103 CFU mL−1 to 1.4 × 107 CFU mL−1. The recovery values for spiked samples were 93.3–110.0%. The obtained results of ART for penicillin, daptomycin, gentamicin, cefoxitin and clindamycin against S. aureus showed agreement with those of traditional broth dilution method. The procedures for bacterial analysis and ART can be accomplished within 20 and 110 min, respectively. The antibiotic-affinity chromatographic test strip showed great promise in point-of-care testing because of its ideal portability and rapidity.

Evaluation of superficially porous particle based zwitterionic chiral ion exchangers against fully porous particle benchmarks for enantioselective ultra-high performance liquid chromatography

Zwitterionic chiral ion-exchange selectors (ZWIX) obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond were immobilized on three different silica particle types, viz. 120 Å 3 μm fully porous particles (FPP), 200 Å 3 μm FPP and 160 Å 2.7 μm superficially porous particles (SPP). Selector densities were determined by elemental analysis and the porosities of packed columns measured by inverse size exclusion chromatography with polystyrene standards. Liquid chromatographic tests with a set of chiral zwitterionic, acidic and basic analytes showed that the surface chemistry was successfully transferred to the distinct particle morphologies. The chromatographic performance of the three columns was evaluated by acquiring van Deemter curves. The results showed that the column packed with the SPP particles gives the best performance and kinetic plots further demonstrated that they represent the most favorable compromise in terms of speed, efficiency and pressure drop. Sub-minute separations could be accomplished at much lower pressure drop on the core-shell column, e.g. 2-amino-2-phenylbutyric acid was baseline separated in less than 15 s on a 5 cm long column. The Maxwell effective medium theory with second order approximation was applied to calculate effective diffusion in the mesoporous zones of SPP and FPP, which allowed eventually to deconvolute the individual peak dispersion contributions (ha, hb, hc,m, hc,s, hc,ads). The efficiency gain of the 160 Å SPP column compared to the 120 Å FPP (benchmark) column was mainly due to lower eddies (ha), smaller c-term accounting for slow adsorption-desorption kinetics in enantioselective chromatography (hc,ads), and also due to lower stationary mass transfer resistance (hc,s). Enhanced effective diffusion (Deff) in the SPP column contributed to a lower longitudinal diffusion (hb). In contrast, the mobile phase mass transfer coefficient was similar in the two columns leading to comparable hc,m contributions. This study discloses some options for improvement of the efficiency of ZWIX-type chiral columns such as replacing narrow pore (120 Å) by wide pore (200 Å) particles, substituting FPP by SPP and reducing the selector density on the surface.

Development of a high performance thin layer chromatography method for the rapid qualification and quantification of phenolic compounds and abscisic acid in honeys

Honey is a natural product with a complex chemical composition consisting of sugars and other bioactive compounds. It is important in many traditional systems of medicine, exhibiting interesting bioactivities, in particular antimicrobial, anti-inflammatory and antioxidant effects. Authentication of botanical origin of honeys is particularly important in this context. Therefore, methods for quality control of honey and detection of its adulteration are very important.A HPTLC method for the quantitative determination of phenolic compounds in honey was developed for the first time. Seven phenolic compounds were detected and determined quantitatively in lime and acacia honey samples. The obtained results show that the HPTLC profiles of phenolic compounds and abscisic acid can be useful for determining the floral origin of honeys. Chromatographic tests were supplemented by statistical analysis (PCA and HCA) led to the successful separation of acacia and lime honey samples.

Isolation and culture of Methanobrevibacter smithii by co-culture with hydrogen-producing bacteria on agar plates

ObjectivesMethanogenic Archaea are considered as extremely oxygen-sensitive organisms, and their culture is fastidious, requiring specific equipment. We report here conditions allowing the cultivation of Methanobrevibacter smithii in an anaerobic chamber without the addition of hydrogen. MethodsWe first enriched the stool sample in an anaerobic liquid medium. To cultivate M. smithii with Bacteroides thetaiotaomicron and other hydrogen-producing bacteria on solid medium in an anaerobic chamber, we divided the agar plates into two compartments and seeded each strain on each compartment. Methane production was assessed by gas chromatography, and the growing colonies were authenticated by MALDI-TOF MS. ResultsWe successfully cultured M. smithii from a liquid culture medium inoculated with stool collected from a healthy donor in an anaerobic chamber. The isolation in pure culture permitted successful culture on agar medium by our performing a co-culture with B. thetaiotaomicron. We also successfully tested the co-cultivation of M. smithii with other known hydrogen-producing bacteria. Gas chromatographic tests showed that these strains produced hydrogen in different amounts. Agar colonies of methanogens were obtained by co-culture with these bacteria, and methane production was detected. ConclusionsWe propose a new approach to isolate and cultivate new strains of M. smithii by using a co-culture–based technique that can facilitate and make available the isolation of new methanogenic Archaea strains in clinical microbiology laboratories.

Occurrence of feline leukemia feline immunodeficiency in domestic cats from the municipality of Mineiros, Goiás

The feline leukemia virus (FeLV) and the feline immunodeficiency virus (FIV) belong to the Retroviridae family and are important agents as they affect the immune system of felines. The objective of this study was to evaluate the occurrence of FIV and FeLV in cats from the municipality of Mineiros, in the Goiás state, randomly selected. Seventy-two cats from the municipality of Mineiros were randomly evaluated using the ALERE™ FIV Ac / FeLV Ag TEST KIT chromatographic test, and of these 12.5% showed reagents to FIV and 2.77% to FeLV, with a higher frequency of infection in male cats, both adults and non-defined, who had free access to the street, which is a very relevant factor in the spread of FIV and FeLV, given their mode of transmission. Studies on the epidemiological investigation of these viral diseases in the region are scarce, but the data obtained in this area provide relevant information to the city's professionals, so that they can guide the owners, seeking control of the diseases in the animals.

Impact of Initial Wettability and Injection Brine Chemistry on Mechanical Behaviour of Kansas Chalk

The injection of seawater-like brines alters stiffness, strength and time-dependent deformation rates for water-saturated chalks. This study deals with the mechanical effects and oil production upon brine injection through wettability-altered samples. The results from two test programs are presented: (a) ‘Wettability determination program’ and (b) ‘triaxial test program’. Kansas chalk samples were saturated by a mixture of oil and water and aged over time at 90 °C. The wettability index of the altered samples was estimated using chromatographic separation tests by co-injecting sulphate ions that adsorb on the water-wet mineral surfaces and non-affine tracer. A good repeatability was observed. In the triaxial test program, unaged water-wet and aged mixed-wet samples were hydrostatically loaded to 1.5 times yield stress so stiffness and strength could be determined. The samples were kept at the same stress level over time to monitor the volumetric creep. After a stagnant flow period of 15 days, MgCl2 brine and seawater were flushed through the samples so the oil production and ion concentration of the effluent water could be obtained. The combined observations of the bulk volume, oil volume and estimated solid volume (from effluent analyses) enabled us to calculate pore volume and thereby oil saturation with time. The mixed-wet samples were found to be stiffer and stronger than the water-wet samples, and when the stress was kept at 1.5 times yield the creep curves overlapped. During the flow-through period, the changes in ion composition are insensitive to the presence of oil, and ongoing water weakening for mixed-wet samples is the same as in the water-wet samples. Further, we found that oil was only produced during the first 2–3 pore volumes (PVs) injected. Afterwards, no oil was produced even though the chemical reactions took place and pore volume reduced.

Comparative molecular characterization, pathogenicity and seroprevalence of avian influenza virus H9N2 in commercial and backyard poultry flocks

This study was conducted to perform the comparative molecular characterization of avian influenza virus (AIV) H9N2, pathogenicity and seroprevalence in commercial and backyard poultry flocks. Fifty commercial poultry flocks were investigated between 2012 and 2015. Eighteen flocks (36%) out of 50 were positive HA. Seven (38.9%) out of 18 were positive by chromatographic strip test for AI common antigen. By Real-time RT-PCR, only two flocks were positive H9. The molecular characterization of two different AI-H9N2 viruses, one isolated from a broiler flock (A/chicken/Egypt/Mansoura-18/2013) and the other from a layer flock (A/chicken/Egypt/Mansoura-36/2015) was conducted on HA gene. Moreover, a higher seroprevalence, using the broiler strain as a known antigen, was shown in backyard chicken flocks 15/26 (57.7%) than duck flocks 9/74 (12.2%). Interestingly, the pathogenicity index (PI) of the H9N2 broiler strain in inoculated experimental chickens ranged from 1.2 (oculonasal route) to 1.9 (Intravenous route). The PI indicated a highly pathogenic effect, with high mortality (up to 100%) in the inoculated chickens correlated with the high mortality (80%) in the flock where the virus was isolated. The firstly recorded clinical signs, including cyanosis in the combs and wattles and subcutaneous haemorrhages in the leg shanks and lesions, as well as histopathology and immunohistochemistry, revealed a systemic infection of the high pathogenicity with the H9N2 virus. Conversely, the H9N2 layer strain showed a low pathogenicity. In conclusion, as a first report, the molecular analysis and pathogenicity of the tested strains confirmed the presence of a high pathogenicity AIV-H9N2 with systemic infections.

CHROMATOGRAPHIC SYSTEM SUITABILITY TESTING: INFLUENCE OF CORRECTION FACTORS AND LATE ELUTING PEAKS OF IMPURITIES ON THE REQUIREMENT FOR THE SIGNAL-TO-NOISE RATIO (REVIEW)

Introduction. In case of methods of impurities determination quite often there are problems connected both with establishment and with check of implementation of the requirement to necessary sensitivity of a chromatographic system. Text. Influence of correction factors and late eluting peaks of impurities on the requirement for the signal-to-noise ratio (S/N) for chromatographic system suitability testing is considered. Conclusion. The recommendations are provided how to detect situations at which the requirement of S/N≥10 is insufficient for reliable detecting peaks of all impurities (related substances) and therewith how to correct concentration of test solution and/or the requirement to S/N for system suitability testing.

Identification of bioactivity markers of Sorbus domestica leaves in chromatographic, spectroscopic and biological capacity tests: Application for the quality control

This work is aimed at establishing analytical and bioactivity markers for the quality control of polyphenol-rich extracts of Sorbus domestica leaves – an antioxidant and anti-inflammatory agent. The potential marker analytes, representative of the main groups of S. domestica polyphenols, were pre-selected by LC–MS/MS analysis of the methanol-water (7:3, v/v) leaf extract (MED). For structural identification, two of them (rare flavonoids) were isolated from MED by preparative HPLC and analysed by 1D and 2D NMR (COSY, HMBC, HMQC) spectroscopy. The in vitro tests revealed that all analytes increase the non-enzymatic antioxidant capacity of plasma, effectively protect human plasma components against oxidative/nitrative damage induced by peroxynitrite (reduce the levels of thiobarbituric acid-reactive substances and 3-nitrotyrosine), and inhibit lipoxygenase and hyaluronidase. Considering the activity parameters and concentration levels, the highest contribution (70–80%) to the activity of the total phenolic fraction of MED was attributed to flavanols represented by (–)-epicatechin, procyanidin B2 and procyanidin C1. However, taking into account the observed synergistic effects, flavonoid and phenolic acid fractions (represented by chlorogenic acid, quercitrin, rutin, quercetin and its 3-O-(2′'-O-β-d-glucopyranosyl)-α-l-rhamnopyranoside and 3-O-(2′'-O-β-d-xylopyranosyl)-α-l-rhamnopyranoside) had also a substantial impact. All nine markers were thus applied as calibration standards, and a simple, accurate, reproducible and fully validated RP-HPLC-PDA method for the standardisation of the target extracts was proposed.

Stable-bond polymeric reversed-phase/weak anion-exchange mixed-mode stationary phases obtained by simultaneous functionalization and crosslinking of a poly(3-mercaptopropyl)methylsiloxane-film on vinyl silica via thiol-ene double click reaction

A polymeric reversed-phase/weak anion exchange (Poly-RP/WAX) mixed-mode stationary phase has been prepared by coating of a poly(3-mercaptopropyl)methylsiloxane film on vinyl-modified silica (100 Å, 5 μm) and simultaneous in situ functionalization with N-(10-undecenoyl)-3-aminoquinuclidine as well as crosslinking to the vinyl silica surface by solventless thiol-ene double click reaction. Such bonding chemistry showed greatly enhanced stability compared to brush-type analogs with bifunctional siloxane bonding to silica. Solid-state 29Si-CP/MAS NMR confirmed the immobilization of the siloxane layer. pH-Dependent ζ-potential determinations revealed a high anion-exchange capacity over the entire pH range with a maximum around pH 5. Oxidation of residual thiols yielded a zwitterionic Poly-RP/WAX/SCX mixed-mode phase with sulfonic acid endcapping and shifted the still net positive surface charge to lower ζ-potentials. It allowed a faster elution of strongly retained anionic species in particular of multiply negatively charged analytes such as oligonucleotides. Chromatographic tests under RPLC and HILIC elution mode with various test substances documented the multimodal utility and complementarity in retention profiles compared to RP, HILIC and commercial mixed-mode phases.